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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.


NF186-GFP does not localize specifically to the AIS. (A) Diagram of experimental timeline for all the four transfection methods attempted (see Table 2); gray line highlights method used for the cells displayed in panel (B,C). (B) Maximum intensity projection of the best observed co-localization between the NF186-GFP construct (cyan) and AnkG (magenta). Asterisks, soma; question marks, putative start and end of NF186-GFP-labeled AIS; arrowheads, AnkG-labeled AIS start and end positions. (C) As in (B), but showing the most commonly observed expression pattern of NF186-GFP.
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Figure 4: NF186-GFP does not localize specifically to the AIS. (A) Diagram of experimental timeline for all the four transfection methods attempted (see Table 2); gray line highlights method used for the cells displayed in panel (B,C). (B) Maximum intensity projection of the best observed co-localization between the NF186-GFP construct (cyan) and AnkG (magenta). Asterisks, soma; question marks, putative start and end of NF186-GFP-labeled AIS; arrowheads, AnkG-labeled AIS start and end positions. (C) As in (B), but showing the most commonly observed expression pattern of NF186-GFP.

Mentions: We used a rat-specific, full-length neurofascin-186 construct tagged with GFP at its C-terminal domain (NF186-GFP), first described by Zhang and Bennett (1998) and used more recently by Dzhashiashvili et al. (2007). We reasoned that over-expression of NF186-GFP starting at a stage at which most AISs have already been established in culture (7 DIV) should not disrupt AIS assembly or disturb cell function. To test its expression pattern we initially transfected cells according to our standard lipofection protocol at 7 DIV (see “Materials and Methods” Section; Table 2), after which at 10 DIV we fixed and stained with an antibody against AnkG to check co-localization with endogenous AIS labeling, and prox1 to confirm DGC identity. We also ran several trials in which: (1) the transfection protocol was started earlier at 4 DIV, and (2) allowed cells a longer developmental time until 14 DIV (Figure 4A; Table 2). In all experiments, we failed to see precise co-localization between NF186-GFP and AnkG antibody label (Figure 4). The NF186-GFP signal tended to be strongly expressed in the cell soma and in a punctate fashion across the dendrites and the axon (Figure 4). Even in the best example of AnkG co-localization, NF186-GFP did not specifically localize to the AIS (Figure 4B), thereby failing to fulfil our first condition as a suitable AIS live-label.


Evaluating Tools for Live Imaging of Structural Plasticity at the Axon Initial Segment
NF186-GFP does not localize specifically to the AIS. (A) Diagram of experimental timeline for all the four transfection methods attempted (see Table 2); gray line highlights method used for the cells displayed in panel (B,C). (B) Maximum intensity projection of the best observed co-localization between the NF186-GFP construct (cyan) and AnkG (magenta). Asterisks, soma; question marks, putative start and end of NF186-GFP-labeled AIS; arrowheads, AnkG-labeled AIS start and end positions. (C) As in (B), but showing the most commonly observed expression pattern of NF186-GFP.
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Figure 4: NF186-GFP does not localize specifically to the AIS. (A) Diagram of experimental timeline for all the four transfection methods attempted (see Table 2); gray line highlights method used for the cells displayed in panel (B,C). (B) Maximum intensity projection of the best observed co-localization between the NF186-GFP construct (cyan) and AnkG (magenta). Asterisks, soma; question marks, putative start and end of NF186-GFP-labeled AIS; arrowheads, AnkG-labeled AIS start and end positions. (C) As in (B), but showing the most commonly observed expression pattern of NF186-GFP.
Mentions: We used a rat-specific, full-length neurofascin-186 construct tagged with GFP at its C-terminal domain (NF186-GFP), first described by Zhang and Bennett (1998) and used more recently by Dzhashiashvili et al. (2007). We reasoned that over-expression of NF186-GFP starting at a stage at which most AISs have already been established in culture (7 DIV) should not disrupt AIS assembly or disturb cell function. To test its expression pattern we initially transfected cells according to our standard lipofection protocol at 7 DIV (see “Materials and Methods” Section; Table 2), after which at 10 DIV we fixed and stained with an antibody against AnkG to check co-localization with endogenous AIS labeling, and prox1 to confirm DGC identity. We also ran several trials in which: (1) the transfection protocol was started earlier at 4 DIV, and (2) allowed cells a longer developmental time until 14 DIV (Figure 4A; Table 2). In all experiments, we failed to see precise co-localization between NF186-GFP and AnkG antibody label (Figure 4). The NF186-GFP signal tended to be strongly expressed in the cell soma and in a punctate fashion across the dendrites and the axon (Figure 4). Even in the best example of AnkG co-localization, NF186-GFP did not specifically localize to the AIS (Figure 4B), thereby failing to fulfil our first condition as a suitable AIS live-label.

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.