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

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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.


Lack of AIS specificity with the NaVβ4-GFP constructs. (A) Diagram of experimental timeline for the three transfection methods attempted (see Table 2); gray line highlights method used for the cells displayed in panels (B,C). (B) Maximum intensity projection of the usual expression pattern of NaVβ4FL-GFP (cyan) and lack of co-localization with AIS marker AnkG (magenta). Asterisks, soma; question marks, presumptive location of NaVβ4-GFP AIS; arrowheads, AnkG AIS start and end positions. (C) As in (B), but for the Na4ΔCT-GFP construct.
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Figure 5: Lack of AIS specificity with the NaVβ4-GFP constructs. (A) Diagram of experimental timeline for the three transfection methods attempted (see Table 2); gray line highlights method used for the cells displayed in panels (B,C). (B) Maximum intensity projection of the usual expression pattern of NaVβ4FL-GFP (cyan) and lack of co-localization with AIS marker AnkG (magenta). Asterisks, soma; question marks, presumptive location of NaVβ4-GFP AIS; arrowheads, AnkG AIS start and end positions. (C) As in (B), but for the Na4ΔCT-GFP construct.

Mentions: NaVβ4 is an auxiliary sodium channel subunit, thought to be responsible for the resurgent Na+ current through the action of its C-terminal tail as an open-channel blocker (Grieco et al., 2005; Bant and Raman, 2010). Due to the high concentration of sodium channels found at the AIS, it was previously shown that overexpression of NaVβ4-GFP labels AISs in fixed tissue from multiple brain regions, including the hippocampus in both slices and dissociated in vitro cultures (Buffington and Rasband, 2013). We tested two constructs where NaVβ4 was fused with GFP: (1) the full-length channel subunit (NaVβ4-FL-GFP, Figure 5B) and (2) a version with a truncation of the functionally important C-terminal tail (NaVβ4-ΔCT-GFP, Figure 5C). DGCs do not express NaVβ4 (Yu et al., 2003; Castelli et al., 2007), so we reasoned that these constructs—especially the functionally NaVβ4-ΔCT-GFP version—might be a benign way of labeling AISs live. We therefore transfected cells with NaVβ4-FL-GFP or NaVβ4-ΔCT-GFP at several stages: either at 7 DIV followed by fixation at 10 DIV as per our normal protocol, or at 11 DIV followed by fixation at 12 or 14 DIV in order to match previously published expression methods (Buffington and Rasband, 2013, Figure 5; Table 2). All cells were immuno-labeled against AnkG and prox1 post fixation. Unfortunately, we were not able to see clear co-localization of either NaVβ4-GFP probe with endogenous AnkG label. Regardless of the transfection protocol used, both constructs were strongly expressed in the cell soma and in a punctate fashion throughout dendrites and axons (Figure 5). We therefore concluded that—at least in our hands—neither of the NaVβ4-GFP plasmids was suitable for use as a live AIS marker.


Evaluating Tools for Live Imaging of Structural Plasticity at the Axon Initial Segment
Lack of AIS specificity with the NaVβ4-GFP constructs. (A) Diagram of experimental timeline for the three transfection methods attempted (see Table 2); gray line highlights method used for the cells displayed in panels (B,C). (B) Maximum intensity projection of the usual expression pattern of NaVβ4FL-GFP (cyan) and lack of co-localization with AIS marker AnkG (magenta). Asterisks, soma; question marks, presumptive location of NaVβ4-GFP AIS; arrowheads, AnkG AIS start and end positions. (C) As in (B), but for the Na4ΔCT-GFP construct.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Lack of AIS specificity with the NaVβ4-GFP constructs. (A) Diagram of experimental timeline for the three transfection methods attempted (see Table 2); gray line highlights method used for the cells displayed in panels (B,C). (B) Maximum intensity projection of the usual expression pattern of NaVβ4FL-GFP (cyan) and lack of co-localization with AIS marker AnkG (magenta). Asterisks, soma; question marks, presumptive location of NaVβ4-GFP AIS; arrowheads, AnkG AIS start and end positions. (C) As in (B), but for the Na4ΔCT-GFP construct.
Mentions: NaVβ4 is an auxiliary sodium channel subunit, thought to be responsible for the resurgent Na+ current through the action of its C-terminal tail as an open-channel blocker (Grieco et al., 2005; Bant and Raman, 2010). Due to the high concentration of sodium channels found at the AIS, it was previously shown that overexpression of NaVβ4-GFP labels AISs in fixed tissue from multiple brain regions, including the hippocampus in both slices and dissociated in vitro cultures (Buffington and Rasband, 2013). We tested two constructs where NaVβ4 was fused with GFP: (1) the full-length channel subunit (NaVβ4-FL-GFP, Figure 5B) and (2) a version with a truncation of the functionally important C-terminal tail (NaVβ4-ΔCT-GFP, Figure 5C). DGCs do not express NaVβ4 (Yu et al., 2003; Castelli et al., 2007), so we reasoned that these constructs—especially the functionally NaVβ4-ΔCT-GFP version—might be a benign way of labeling AISs live. We therefore transfected cells with NaVβ4-FL-GFP or NaVβ4-ΔCT-GFP at several stages: either at 7 DIV followed by fixation at 10 DIV as per our normal protocol, or at 11 DIV followed by fixation at 12 or 14 DIV in order to match previously published expression methods (Buffington and Rasband, 2013, Figure 5; Table 2). All cells were immuno-labeled against AnkG and prox1 post fixation. Unfortunately, we were not able to see clear co-localization of either NaVβ4-GFP probe with endogenous AnkG label. Regardless of the transfection protocol used, both constructs were strongly expressed in the cell soma and in a punctate fashion throughout dendrites and axons (Figure 5). We therefore concluded that—at least in our hands—neither of the NaVβ4-GFP plasmids was suitable for use as a live AIS marker.

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.