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

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 Na_Vβ4-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-Na_VII-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.

Keywords: axon initial segment; dentate granule cell; imaging; plasticity.

Figure 1

Schematic of candidate methods for live labeling the axon initial segment (AIS) in vitro. (1) Antibody targeted against the neurofascin extracellular domain (extNF ab). (2) Plasmid DNA fusion construct of neurofascin and GFP (NF186-GFP). (3) Plasmid DNA fusion construct of 270kDa-ankyrin-G (AnkG) and GFP (270kDa-AnkG-GFP). (4) Plasmid DNA fusion construct of the sodium channel β4 subunit and GFP (Na_Vβ4-GFP). (5) Plasmid DNA fusion construct of the sodium channel loop between the II and III α subunits and YFP (YFP-Na_VII–III).

Figure 2

Live AIS label with a neurofascin antibody does not report rapid structural plasticity. (A) Diagram of the experimental timeline used to test the extNF antibody as an AIS live label method. (B) Example maximum intensity projections of control or 3 h/6 h depolarized dentate granule cells (DGCs) bearing extNF live-labeled AISs (cyan) and stained post fixation with antibodies against AnkG (magenta, axonal) or prox1 (magenta, nuclear, white asterisks; cyan nuclear, black asterisks—due to secondary antibody contamination); arrowheads, DGC AIS start and end positions. (C) Plot shows distributions of AIS lengths measured with extNF applied live for either 3 or 6 h (blue) and AnkG (magenta) from control (Con) or depolarized (Dep) DGCs. Each point represents a single cell; black lines show mean ± SEM; orange symbols, values from example cells displayed in (B); Bonferroni post-test after two-way repeated measures ANOVA; ns, non-significant; **p < 0.01; ****p < 0.0001. (D) Correlation analysis for AISs labeled with both extNF and AnkG in the same neuron under control (dotted symbols) or depolarized (triangle symbols) conditions in three separate experiments: extNF post fixation (large open circles and triangles), extNF applied live for the duration of a 3 h treatment (medium sized light gray filled dots and triangles), extNF applied live for the duration of a 6 h treatment (small, dark gray filled dots and triangles). Lines show best linear regression fit for extNF applied post fixation (blue), live for 3 h (orange) or live for 6 h (red); Pearson’s correlation; **p = 0.008; ****p < 0.0001; ns, non-significant.

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.

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.

Figure 5

Lack of AIS specificity with the Na_Vβ4-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 Na_Vβ4FL-GFP (cyan) and lack of co-localization with AIS marker AnkG (magenta). Asterisks, soma; question marks, presumptive location of Na_Vβ4-GFP AIS; arrowheads, AnkG AIS start and end positions. (C) As in (B), but for the Na4ΔCT-GFP construct.

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.

Figure 7

YFP-Na_VII–III has a high degree of correlation with AnkG and is able to accurately track AIS plasticity following a 6 h depolarization treatment. (A) Diagram of the experimental timeline used to test the YFP-Na_VII–III construct as an AIS live label. (B) Maximum intensity projections of control or 3 h and 6 h depolarized DGCs bearing YFP-Na_VII–III live-labeled AISs (cyan) and stained post fixation with antibodies against AnkG (magenta) or prox1 (dark blue). Asterisks, soma; arrowheads, DGC AIS start and end positions. (C) Distributions of AIS lengths determined from YFP-Na_VII–III (blue) and AnkG (magenta) from control (Con) or depolarized (Dep) DGCs. Each symbol represents a single cell; black lines show mean ± SEM; orange symbols represent cells from panel (B); Bonferroni post-test after two-way repeated measures ANOVA; ns, non-significant; **p < 0.01; ****p < 0.0001. (D) Correlation analysis for AISs labeled with YFP-Na_VII–III and AnkG under control (dotted symbols) or depolarized (triangle symbols) conditions. Lines show best linear regression fit for a 3 h (orange) or 6 h treatment (red); orange symbols represent cells from panel (B); Pearson’s correlation; ns, non-significant; ***p < 0.001; ****p < 0.0001.

Figure 8

YFP-Na_VII–III labeled cells exhibit normal spike firing properties. (A) Brightfield image of a control untransfected neuron, and a maximum intensity projection of a cell expressing YFP-Na_VII–III (cyan), both patched in whole-cell mode. Asterisks, nucleus; arrowheads, DGC AIS start and end positions. (B) Example whole-cell current-clamp recordings of threshold APs fired to 10-ms somatic current injection from neurons displayed in (A); orange asterisk, V max; orange dotted lines, AP height and width calculation. (C) Single action potential current threshold; left, each dot represents a single cell from either the control untransfected group (black) or YFP-Na_VII–III expressing (cyan) group; orange colored symbols represent values from the example cells presented in panel (A); bars show mean ± SEM; unpaired t-test p = 0.76. Right, correlation of current threshold vs. AIS length revealed by live label with YFP-Na_VII-III; each dot shows one cell; orange dot shows the example cell from panel (A); line shows best fit linear regression; **p < 0.01. (D) Single action potential voltage threshold; details same as panel (C); left, p = 0.37; right, *p < 0.05. (E) Action potential height; details same as panel (C); left, p = 0.1; right, *p < 0.05. (F) Action potential width at half height; details same as panel (C); left, p = 0.1; right, ns, non-significant. (G) Example traces of maximum firing elicited via a 500 ms duration current injection used to probe repetitive spiking. (H) Maximum number of spikes fired to a 500 ms current injection pulse; details same as panel (C); left, p = 0.2; right, ns, non-significant.

Figure 9

Diagram summarizing the expression pattern of all five live in vitro AIS markers tested. (1) The live extNF antibody localized at the presumptive AIS endogenous site; however, it did not recapitulate the plasticity phenotype. The same was true for a version of the label conjugated with a 488 nm secondary antibody. (2) The NF186-GFP construct did not localize specifically to the AIS. (3) The 270kDa-Na_Vβ4-GFP construct, in both the full length or the C-terminal tail deletion versions, did not localize specifically to the AIS. (4) The 270kDa-AnkG-GFP construct did become localized specifically; however, it artificially lengthened the AIS. (5) The YFP-Na_VII–III fusion construct was localized specifically at the AIS, displayed a shortening phenotype following a 6 h depolarization treatment, and was also shown not to alter neuronal excitability.