Imaging of the Axon Initial Segment

Abstract

The axon initial segment (AIS) is the first 20- to 60-μm segment of the axon proximal to the soma of a neuron. This highly specialized subcellular domain is the initiation site of the action potential and contains a high concentration of voltage-gated ion channels held in place by a complex nexus of scaffolding and regulatory proteins that ensure proper electrical activity of the neuron. Studies have shown that dysfunction of many AIS channels and scaffolding proteins occurs in a variety of neuropsychiatric and neurodegenerative diseases, raising the need to develop accurate methods for visualization and quantification of the AIS and its protein content in models of normal and disease conditions. In this article, we describe methods for immunolabeling AIS proteins in cultured neurons and brain slices as well as methods for quantifying protein expression and pattern distribution using fluorescent labeling of these proteins. © 2019 by John Wiley & Sons, Inc.

Figure 1.

A. Schematic of a neuron depicting soma, dendrites, axon and axon initial segment (AIS); presynaptic inputs and synaptic boutons are shown in light purple and green and the excitatory postsynaptic potential (EPSP) and action potentials are shown in red. The inset is a zoom of the AIS depicting myelin sheath and a node of Ranvier. B. Confocal microscopy of a primary rat hippocampal neuron, labeled with antibodies against voltage-gated sodium channel (PanNav; red) and microtubule associated protein 2 (MAP2; blue) antibody visualizing the AIS and the somatodendritic compartment, respectively. Figure was previously published in Hsu, Nilsson & Laezza, 2014.

Figure 2.

Rat primary hippocampal culture stained with A,D. MAP2 (white) and B,E. FGF14 (white), as described in basic protocol 1. C. Neurons were treated with 0.25% dimethyl sulfoxide (DMSO) before fixation, then one axon (the ~20 μm region indicated by the white arrows) and one dendrite (the ~20 μm region indicated by the yellow arrows) were identified, based on MAP2 staining (basic protocol 3). F. Neurons were treated with 4,5,6,7-tetrabromobenzotriazole (TBB) before fixation, then one axon (white arrows) and one dendrite (yellow arrows) were identified, based on MAP2 staining. This data set was previously reported in Hsu et al., 2016.

Figure 3.

A. Staining of the AIS protein β-IV Spectrin in the granule cells layer of the dentate gyrus (DG) in a fresh-frozen mouse hippocampus preparation, as described in basic protocol 2. B. Staining of the AIS sodium channels (PanNav) in the same preparation of A. C. Corresponding merge of β-IV Spectrin (A), PanNav (B), and TO-PRO-3 counterstain.

Figure 4.

Select the segmented line tool from the ImageJ toolbar. The brightness and contrast of the MAP2 channel (shown) have been adjusted in order to see all neurites.

Figure 5.

Set the segmented line tool width to 5 pixel width, in order to trace the full width of the AIS in the MAP2 channel.

Figure 6.

Use the segmented line tool to trace the neurite with the least MAP2 staining that is positive for the AIS marker. This neurite may not be visible until after brightness and contrast have been adjusted.

Figure 7.

Bring up the ROI manager using control + T.

Figure 8.

Overlay the ROI onto the channel of interest, which in this figure is the FGF14 channel. Brightness and contrast have been adjusted for display of this image. Do not adjust brightness or contrast for this image during actual analysis.

Figure 9.

Use control + k to create a graph of the intensity data of the FGF14 channel in ImageJ.

Figure 10.

Select “Data”>”Copy All Data” to select data to paste the FGF14 channel intensity data into an excel file.

Figure 11.

A. Fluorescent intensity of FGF14 at AIS of DMSO treated neuron as described in the statistical analysis. B. Fluorescent intensity of FGF14 at AIS of TBB treated neuron as described in the statistical analysis. C. Fluorescent intensity of FGF14 at a representative dendrite of a DMSO treated neuron as described in statistical analysis. D. Fluorescent intensity of FGF14 at a representative dendrite of a TBB treated neuron as described in statistical analysis. This analysis shows a total decrease in FGF14 fluorescent intensity but can also show shifts in protein expression along the AIS. As previously published in Hsu et al., 2016., treatment with TBB decreases expression of FGF14 at the AIS.

Figure 12.

A. Fluorescent intensity of FGF14 expressed as an average of fluorescent intensity along AIS, with control (DMSO-treated neuron) and experimental group (TBB-treated neuron) displayed on the x-axis, as described in the statistical analysis. B. Fluorescent intensity of FGF14 expressed as an average of fluorescent intensity along the dendrite. C. Fluorescent intensity of FGF14 expressed as the sum of fluorescent intensity along AIS, with control (DMSO-treated neuron) and experimental group (TBB-treated neuron) displayed on the x-axis, as described in the statistical analysis. D. Fluorescent intensity of FGF14 expressed as the sum of fluorescent intensity along the dendrite. As described in Hsu et al., 2016., treatment with TBB decreases intensity of FGF14 at the AIS and increases intensity at the dendrite, indicating loss of FGF14 axonal polarity in neurons.

Figure 13.

Automated image processing using NeuroTreeTracer of representative images from Figure 2. Input images are sum projection views of confocal images of neurons treated with DMSO A-C., or CK2 Inhibitor TBB D-E. The software automatically generates the following outputs: A,D. binary image segmentation; B,E. detection of the soma region; C,F. computation of centerline traces of each neurite emanating from the soma. Dendrites are indicated with yellow arrow and AIS are indicated with white arrows. Algorithm details are described in the basic protocol 4.

Figure 14.

A. Fluorescent intensity of FGF14 at AIS of DMSO treated neuron automatically traced with NeuroTree Tracer. B. Fluorescent intensity of FGF14 at the AIS of a TBB treated neuron automatically traced with NeuroTree Tracer. C. Fluorescent intensity of FGF14 at the same representative dendrite of DMSO treated neuron shown in Figure 2 automatically traced with NeuroTree Tracer. D. Fluorescent intensity of FGF14 at the same representative dendrite of TBB treated neuron shown in Figure 2 automatically traced with NeuroTree Tracer. This analysis shows a total decrease in FGF14 fluorescent intensity but can also show shifts in protein expression along the AIS. As previously published in Hsu et al., 2016., treatment with TBB decreases expression of FGF14 at the AIS.

Figure 15.

A. Fluorescent intensity of FGF14 expressed as an average of fluorescent intensity along AIS, with control (DMSO-treated neuron) and experimental group (TBB-treated neuron) displayed on the x-axis from neurons automatically traced with NeuroTree Tracer. B. Fluorescent intensity of FGF14 expressed as an average of fluorescent intensity along the dendrite from neurons automatically traced with NeuroTree Tracer. C. Fluorescent intensity of FGF14 expressed as the sum of fluorescent intensity along AIS, with control (DMSO-treated neuron) and experimental group (TBB-treated neuron) displayed on the x-axis from neurons automatically traced with NeuroTree Tracer. D. Fluorescent intensity of FGF14 expressed as the sum of fluorescent intensity along the dendrite from neurons automatically traced with NeuroTree Tracer. As described in Hsu et al., 2016., treatment with TBB decreases intensity of FGF14 at the AIS and increases intensity at the dendrite, indicating a shift in FGF14 polarity in neurons.

Figure 16.

Automated image analysis using NeuroTreeTracer. The software automatically processes A. a fluorescent image in 2-dimensional projection view and B. correspondingly generates the segmented image, C. detects the somas and D. computes the centerline traces corresponding to each cell; neurites corresponding to the same cell are displayed in matching color.