Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy (3D-SIM)

Summary

The present protocol describes a method to visualize and measure actin rings and other components of the membrane periodic skeleton of the axon initial segment using cultured rat hippocampal neurons and 3D-structured illumination microscopy (3D-SIM).

Abstract

The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to the maintenance of neuronal polarity by sorting somato-dendritic cargo. A membrane periodic skeleton (MPS) comprising periodic actin rings provides a scaffold for anchoring various AIS proteins, including structural proteins and different ion channels. Although recent proteomic approaches have identified a considerable number of novel AIS components, details of the structure of the MPS and the roles of its individual components are lacking. The distance between individual actin rings in the MPS (~190 nm) necessitates the employment of super-resolution microscopy techniques to resolve the structural details of the MPS. This protocol describes a method for using cultured rat hippocampal neurons to examine the precise localization of an AIS protein in the MPS relative to sub-membranous actin rings using 3D-structured illumination microscopy (3D-SIM). In addition, an analytical approach to quantitively assess the periodicity of individual components and their position relative to actin rings is also described.

Introduction

The axon initial segment (AIS) is a short, uniquely specialized region of the proximal axon of vertebrate neurons¹. The AIS comprises a transport filter and diffusion barrier essential in maintaining neuronal polarity by sorting somato-dendritic cargo^2,3,4,5,6,7. In addition, the unique structure of the AIS allows it to accommodate clusters of voltage-gated ion channels that facilitate its function as the site of action potential initiation

Protocol

Primary hippocampal neurons used in these experiments were harvested¹⁶ from embryonic day 17 Wistar rat embryos of either sex under the ethical guidelines of the University of Helsinki and Finnish law.

1. Sample preparation

1. On high-fidelity glass coverslips, allow rat hippocampal neurons to grow in sparse culture conditions (~5,000-10,000 cells/cm²) for 14 days (14 days-in-vitro).
   NOTE: Using sparse cultures reduces the chances of overlapping neurites, which helps quantify the MPS.
2. Fix neurons using 4% paraformaldehyde for 12 min at room temperature. Wash the c....

Results

Using cultured rat hippocampal neurons and 3D-SIM, a protocol is described to visualize and measure actin rings and other components of the MPS in the AIS. Reconstructions of image stacks showed clear periodicity of actin rings (Figure 2A). In our hands, the mean inter-peak distance of actin rings in the MPS, visualized using Alexa 488-tagged phalloidin, was 190.36 ± 1.7 nm (mean ± SEM). This is in line with the previously reported average distance of ~190 nm between actin rings in the MPS. .......

Discussion

The protocol described here provides a method for visualizing and measuring MPS proteins using the super-resolution technique. As actin rings and other MPS components display a periodicity of ~190 nm^9,10, conventional diffraction-limited imaging approaches cannot reveal the details of the MPS. Several microscopy setups may resolve diffraction-limited structures in super-resolution, and SIM represents a robust and uncomplicated option. Importantly, SIM is compatib.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24-well plates	Corning	3524
4% Paraformaldehyde
Alexa-488 Phalloidin	ThermoFisher	A12379
Alexa-647 anti-mouse	ThermoFisher	A31571
Anti-Ankyrin G antibody	UC Davis/NIH NeuroMab Facility, Clone 106/36	75-146
Anti-MAP2 antibody	Merck Millipore	AB5543
B-27	Invitrogen	17504044
Bovine Serum Albumin (BSA)	BioWest	P6154
Deltavision OMX SR	GE Healthcare Life Sciences	N/A
Fiji software package	ImageJ
GNU Octave	GNU
High performance coverslips	Marienfeld	117530
Immersion Oil Calculator	Cytiva Life Sciences		https://tinyurl.com/ImmersionOilCalculator
L-Glutamine	VWR	ICNA1680149
MATLAB R2020a	Mathworks
Neurobasal media	Invitrogen	21103049
OMX SR	Delta Vision OMX
Primocin	InvivoGen	ant-pm-1
ProLong Gold mounting media	Invitrogen	P10144
softWoRx Deconvolution	Cytiva Life Sciences
Superfrost Slides	Epredia	ISO 8037/1
TetraSpeck microspheres 0.1 µm	ThermoFisher	T7279
Triton-X	Sigma	X100