Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Summary

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Abstract

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.

Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

Introduction

A dendritic spine is a small protrusion of the neuron membrane. This characteristic structure is specialized to typically receive input from a single synapse and represents the physical contact area between two neurons. Most functionally mature dendritic spines consist of a globular tip, termed head, and a thin neck that connects the head to the dendritic shaft. However, spines are not static and actively move and change their morphology continuously even in the adult brain ². Within a 2 week period of time, rat primary hippocampal neuron cultures derived from late embryonic or early postnatal time develop complex dendritic arbors with numerous membrane pro....

Protocol

All experimental procedures involving animals were optimized to reduce animal suffering and were approved by the Commission for Animal Experimentation, University of Amsterdam, DEC protocol # DED204 and DED250.

1. Coverslip Preparation

1. Cut down the coverslips to a size of 15 mm x 15 mm using a carbide or diamond scribe, so that they fit into the wells of a 12-well plate.
2. While working in a fume hood, start coverslip coating by fully submerging coverslips in concentrated nitric acid solution (70% wt/wt) in a glass container. To ensure even distribution shake, then incubate for a minimum of 4 hr. It is possible to reuse ....

Results

Described here is a standardized working protocol for imaging dendritic spines from rat primary hippocampal neurons in vitro using SIM. The protocol workflow and its crucial steps are shown in Figure 1. Overall, the protocol takes approximately 2 weeks of experimental work separated in a first phase of sample preparation, including culture, development and transfection of rat primary hippocampal neurons and immunohistochemistry, and second phase of sample imaging using SIM. The rat primary hippo.......

Discussion

In this article a working protocol to image dendritic spines from rat primary hippocampal neurons cultured in vitro using SIM is described. The primary hippocampal neuron culture method is an adaptation of the original method described by Kaech and Banker ¹⁸. The main differences are the use of Neurobasal/B27 culture medium, which eliminates the requirement of astroglial feeder cultures, and the addition of the mitotic inhibitor FUDR on day 3 which promotes neuronal survival while suppressing glial pr.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fine forceps
Big forceps
Fine scissor
Big scissor
Blunt spatula
Dissecting microscope with illumination
Light microscope
37 °C water bath
Laminar flow cell culture hood
High-temperature dry-oven
Bunsen burner
Cell culture incubator (5% CO2, 37 °C)
Microcentrifuge
Orbital shaker
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Nikon structured illumination microscope setup consisting of:
Nikon Eclipse Ti research inverted microscope with Perfect Focus System
Nikon CFI Apo TIRF 100x oil objective lens (N.A. 1.49)
4 Coherent Sapphire Lasers (458, 488, 514 and 561 nm exitation wavelength)
SIM Illuminator
Nikon Stage Controller
MCL Nano-Drive piezo controller
Nikon Intensilight C-HGFIE mercury lamp
SIM Microscope Enclosure temperature control
Andor EM-CCD Camera iXon DU897
PC with Microsoft Windows 7 Home Edition
Nikon’s NiS Elements 6.14 SIM software package
Nikon type A immersion oil