Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Summary

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Abstract

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images. Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3]. Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons. One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, and maintaining general cell health. Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons. A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde. Our approach to imaging GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events. The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Protocol

Part 1: Transfection of neurons using Lipofectamine 2000 (Invitrogen)

Equipment set-up:

Rat or mouse hippocampal neurons are cultured according to Kaech and Banker, 2006 [4]. The typical cell density used for transfections is 250,000 cells/6-cm dish. Required reagents and equipment include 50 mM kynurenic acid, regular benchtop tube holder, a benchtop cooler (best to keep Lipofectamine reagent cold),
Lipofectamine reagent, MEM, microfuge tubes, micropipetters, and sterile forceps.

Procedure:

1. For each transfection label two 1.5 mL tubes; one to contain the plasmid DNA, one to ....

Discussion

Live cell imaging is a challenging, but powerful technique for the direct observation of organelle transport in cultured neurons. Difficulties can arise upstream of the procedure with poor neuron health due to culture complications. Therefore, cell health (for examples see ref. 4) should be assessed before and after transfection by observing the coverslips while in growth medium using a tissue culture light microscope. The process of transferring neuron-containing coverslips to the imaging chamber can be stressful to.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MEM-Eagle with Earle salts and L-glutamine	Reagent	Mediatech, Inc.	10-010-CV
Lipofectamine 2000	Reagent	Invitrogen	11668-027	Transfection reagent
10X Hanks with Ca2+ and Mg2+	Reagent	GIBCO, by Life Technologies	14185-052	Live-imaging medium
1M HEPES	Reagent	GIBCO, by Life Technologies	15630-130	Live-imaging medium