Real-Time In Vitro Imaging of Axonal Branching in Mouse Cortical Neurons

Abstract

Source: Winkle, C. C., et al. Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis. J. Vis. Exp. (2016)

This video demonstrates a live-imaging technique using differential interference contrast (DIC) microscopy to observe real-time axonal branching in cortical neurons. Time-lapse videos capture the effects of netrin-1 treatment, a signaling molecule that induces localized exocytosis and cytoskeletal rearrangements in axons. These processes initiate membrane protrusions, which develop into mature axonal branches, providing insights into neuronal development.

Protocol

1. Differential Interference Contrast (DIC) Time-lapse Microscopy of Axon Branching

Note: While this protocol utilizes DIC, other transmitted light microscopy methods may be used for the same purposes (for example: phase contrast).

1. At 2 days in vitro (DIV), place glass bottom imaging dish containing untransfected neurons in pre-warmed environmental chamber to maintain a humid environment with 5% CO2 and 37 °C. Utilize a microscope equipped with a 60X Plan-Apochromat, 1.4 numerical aperture (NA) DIC objective lens and high NA condenser for best image quality and resolution.
2. Adjust the focal plane to find neurons through the oculars using transmitted light illumination.
3. Using the multi area acquisition function on imaging software, find and save the XYZ locations of at least 6 cells. Position the stage so that neurons of interest fit within the field of view for best results.
   1. Stimulate with 250 ng/ml netrin-1 and press 'start multi area acquisition'. Sequentially acquire images at each position every 20 sec for 24 hr, pausing acquisition and refocusing as necessary.
4. Review images in imaging software using Apps>Review Multi Dimensional Data>open file name. Identify stable axon branches (20 µm long) that form during the imaging session. Use the line draw tool to measure a stable axon branch from the base of the axon to the tip to ensure the 20 µm length qualification is met.
   1. In a spreadsheet, record the frame number after netrin stimulation when membrane protrusions initiate in areas where branches later form, as well as the frame number after netrin stimulation when nascent branches reach 20 µm in length.
   2. Multiply the number of frames between protrusion and branch formation by 20 sec to calculate the formation time per branch.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35mm Glass bottom live cell imaging dishes	Matek Corporation	p356-1.5-14-C	must be coated with 1mg/mL Poly-d-lysine and rinsed prior to plating cells
Olympus IX81-ZDC2 inverted microscope	Olympus
Lambda LS xenon lamp	Sutter Instruments Company
Environmental Stage top incubator	Tokai Hit
100x 1.49 NA TIRF objective	Olympus
Andor iXon EM-CCD	Andor
Odyssey Licor Infrared Imaging System	LI-COR	Odyssey CL-X	Used for scanning blots
Image studio software suite	LI-COR		Used for scanning on the Odyssey Infrared system; Image studio lite used for offline analysis of blots
Metamorph for Olympus	Molecular devices, LLC	version 7.7.6.0	Software used for all imaging and the analysis of DIC timelapse
CELL TIRF control software	Olympus		Software used to control lasers for TIRF imaging
Fiji (Image J)	NIH	ImageJ Version 1.49t
60x Plan Apochromat 1.4 NA objective	Olympus
40x 1.4 NA Plan Apochromat objective	Olympus
Neurobasal media	GIBCO	21103-049	Base solution for both serum free and trypsin quenching media
Supplement B27	GIBCO	17504-044	500ml/50mLs Serum free media and Trypsin Quenching media
L-Glutamine		35050-061	1mL/50mLs Serum free media
Fetal bovine serum	Corning/CELLGRO	35-010-CV
Hank's Balanced Salt Solution (HBSS)	Corning/CELLGRO	20-021-CV
Poly-D-Lysine	Sigma	p-7886