fluorescence photoactivation imaging of neurofilament transport in myelinated mouse axons 

Fluorescence Photoactivation Imaging of Neurofilament Transport in Myelinated Mouse Axons 

Grasp the proximal end of the nerve and slowly lay it down onto a cover slip. Then, straighten it under gentle tension. Place the micro aqueduct slide over the nerve with the grooved slide down and position the direction of flow parallel to the nerve. Flip the cover slip and micro aqueduct assembly over and place it in the perfusion chamber housing with the slide opposed to the outer gasket.To secure the perfusion chamber, place it in the metal housing and rotate the locking ring. Slowly depress the saline syringe plunger and fill the perfusion chamber. Keep the inlet and outlet tubing, outlet flask, and syringe elevated above the chamber itself at all times during the setup and imaging. Transfer the perfusion assembly to an inverted microscope stage and mount the saline syringe into the syringe pump. Then, start the motor and adjust the speed for a flow rate of 0.25 millilitres per minute.Connect the inline solution heater and set it to 37 degrees Celsius. Connect the objective heater and set it to 37 degrees Celsius. Apply oil to the objective and insert the perfusion chamber into the stage mount. Apply oil to the chamber heater pad and attach it to the perfusion chamber. Then, connect it and set it to 37 degrees Celsius.Lock the perfusion chamber into the stage adapter and bring the objective oil into contact with the cover slip on the underside of the chamber. Use brightfield illumination to focus on the layer of axons on the bottom surface of the nerve closest to the cover slip surface. Myelinated axons can be identified by the presence of a myelin sheath, which is visible under brightfield-transmitted light illumination without contrast enhancement.Acquire a brightfield reference image, recording the directionality of the nerve. Then acquire a confocal image using a 488 nanometer laser and an emission filter appropriate for photoactivatable GFP. Set the laser power to approximately five times normal imaging power and acquire an image with an exposure time of three to four minutes. Then acquire another confocal image to record the pre-activation autofluorescence after the bleaching step.Draw a line parallel to the axons with a length equal to the desired activation window size of the brightfield image. Using this line as a guide, draw a rectangular region of interest across the field of view perpendicular to the axons. Activate the GFP fluorescence in this region by pattern excitation with the 405 nanometer light, making sure that an image is acquired just prior to and immediately following activation. As the activation finishes, start a one-minute timer. When it goes off, start acquisition of a time-lapse series.

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Source:&#160;<a style='text-decoration:none;' href='https://app.jove.com/v/61264/imaging-and-analysis-of-neurofilament-transport-in-excised-mouse-tibial-nerve'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Boyer, N. P.&#160;et. al.,<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2020)</a> &#160;This video demonstrates the fluorescence photoactivation method to analyze neurofilament transport in myelinated axons of peripheral nerves. By tracking photoactivated green fluorophore-tagged neurofilaments, the technique provides insights into axonal transport in neurodegenerative diseases.

Source:&#160;Boyer, N. P.&#160;et. al., Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve.&#160;J. Vis. Exp. (2020)&#160;This ...

Begin with a perfusion assembly placed on the stage of an inverted microscope.The perfusion assembly contains a dissected peripheral nerve secured between a coverslip and a micro aqueduct slide with continuous saline perfusion.The nerve&#8217;s myelinated axons express the green fluorophore-tagged neurofilaments.Using brightfield illumination, focus on the myelinated axons.Switch to confocal mode, photobleach with high laser power, then capture a low-autofluorescence reference image using low laser power.Draw parallel lines along the axon to define the photoactivation window length. Use this as a guide to create a rectangular activation region.Illuminate this region with a specific wavelength to activate the green fluorophore-tagged neurofilaments, causing them to fluoresce. Acquire the image.After activation, capture time-lapse images.Over time, the fluorescent neurofilaments move in both directions and spread into adjacent flanking regions.The appearance of fluorescence in the flanking regions adjacent to the activation region confirms neurofilament transport.

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Video: Fluorescence Photoactivation Imaging of Neurofilament Transport in Myelinated Mouse Axons  - Experiment

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

JoVE Journal

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

Fluorescence Photoactivation Imaging of Neurofilament Transport in Myelinated Mouse Axons

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Fluorescence Photoactivation Imaging of Neurofilament Transport in Myelinated Mouse Axons