Name: Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve (Video) | JoVE Journal
Uploaded: 2020-08-31T13:00:00.000+00:00
Duration: 9 min 52 s
Description: Learn fluorescence photoactivation methods to analyze neurofilament transport dynamics in myelinated axons from transgenic mouse peripheral nerves. This protocol enables precise measurement of transport velocity and directionality.

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The authors would like to thank Paula Monsma for instruction and assistance with confocal microscopy and tibial nerve dissection and Dr. Atsuko Uchida, Chloe Duger and Sana Chahande for assistance with mouse husbandry. This work was supported in part by collaborative National Science Foundation Grants IOS1656784 to A.B. and IOS1656765 to P.J., and National Institutes of Health Grants R01 NS038526, P30 NS104177 and S10 OD010383 to A.B. N.P.B. was supported by a fellowship from the Ohio State University President&#8217;s Postdoctoral Scholars Program.

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</ol>

The authors have nothing to disclose.

Care must be taken in the analysis of pulse-escape and pulse-spread experiments because there is significant potential for the introduction of error during the post-processing, principally during the flat-field correction, image alignment and bleach correction. Flat-field correction is necessary to correct for non-uniformity in the illumination, which results in a fall-off in intensity across the field of view from center to periphery. The extent of non-uniformity is wavelength-dependent and thus, should always be perfor...

The axonal transport of neurofilaments was first demonstrated in the 1970s by radioisotopic pulse-labeling1. This approach has yielded a wealth of information about neurofilament transport in vivo, but it has relatively low spatial and temporal resolution, typically on the order of millimeters and days at best2. Moreover, radioisotopic pulse-labeling is an indirect approach that requires the injection and sacrifice of multiple animals to generate a single time course. With the discovery of fluorescent proteins and advances in fluorescence microscopy in the 1990s, it subsequently became possible to image neurofilament transport directly in cultured neurons on a time scale of seconds or minutes and with sub-micrometer spatial resolution, affording much greater insight into the mechanism of movement3. These studies have revealed that neurofilament polymers in axons move rapidly and intermittently in both anterograde and retrograde directions along microtubule tracks, propelled by microtubule motor proteins. However, neurofilaments are diffraction-limited structures just 10 nm in diameter that are typically spaced apart from their neighbors by only tens of nanometers; therefore, the polymers can only be tracked in cultured neurons that contain sparsely distributed neurofilaments so that the moving polymers can be resolved from their neighbors4. Thus, it is not presently possible to track single neurofilaments in axons that contain abundant neurofilament polymers, such as myelinated axons.
To analyze the axonal transport of neurofilaments in neurofilament-rich axons using fluorescence microscopy, we use a fluorescence photoactivation pulse-escape method that we developed to study the long-term pausing behavior of neurofilaments in cultured nerve cells4,5. Neurofilaments tagged with a photoactivatable fluorescent neurofilament fusion protein are activated in a short segment of axon, and then the rate of departure of those filaments from the activated region is quantified by measuring the fluorescence decay over time. The advantage of this approach is that it is a population-level analysis of neurofilament transport that can be applied on a time-scale of minutes or hours without the need to track the movement of individual neurofilament polymers. For example, we have used this method to analyze the kinetics of neurofilament transport in myelinating cultures6.
Recently, we described the development of an hThy1-paGFP-NFM transgenic mouse that expresses low levels of a paGFP-tagged neurofilament protein M (paGFP-NFM) in neurons under the control of the human neuron-specific Thy1 promoter7. This mouse permits the analysis of neurofilament transport in situ using fluorescence microscopy. In this article, we describe the experimental approaches for analyzing neurofilament transport in myelinated axons of tibial nerves from these mice using two approaches. The first of these approaches is the pulse-escape method described above. This method can generate information about the pausing behavior of the neurofilaments, but is blind to the direction in which the filaments depart the activated region, and therefore does not permit measurement of the net directionality and transport velocity8. The second of these approaches is a new pulse-spread method in which we analyze not just the loss of fluorescence from the activated region, but also the transient increase in fluorescence in two flanking windows through which the fluorescent filaments move as they depart the activated region in both anterograde and retrograde directions. In both approaches, parameters of neurofilament transport such as the average velocity, net directionality and pausing behavior can be obtained by using mathematical analysis and modeling of the changes in fluorescence in the measurement windows. Figure 3&#160;illustrates these two approaches.
This protocol demonstrates&#160;dissection and preparation of the nerve, activation and imaging of the paGFP fluorescence, and quantification of neurofilament transport from the acquired images using the FIJI distribution package of ImageJ9. We use the tibial nerve because it is long (several cm) and does not branch; however, in principle any nerve expressing paGFP-NFM is appropriate for use with this technique if it can be dissected and de-sheathed without damaging the axons.

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imaging and analysis of neurofilament transport in excised mouse tibial nerve

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

Figure 3 shows representative images from pulse-escape and pulse-spread experiments. We have published several studies that describe data obtained using the pulse-escape method and our methods for the analysis of those data5,6,7,8,17. Below, we show how the pulse-spread data can yield information on the directionality and velocity...

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...

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

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Learn fluorescence photoactivation methods to analyze neurofilament transport dynamics in myelinated axons from transgenic mouse peripheral nerves. This protocol enables precise measurement of transport velocity and directionality.

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

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