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.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of The Ohio State University.
1. Preparation of nerve saline solution
<ol>
	<li>Make 100 mL of Breuer&#8217;s saline10: 98 mM NaCl, 1 mM KCl, 2 mM KH2PO4, 1 mM MgSO4, 1.5 mM CaCl2, 5.6% D-glucose, 23.8 mM NaHCO3 in double-distilled water.</li>
	<li>Bubble 95% oxygen/5% carbon dioxide (carbogen) through the sali...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+Spatial+Variations+in+Slow+Axonal+Transport+Velocity+Along+Peripheral+Motoneuron+Axons.">Temporal and Spatial Variations in Slow Axonal Transport Velocity Along Peripheral Motoneuron Axons.</a> Neuroscience. 102 (1), 193-200 (2001).</li><li>Jung, P., Brown, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+the+Slowing+of+Neurofilament+Transport+Along+the+Mouse+Sciatic+Nerve.">Modeling the Slowing of Neurofilament Transport Along the Mouse Sciatic Nerve.</a> Physical Biology. 6 (4), 046002 (2009).</li><li>Cohen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+size+index:+d.">The effect size index: d.</a> Statistical Power Analysis for the Behavioral Sciences 2nd ed. , 20-26 (1988).</li><li>Misgeld, T., Kerschensteiner, M., Bareyre, F. M., Burgess, R. W., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+axonal+transport+of+mitochondria+in+vivo.">Imaging axonal transport of mitochondria in vivo.</a> Nature Methods. 4 (7), 559-561 (2007).</li><li>Gilley, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+axonal+transport+and+locomotor+changes+and+tau+hypophosphorylation+in+a+"P301L"+tau+knockin+mouse.">Age-dependent axonal transport and locomotor changes and tau hypophosphorylation in a "P301L" tau knockin mouse.</a> Neurobiology of Aging. 33 (3), 1-15 (2012).</li><li>Marinkovic, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+deficits+and+degeneration+can+evolve+independently+in+mouse+models+of+amyotrophic+lateral+sclerosis.">Axonal transport deficits and degeneration can evolve independently in mouse models of amyotrophic lateral sclerosis.</a> Proceedings of the National Academy of Science of the United States of America. 109 (11), 4296-4301 (2012).</li><li>Milde, S., Adalbert, R., Elaman, M. H., Coleman, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+declines+with+age+in+two+distinct+phases+separated+by+a+period+of+relative+stability.">Axonal transport declines with age in two distinct phases separated by a period of relative stability.</a> Neurobiology of Aging. 36 (2), 971-981 (2015).</li><li>Gibbs, K. L., Kalmar, B., Sleigh, J. N., Greensmith, L., Schiavo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+axonal+transport+in+murine+motor+and+sensory+neurons.">In vivo imaging of axonal transport in murine motor and sensory neurons.</a> Journal of Neuroscience Methods. 257, 26-33 (2016).</li></ol>

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.

<table>
	<tbody>
		<tr>
			<td>14 x 22 Rectangle Gasket 0.1mm</td>
			<td>Bioptechs</td>
			<td>1907-1422-100</td>
			<td>inner gasket</td>
		</tr>
		<tr>
			<td>2-deoxy-D-glucose</td>
			<td>Sigma</td>
			<td>D6134</td>
			<td></td>
		</tr>
		<tr>
			<td>30mm Round Gasket w/ Holes</td>
			<td>Bioptechs</td>
			<td>1907-08-750</td>
			<td>outer gasket</td>
		</tr>
		<tr>
			<td>35 x 10mm dish</td>
			<td>Thermo Fisher</td>
			<td>153066</td>
			<td>dissection dishes</td>
		</tr>
		<tr>
			<td>40mm round coverslips</td>
			<td>Bioptechs</td>
			<td>40-1313-0319</td>
			<td></td>
		</tr>
		<tr>
			<td>60mL syringe - Luer-lock tip</td>
			<td>BD</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>Andor Revolution WD spinning-disk confocal system</td>
			<td>Andor</td>
			<td></td>
			<td>outfitted with Perfect Focus and FRAPPA systems</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher</td>
			<td>C79</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisher</td>
			<td>12-541-B</td>
			<td>for fluorescein slide</td>
		</tr>
		<tr>
			<td>D-(+)-glucose solution</td>
			<td>Sigma</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting pins</td>
			<td>Fine Science Tools</td>
			<td>26001-70</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td>fine tipped forceps</td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Zeiss</td>
			<td>47 50 03</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection pan with wax</td>
			<td>Ginsberg Scientific</td>
			<td>568859</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>Fine Science Tools</td>
			<td>14061-09</td>
			<td>initial dissection scissors</td>
		</tr>
		<tr>
			<td>FCS2 perfusion chamber</td>
			<td>Bioptechs</td>
			<td>060319-2-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein sodium</td>
			<td>Fluka</td>
			<td>46960</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline solution heater</td>
			<td>Warner Instruments</td>
			<td>SH27-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminectomy forceps</td>
			<td>Fine Science Tools</td>
			<td>11223-20</td>
			<td>initial dissection forceps</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Microaqueduct slide</td>
			<td>Bioptechs</td>
			<td>130119-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher</td>
			<td>12-544-3</td>
			<td>for fluorescein slide</td>
		</tr>
		<tr>
			<td>Microscope stage insert</td>
			<td>Applied Scientific Instrumentation</td>
			<td>I-3017</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective heater system</td>
			<td>Okolab</td>
			<td></td>
			<td>Oko Touch with objective collar</td>
		</tr>
		<tr>
			<td>Objective oil - type A</td>
			<td>Nikon</td>
			<td>discontinued</td>
			<td></td>
		</tr>
		<tr>
			<td>Plan Apo VC 100x 1.40 NA objective</td>
			<td>Nikon</td>
			<td>MRD01901</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Fisher</td>
			<td>P217</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6297</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium iodoacetate</td>
			<td>Sigma-Aldrich</td>
			<td>I2512</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Sage Instruments</td>
			<td>Model 355</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing adapter - female</td>
			<td>Small Parts Inc.</td>
			<td>1005109</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing adapter - male</td>
			<td>Small Parts Inc.</td>
			<td>1005012</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon tubing</td>
			<td>Bioptechs</td>
			<td></td>
			<td>1/16&#34; ID, 1/32&#34; wall thickness</td>
		</tr>
		<tr>
			<td>Vannas spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15018-10</td>
			<td>fine scissors</td>
		</tr>
	</tbody>
</table>

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...

Watch this Scientific Journal Video about Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve at JoVE.com

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

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 ...

This protocol allows direct imaging of the neurofilament population in myelinated axons from juvenile or adult mice using fluorescence microscopy, making it possible to measure the directionality and velocity of neurofilament transport in isolated nerve segments. This method depends on the availability of mice expressing a photoactivatable fluorescent fusion of the protein of interest, but can be adapted for use in any readily dissected nerve. To begin, pour oxygenated saline into a 60 milliliter syringe and ensure that there is minimal air remaining in the syringe.Connect the syringe to the tubing, then place the outflow tube into a waste flask. Place the outer gasket into the perfusion chamber housing, ensuring that the flow inlet and outlet posts are aligned with the holes in the gasket, then lay the inner gasket on a number 1.5 circular coverslip and carefully smooth out any wrinkles in the gasket to ensure a tight seal. Place the coverslip and gasket on a paper towel or a task wipe with the gasket facing up.Use a pair of large dissection scissors to make a dorsal incision in the skin near the middle of the spine and continue to cut around the ventral aspect of the animal. Slowly pull the skin away from the muscle and cut the fascia. Next, use microdissection scissors to make an incision in the thigh muscles midway between the tail and knee to expose the sciatic nerve.Make sure that the nerve, which is visible through the muscle, is not cut. Extend the incision dorsally and ventrally to remove the muscle, then remove the muscles of the calf, keeping the cuts shallow and short to avoid damaging nerves. Continue removing the muscles until the tibial nerve is fully exposed from the point where it branches from the sciatic nerve to the heel.Grasp the tibial nerve at the spine proximal end with a pair of forceps and cut it with a pair of microdissection scissors, making sure to not apply too much tension to the nerve. Carefully lift it away from the muscle and cut any attachments, taking care not to put tension on the nerve. Cut the spine distal end of the tibial nerve and transfer it to a small Petri dish of room temperature oxygenated saline.Starting from the proximal end of the nerve, gently grasp the exposed axon ends with a pair of very fine tip forceps. With a second pair of forceps, grasp the nerve sheath proximally and slowly pull towards the distal end of the nerve making sure that no undue tension is applied during this process. Grasp the proximal end of the nerve and slowly lay it down onto a coverslip, then straighten it under gentle tension.Place the microaqueduct slide over the nerve with the grooved slide down and position the direction of flow parallel to the nerve. Flip the coverslip and microaqueduct assembly over and place it in the perfusion chamber housing with the slide opposed to the outer gasket. To secure the profusion 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 profusion 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 milliliters 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 profusion 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 profusion chamber into the stage adapter and bring the objective oil into contact with the coverslip 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 coverslip 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 timelapse series. Add 250 milligrams of fluorescein powder to 0.5 milliliters of double distilled water and mix it until there are no visible particles, then spin the solution for 30 seconds in a tabletop centrifuge to sediment any undissolved material.Add eight microliters of fluorescein solution to a slide and apply a number 1.5 coverslip. Blot the excess liquid, seal the coverslip with nail polish, and allow the slide to dry. Place the slide coverslip side down on the inverted microscope stage and adjust the focus on the thin plane of fluorescence at the surface of the coverslip.Move the slide around to find a field of view that does not contain air bubbles or large fluorescein particles and acquire a Z-stack spanning six micrometers at 0.2 micrometer intervals such that the middle image is the original focus plane. Close all light path shutters including the camera shutter and set the laser power and the exposure time to zero seconds. Acquire a stack of 100 images with these settings, which will be averaged to generate the dark field image.Representative images from pulse-escape and pulse-spread experiments are shown here. For the pulse-escape method, the fluorescence decay kinetics in the activated region can yield information on long-term and short-term pausing behavior. The activated region can be short for these experiments.A longer activated region is used for the pulse-spread method in order to provide a larger pool of fluorescent neurofilaments. This lengthens the time over which the fluorescence increase in the flanking regions remains linear. 15 micrometer flanking windows are used here.The total fluorescence from the measuring window was quantified. The decay is biphasic for the pulse-escape method with an initial exponential decay representing the departure of on-track neurofilaments and a second slower exponential decay at 10 to 20 minutes that represents the departure of off-track filaments. For the pulse-spread strategy, the calculations of the velocity and directionality of the movement are dependent only on the slopes in the flanking windows.For the window lengths used here, the linear phase extends for about five minutes. A timelapse from a glycolytically inhibited nerve is shown here, demonstrating an apparent reduction in transport out of the activated region. Indeed, the distal and proximal slopes are significantly lower in nerves treated with inhibitor.There is also a significant decrease in the population velocity between these two conditions. When attempting this protocol, remember that it is crucial to be hasty in conducting the dissection and chamber assembly portions as nerve health declines following sacrifice of the animal. Neuronal cell culture can allow measurement and analysis of the movement of individual neurofilaments, which would supplement the population scale data gathered using this protocol.

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Research

JoVE Journal

Neuroscience

5.9K visualizações.  The Ohio State University. Este protocolo permite a imagem direta da população de neurofilamento em axônios mieliados de camundongos juvenis ou adultos usando microscopia de fluorescência, possibilitando medir a direcionalidade e a velocidade do transporte de neurofilamento em segmentos nervosos isolados. Este método depende da disponibilidade de camundongos expressando uma fusão fluorescente fotoativa da proteína de interesse, mas pode ser adaptado para uso em qualquer nervo prontamente dissecado. Para começar...