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1. Imaging Vesicle Motility
<ol>
	<li>Prepare the plasmids that will label each type of vesicle. For instance, use EGFP-Rab5 for early endosomes, EGFP-Rab7 for late endosomes, LAMP-EGFP for lysosomes, and EGFP-LC3 for autophagosomes. 
	NOTE: These plasmids are available on request from tsuruta.fuminori.fn@u.tsukuba.ac.jp. Typically, 3 -5 days in vitro (Division or DIV) neurons can be transfected with these plasmids using transfection reagents.</li>
	<li>Mix 4.0 &#181;g of plasmids with 200 &#181;L of serum-free medium by pipetting. In a separate tube, dilute 8 &#181;L of transfection reagent (see Table of Materials) in 200 &#181;L of serum-free medium. Incubate for 5 minutes at room temperature (RT).</li>
	<li>Mix the plasmid solution and transfection regent solution in step 1.2 by pipetting. Incubate for 20 min at RT.</li>
	<li>Add serum-free medium and 400 &#181;L of the plasmid solution from step 1.3 to each coated glass-bottom dish containing cortical neurons cerebral cortical culture medium. Incubate the neurons at 37 &#176;C in a 5% CO2 incubator for 30 min. Replace the medium with 2 mL of cerebral cortical culture medium. Incubate the neurons at 37 &#176;C in a 5% CO2 incubator for 1-2 days. 
	NOTE: Prepare a cerebral cortical culture medium consisting of neuron basal medium supplemented with 1x B-27 supplements, 2 mM L-glutamine, and 100 U/mL penicillin-streptomycin.</li>
	<li>After 1 - 2 days of transfection, select the transfected cells for image acquisition. 
	NOTE: Premature neurons less than 7 DIV have a tendency to exhibit dynamic vesicle motility. Select neurons that moderately express the fluorescence probe because clear images cannot be obtained when a highly expressing neuron is selected. Also, select a typical neuronal shape, such as pyramidal neurons, which have long axons and intricate dendrites, for ease of identification of the axon and dendrites (see Figure 1A).</li>
	<li>Image acquisition using a time-lapse imaging system: For imaging, use a fluorescence microscope equipped with a Charge-Coupled Device (CCD) camera with 40X Plan Apo 0.95NA or 100X Plan Apo VC NA1.4 objective lenses. Control the temperature at 37 &#176;C using an incubation system. Acquire neuron images at one frame/5 s over a 100 s period controlled by the image acquisition software. Alternatively, acquire images at shorter intervals and for longer time periods, such as at one frame per 2 s over a 300-s period. 
	NOTE: A variety of applications are available for taking sequential images, many of which would be suitable for this method. Therefore, no particular application is recommended; instead, each researcher should use whatever application is available.</li>
</ol>
2. Image Analysis
<ol>
	<li>Open all sequential images in ImageJ software with Manual Tracking. 
	NOTE: To date, Manual Tracking has been widely used in tracking experiments. Manual Tracking was developed by Dr. Fabrice Cordeli&#232;res. Detailed written instructions are available online. For the analysis, the quality of the imaging data is improved with a sufficient number of images. It is recommended to use more than 20 images.</li>
	<li>To combine the sequential images, choose&#8594; &#8594; . Click .</li>
	<li>Choose &#8594; ; a tracking window will pop up.</li>
	<li>Click on the checkbox of and define the tracking parameters in the parameters section. 
	NOTE: Several parameters can be set, including time interval, x/y calibration, z calibration, search square size for centering, dot size, line width, and font size. In this simplified assay, both time interval and x/y calibration were defined. Under other experimental circumstances, it may be necessary to define other parameters as well. The parameters used here are as follows: the is 5 s, and the is either 0.26642 &#181;m for a 40X Plan Apo 0.95NA objective lens or 0.10657 &#181;m for a 100X Plan Apo VC NA1.4 objective lens.</li>
	<li>After the parameters are defined, click on to start a new track.</li>
	<li>After determining the vesicles of interest (e.g., the vesicles indicated by blue and red arrowheads in Figure 1A (right panel)), click on the center of the signals in the sequential images to record the xy coordinates. The results of the recorded xy coordinates, distance, and velocity show up in a new window. 
	NOTE: In the case of lysosomes, large bright fluorescent signals (e.g., Figure 1A (right panel), orange arrowhead) are often observed in neurons. These signals occasionally exhibit either accumulated vesicles or pathological varicosities, so these signals should be avoided for quantifying motility.</li>
	<li>Repeat step 2.6 to collect the xy coordinates of vesicles from all sequential images; after the reference point is selected, each image automatically advances to the successive image.</li>
	<li>Export these data to a new table (e.g., a spreadsheet). Use appropriate software to create a suitable graph (see Table of Materials). 
	NOTE: To analyze the data, select the column, sum up all values of this column, and show a motility distance as a bar graph, such as in Figure 1B. Additionally, repeat this step, calculate the average of the total motility distance, and show as a bar graph, as in Figure 1C. The software to create a bar graph and waveform data is shown in the Table of Materials.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Neurobasal medium</td>
			<td>Thermo Fisher</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher</td>
			<td>31985070</td>
			<td>Serum free medium</td>
		</tr>
		<tr>
			<td>Penicilin Streptmycin</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Thermo Fisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplements</td>
			<td>Thermo Fisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-D-lysine hydrobromide</td>
			<td>Sigma</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-ornithine</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Thermo Fisher</td>
			<td>11668027</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>Polyethyleneimine &#34;Max&#34;</td>
			<td>polysciences</td>
			<td>24765</td>
			<td>Transfection reagent. As an alternative to Lipofectamine2000, 0.1mg/ml Polyethyleneimine dissolved in sterilized water is available. But it is low efficiency and high toxicity.</td>
		</tr>
		<tr>
			<td>BIOREVO BZ-9000</td>
			<td>Keyence</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubation system INUG2-K13</td>
			<td>Tokay Hit</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism version 6.0</td>
			<td>GraphPad Software</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel version 15</td>
			<td>Microsoft</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ verion 1.47</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of vesicle motilities in neurons using fluorescence imaging

Source: Tsuruta, F., et al. <a href="https://app.jove.com/v/55488">Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes.</a> J. Vis. Exp. (2017).
This video demonstrates how neuronal vesicles are labeled with specific fluorescent proteins to visualize their movement. First, a plasmid coding for a fluorescent protein is incubated with a transfection agent and then introduced into a culture of neurons. Subsequently, images of the neurons are acquired and analyzed to track the vesicles&#39; movement.

<img alt="Figure 1" src="/files/ftp_upload/22989/22989_Figure1.jpg" /> 
Figure 1: Lysosome Motility in Neuronal Dendrites. (A) Mouse cortical neurons (4 DIV) were transfected with LAMP-EGFP plasmid for 2 days. This image shows the motility of LAMP-EGFP-containing vesicles in the dendrites. The inset shows the time-lapse image of the LAMP-EGFP-positive vesicles. The red arrowhead indicates a moving vesicle, and the blue arrowhead indicates a resting vesicle. The orang...

Visualization of Vesicle Motilities in Neurons Using Fluorescence Imaging

1. Imaging Vesicle Motility

	Prepare the plasmids that will label each type of vesicle. For instance, use EGFP-Rab5 for early endosomes, EGFP-Rab7 for ...

Take the desired plasmid in serum-free media. This plasmid encodes vesicle-specific proteins with fluorescent tags.
&#160;In another tube, take the transfection reagent diluted in serum-free media.
Mix the two solutions and incubate.
The transfection reagent combines with the plasmid, forming a complex.
Take a well containing adherent neurons in the media with serum. Add the complexes and incubate. The complex facilitates the plasmid&#39;s entry into the cell.
Discard the media to remove non-internalized complexes.
Add fresh media and then incubate.
The plasmid enters the nucleus to be transcribed and is then translated into vesicle-specific fluorescent proteins that label the vesicles in the neurons.
Using a fluorescence microscope under controlled temperature, acquire images at specified time intervals.
To analyze the images, define tracking parameters.
Identify the vesicle of interest and record its coordinates in each of the images.
Compile the data to visualize the vesicle&#39;s movement.

visualization-vesicle-motilities-neurons-using-fluorescence

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Developmental and Pediatric Disorders

32 次观看. 来源:Tsuruta， F. 等人 使用荧光探针定量培养神经元中的内体和溶酶体运动性。J. Vis. Exp. （2017 年）。 该视频演示了如何用特异性荧光蛋白标记神经元囊泡以可视化其运动。首先，将编码荧光蛋白的质粒与转染试剂一起孵育，然后引入神经元培养物中。随后，获取并分析神经元的图像以跟踪囊泡的运动。 

视频: 使用荧光成像可视化神经元中的囊泡运动 - 概念

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

可视化和分析星形细胞中细胞内器官和其他货物的细胞内传输

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

科研

JoVE Journal

神经科学

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.

使用微流体腔系统在运动神经元培养物中有机细胞的斧头迁移

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.

Visualization of Vesicle Motilities in Neurons Using Fluorescence Imaging

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Visualization of Vesicle Motilities in Neurons Using Fluorescence Imaging