DNL was supported by the University of North Carolina at Chapel Hill (UNC) School of Medicine as a Simmons Scholar. TWR was supported by UNC PREP Grant R25 GM089569. Work using the UNC Neuroscience Center Microscopy Core Facility was supported, in part, by funding from the NIH-NINDS Neuroscience Center Support Grant P30 NS045892 and the NIH-NICHD Intellectual and Developmental Disabilities Research Center Support Grant U54 HD079124.

All animal procedures were performed with the approval of the University of North Carolina at Chapel Hill Animal Care and Use Committee (IACUC).
1. Brain dissection and culture of primary mouse astrocytes
NOTE: The following protocol was adapted from published methods, which follows the original procedure developed by McCarthy and deVellis13,14,15. Mixed cell cultures of McCa...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glia+in+mammalian+development+and+disease.">Glia in mammalian development and disease.</a> Development. 142 (22), 3805-3809 (2015).</li><li>Allaman, I., B&#233;langer, M., Magistretti, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-neuron+metabolic+relationships:+for+better+and+for+worse.">Astrocyte-neuron metabolic relationships: for better and for worse.</a> Trends in Neurosciences. 34 (2), 76-87 (2011).</li><li>Chung, W. S., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+glial+cells+in+synapse+elimination.">The role of glial cells in synapse elimination.</a> Current Opinion in Neurobiology. 22 (3), 438-445 (2012).</li><li>Chung, W. S., Allen, N. J., Eroglu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+Control+Synapse+Formation,+Function,+and+Elimination.">Astrocytes Control Synapse Formation, Function, and Elimination.</a> Cold Spring Harbor Perspectives in Biology. 7 (9), 020370 (2015).</li><li>Shin, J. W., 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=Distribution+of+glutamate+transporter+GLAST+in+membranes+of+cultured+astrocytes+in+the+presence+of+glutamate+transport+substrates+and+ATP.">Distribution of glutamate transporter GLAST in membranes of cultured astrocytes in the presence of glutamate transport substrates and ATP.</a> Neurochemistry Research. 34 (10), 1758-1766 (2009).</li><li>Potokar, M., 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=Cytoskeleton+and+vesicle+mobility+in+astrocytes.">Cytoskeleton and vesicle mobility in astrocytes.</a> Traffic. 8 (1), 12-20 (2007).</li><li>Potokar, M., 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=Regulation+of+AQP4+surface+expression+via+vesicle+mobility+in+astrocytes.">Regulation of AQP4 surface expression via vesicle mobility in astrocytes.</a> Glia. 61 (6), 917-928 (2013).</li><li>Potokar, M., Lacovich, V., Chowdhury, H. 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T., 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=HYS-32-Induced+Microtubule+Catastrophes+in+Rat+Astrocytes+Involves+the+PI3K-GSK3beta+Signaling+Pathway.">HYS-32-Induced Microtubule Catastrophes in Rat Astrocytes Involves the PI3K-GSK3beta Signaling Pathway.</a> PLoS ONE. 10 (5), 0126217 (2015).</li><li>Basu, R., Bose, A., Thomas, D., Das Sarma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule-assisted+altered+trafficking+of+astrocytic+gap+junction+protein+connexin+43+is+associated+with+depletion+of+connexin+47+during+mouse+hepatitis+virus+infection.">Microtubule-assisted altered trafficking of astrocytic gap junction protein connexin 43 is associated with depletion of connexin 47 during mouse hepatitis virus infection.</a> Journal of Biological Chemistry. 292 (36), 14747-14763 (2017).</li><li>Schildge, S., Bohrer, C., Beck, K., Schachtrup, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+mouse+cortical+astrocytes.">Isolation and culture of mouse cortical astrocytes.</a> Journal of Visual Experiments. (71), e50079 (2013).</li><li>McCarthy, K. D., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+separate+astroglial+and+oligodendroglial+cell+cultures+from+rat+cerebral+tissue.">Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.</a> The Journal of Cell Biology. 85 (3), 890-902 (1980).</li><li>Tedeschi, B., Barrett, J. N., Keane, R. 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Here, we describe an experimental approach to express, visualize, and track fluorescently tagged organelles and membrane proteins of interest using time-lapse video microscopy in high-purity primary mouse cortical MD astrocytes. We also outline a methodology for measuring particle dynamics. Direct visualization of protein and organelle dynamics in primary astrocytes provides a powerful tool to study the regulation of intracellular transport in these cells in vitro.
The methodology for establis...

Astrocytes are the most abundant cells in the adult central nervous system, where they perform unique developmental and homeostatic functions1. Astrocytes modulate synaptic development through direct contact with pre- and postsynaptic terminals as part of the tri-partite synapse, which contains neurotransmitter receptors, transporters, and cell adhesion molecules that facilitate synapse formation and neuron-astrocyte communication2. In addition, astrocytes actively control synaptic transmission and prevent neuronal excitotoxicity by rapidly removing excitatory neurotransmitters from the synaptic cleft, recycling neurotransmitters, and participating in synaptic pruning3,4,5,6. To enable these highly interactive functions, astrocytes communicate among themselves, with other glial cells, and with neurons through specialized membrane proteins, including cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. Astrocytes actively change the surface levels of these proteins in response to fluctuations in their intra- and extracellular environment7. Furthermore, changes in the levels and distribution of mitochondria, lipid droplets, and degradative and recycling organelles modulate energy supply, metabolite availability, and cellular clearing processes that are essential for astrocyte function and survival.
The dynamic changes in membrane protein and organelle trafficking and positioning in astrocytes are facilitated by the concerted function of motor proteins and adaptors that promote cargo motility8,9. Similarly, surface levels of membrane proteins are modulated through internalization and recycling events10. These cargos are transported via an intricate network of actin, microtubules, and possibly intermediate filaments tracks8. Studies based on immunofluorescence staining of end-binding protein 1 (EB1), which accumulates at the growing microtubule plus ends, suggest that in astrocytes bundles of microtubules radiate out from the perinucleus and extend their plus end towards the periphery11. However, a comprehensive examination of the organization and polarity of microtubules and other cytoskeletal elements using live-cell imaging is still lacking. While many of the mechanisms underlying the dynamics of organelles and membrane proteins have been extensively studied in neurons and other cell types, cargo motility in astrocytes is less well understood. Most of our current knowledge about changes in protein and organelle distribution in astrocytes is based on traditional antibody-based labeling of fixed preparation, which precludes precise spatial and temporal examination of cargo dynamics7,12.
Here, we describe a method to label membrane proteins and organelles for live imaging in high purity primary mouse astrocyte cultures. Using this protocol, we provide examples in which we track the dynamic localization of green fluorescent protein (GFP)-tagged membrane proteins in transfected astrocytes, including the gap junction protein connexin 43 (Cx43-GFP) and the excitatory amino acid transporter 1 (EAAT1-GFP). We also describe the use of a fluorescent acidotropic probe to visualize acidic organelles and follow their trafficking dynamics in live astrocytes. Finally, we demonstrate how to analyze the time-lapse data to extract and evaluate transport parameters for individual cargos.

<table>
	<tbody>
		<tr>
			<td>2.5% Trypsin (10x)</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75-203-637</td>
			<td>Sorvall ST8 Centrifuge</td>
		</tr>
		<tr>
			<td>Cell Culture Grade Water</td>
			<td>Gen Clone</td>
			<td>25-511</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Microscope</td>
			<td>Zeiss</td>
			<td>WSN-AXIOVERT A1</td>
			<td>Vert.A1 inverted scope, Inverted tissue culture microscope with fluorescent capabilities</td>
		</tr>
		<tr>
			<td>Cytosine Arabinoside</td>
			<td>Sigma</td>
			<td>C1768-100MG</td>
			<td>(AraC) Dissolve lyophilized powder in sterile cell culture grade water to make a 10mM stock, aliquot and freeze for long term storage</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542-5MG</td>
			<td>Dissolve lyophilized powder in deionized water to a maximum concentration of 20 mg/ml, heat or sonicate as necessary. Use at 300 nM for counterstaining.</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Stemi 305</td>
		</tr>
		<tr>
			<td>Dissecting Scissors</td>
			<td>F.S.T</td>
			<td>14558-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gen Clone</td>
			<td>25-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gemini</td>
			<td>100-106</td>
			<td>Heat-Inactivated</td>
		</tr>
		<tr>
			<td>FIJI (Fiji is Just Image J)</td>
			<td>NIH</td>
			<td>Version 1.52i</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Tip Tweezers</td>
			<td>F.S.T</td>
			<td>11254-20</td>
			<td>Style #5</td>
		</tr>
		<tr>
			<td>Fluorescence light source</td>
			<td>Excelitas</td>
			<td>012-63000</td>
			<td>X-Cite 120Q</td>
		</tr>
		<tr>
			<td>GFAP antibody</td>
			<td>Cell Signaling</td>
			<td>3670S</td>
			<td>GFAP (GA5) mouse monoclonal antibody</td>
		</tr>
		<tr>
			<td>Glass Bottom Dishes</td>
			<td>Mattek corporation</td>
			<td>P35G-1.5-14-C</td>
			<td>35 mm Dish | No. 1.5 Coverslip | 14 mm Glass Diameter | Uncoated</td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>F.S.T</td>
			<td>11054-10</td>
			<td>Graefe Iris Forceps with curved tips</td>
		</tr>
		<tr>
			<td>Green fluorescent dye that stains acidic compartments (late endosomes and lysosomes)</td>
			<td>Life Technologies</td>
			<td>L7526</td>
			<td>LysoTracker Green DND 26. Pre-dissolved in DMSOS to a 1mM stock solution. Dilute to the final working concentration in the growth medium or buffer of choice.</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (10x)</td>
			<td>Gibco</td>
			<td>14065-056</td>
			<td>Magnesium and calcium free</td>
		</tr>
		<tr>
			<td>Imaging Media</td>
			<td>Life Technologies</td>
			<td>A14291DJ</td>
			<td>Live Cell Imaging Solution</td>
		</tr>
		<tr>
			<td>Inverted Confocal Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>LSM 780</td>
		</tr>
		<tr>
			<td>KymoToolBox</td>
			<td></td>
			<td></td>
			<td>https://github.com/fabricecordelieres/IJ_KymoToolBox</td>
		</tr>
		<tr>
			<td>Lipofection Enhancer Reagent</td>
			<td>Life Technologies</td>
			<td>11514015</td>
			<td>Plus Reagent</td>
		</tr>
		<tr>
			<td>Lipofection Reagent</td>
			<td>Life Technologies</td>
			<td>15338100</td>
			<td>Lipofectamine LTX reagent</td>
		</tr>
		<tr>
			<td>Orbital shaking incubator</td>
			<td>New Brunswick Scientific</td>
			<td>8261-30-1008</td>
			<td>Innova 4230 , orbital shaking incubator with temperature and speed control</td>
		</tr>
		<tr>
			<td>Penicillin/Strepomycin solution (100x)</td>
			<td>Gen Clone</td>
			<td>25-512</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (10x)</td>
			<td>Gen Clone</td>
			<td>25-507x</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine Hydrobromide</td>
			<td>Sigma</td>
			<td>P7405</td>
			<td>Dissolve in Tris buffer, pH 8.5, at 1mg/mL. Freeze for long term storage. Avoid cycles of freezing and thawing</td>
		</tr>
		<tr>
			<td>Reduced serum medium</td>
			<td>Gibco</td>
			<td>31985-062</td>
			<td>OPTI-MEM</td>
		</tr>
		<tr>
			<td>Tissue Culture Flasks</td>
			<td>Olympus Plastics</td>
			<td>25-209</td>
			<td>75 cm^2. 100% angled neck access, 0.22um hydrophobic vented filter cap</td>
		</tr>
		<tr>
			<td>Tissue culture incubator</td>
			<td>Thermo Scientific</td>
			<td>51030285</td>
			<td>HERAcell VIOS 160i, tissue culture incubator with temperature, humidity, and CO2 control</td>
		</tr>
		<tr>
			<td>Tris-Base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td>8.402 g dissolved to one liter in water with 4.829 g Tris HCl to make 0.1 M Tris buffer, pH 8.5</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma</td>
			<td>T3253</td>
			<td>4.829 g dissolved to one liter in water with 8.402 g Tris Base to make 0.1 M Tris buffer, pH 8.5</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum-Driven Filter Systems</td>
			<td>Olympus Plastics</td>
			<td>25-227</td>
			<td>500 ml, PES membrane, 0.22 &#181;m</td>
		</tr>
		<tr>
			<td>Vannas scissors straight</td>
			<td>Roboz</td>
			<td>RS-5620</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The protocol for establishing primary mouse MD astrocytes outlined above should yield reproducible, high quality cultures. Although cultures initially contain a mix of astrocytes, fibroblasts, and other glial cells, including microglia and oligodendrocytes (Figure 1Bi,Biv; red arrowheads), the addition of AraC to the mixed culture between DIV5-DIV7 minimizes the proliferation of these contaminant cells. The combined AraC treatment and shaking-based purification strategy enri...

Watch this Scientific Journal Video about Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes at JoVE.com

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

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.

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 ...

Our protocol can be used to investigate the motility and localization of astrocytic organelles or proteins under controlled extracellular environments and mild disease states. Unlike the MHA fixed preparations, which provide single snapshots of protein and organelle localization, our live imaging technique can be used to analyze particle dynamics in individual live astrocytes. The day before the transfection seed astrocytes at the appropriate density for imaging at 24 to 48 hours post transfection.Dilute the lipofection reagent in reduced serum medium to the appropriate experimental concentration. Dilute five micrograms of high purity DNA in 250 microliters of reduced serum medium and add five microliters of lipofection enhancer reagent to the tube. Mix the lipofection reagent with an equal volume of the DNA lipofection enhancer mix and incubate the solution for 15 minutes at room temperature.At the end of the incubation, remove the supernatant from the astrocyte culture and add 100 microliters of the transfection mix dropwise to the cells. After six hours at 37 degrees Celsius and 5%carbon dioxide, replace the transfection complex with an appropriate volume of astrocyte culture medium and return the cells to the cell culture incubator for an additional 24 to 72 hours. 30 minutes before the imaging, dilute a suitable lysosomal labeling probe in 200 microliters of astrocyte culture medium to a working concentration of one micromolar and label the astrocytes with a probe for 30 minutes at 37 degrees Celsius.Then wash the cells one time with warm astrocyte culture medium and replace the wash with imaging medium. Immediately after the probe labeling, place the astrocyte culture container into the appropriate adapter on the microscope stage and using EBI fluorescence light, locate the cells that express fluorescent proteins or probe. Use the digital camera to adjust the fluorescent sample illumination to visualize the selected cells and adjust the focus and zoom in to a single cell.Then use the Zoom and Definite Focus functions to acquire a single Z-stack time-lapse series at a frequency of one frame every two seconds for time intervals ranging between 300 and 500 seconds. Save and export the time-lapse images as AVI or TIFF stack files. To analyze the time-lapse images, open the time-lapse image sequence in ImageJ or Fiji and use the Split Channel tool to split the channels.In the eight bit green channel image, use the Segmented Line tool to trace a line along the trajectories of the particles using the same desired directional convention for all of the movies so that the polarity is consistent within and across all of the cells being studied. Double click the Line tool to adjust the line width to match the thickness of the track and run the Draw Kymo macro of the Kymo ToolBox plugin using a line width of 10. A prompt asking to calibrate the image in time and space will appear.Once calibrated, kymographs will be generated and can be saved as TIFF files. To assign particle trajectories, use the Segmented Line tool to manually track each particle in the kymograph for the full duration of the acquisition and use the ROI manager to record each particle trajectory as a region of interest. Save all of the regions of interest per each time-lapse video for further analysis and run the Analyze Kymo macro of the Kymo ToolBox plugin.A window will open asking to define the outward direction of particle motion from the nucleus of the cell of interest from the dropdown menu. The limit speed should be defined according to the sensitivity of the software for each cargo of interest and the line width should be adjusted to match the thickness of each particle trajectory. The Log All Data and Log Extrapolated Coordinates should be for calculation of the various cargo transport parameters.Click Okay. The data calculated for the individual tracks will then be pooled per kymograph and saved in text files specific to each image. Combining cytosine arabinoside treatment with the shaking based purification strategy enriches the purity of the astrocyte cultures over traditional protocols that include the purification step only.Lipofection based transfection allows the transient expression of proteins at levels that are optimal for live cell imaging without causing toxicity or affecting astrocyte viability. Similarly, the use of a fluorescent probe permits the rapid and efficient labeling of acidic endolysosomal organelles for the tracking of organelle dynamics and astrocytes. The time-lapse data from the imaging can be used to generate kymographs that track cargo motion in time and space.In these kymographs, anterograde movement of the indicated cargo is represented by trajectories with negative slopes, while retrograde movement is represented by trajectories with positive slopes. Stationary vesicles appear as vertical trajectories. In this analysis, quantification of the flux of a specific cargo through an area of the astrocyte revealed differences in the percentage of modal particles among cargoes that are likely representative of their normal base line motility in the region of the astrocyte within which the movies were acquired.The precise mapping of the change in the X, Y position along the full timescale for each particle obtained from the kymograph can also be used to evaluate other motion parameters, such as cargo velocity and run length. As this protocol requires high quality astrocyte cultures and efficient cargo labeling, the transfection reagent incubation time and acquisition timeline should be adjusted for each plasmid or cargo of interest. This protocol can be modified to characterize transport events in astrocytes in response to cell damage, toxicity, pathogenic mutations, synaptic activity, or changes in the intra or extracellular environment.

visualizing-analyzing-intracellular-transport-organelles-other-cargos

Research

JoVE Journal

Neuroscience

7.6K Views.  University of North Carolina at Chapel Hill. Our protocol can be used to investigate the motility and localization of astrocytic organelles or proteins under controlled extracellular environments and mild disease states. Unlike the MHA fixed preparations, which provide single snapshots of protein and organelle localization, our live imaging technique can be used to analyze particle dynamics in individual live astrocytes. The day before the transfection seed astrocytes at the appropriate density for imaging at 24 to 48 hours post transfe...