Name: analyzing the size, shape, and directionality of networks of coupled astrocytes
Uploaded: 2018-10-04T13:00:00
Description: Watch this Scientific Journal Video about Analyzing the Size, Shape, and Directionality of Networks of Coupled Astrocytes at JoVE.com

This work is funded by the Canadian Institutes of Health Research, Grant/Award Number: 14392.

All procedures abode by the Canadian Institutes of Health Research rules and were approved by the University of Montreal Animal Care and Use Committee.
1. Preparation of Rat Brain Slices
<ol>
	<li>Prepare 1 L of a sucrose-based solution (Table 1) and 1 L of standard artificial cerebral-spinal fluid (aCSF) (Table 2).</li>
	<li>Bubble the sucrose-based solution with a mix of 95% O2 and 5% CO2 (carbogen) for 30 min before placing it in -80...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+hemichannels+in+astrocytes:+a+novel+mechanism+of+glutamate+release.">Functional hemichannels in astrocytes: a novel mechanism of glutamate release.</a> Journal of Neuroscience. 23 (9), 3588-3596 (2003).</li><li>Ma, B., 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=Gap+junction+coupling+confers+isopotentiality+on+astrocyte+syncytium.">Gap junction coupling confers isopotentiality on astrocyte syncytium.</a> Glia. 64 (2), 214-226 (2016).</li><li>Meme, W., Vandecasteele, M., Giaume, C., Venance, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+coupling+between+hippocampal+astrocytes+in+rat+brain+slices.">Electrical coupling between hippocampal astrocytes in rat brain slices.</a> Neuroscience Research. 63 (4), 236-243 (2009).</li><li>Ransom, B. 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A number of electrophysiological methods exist to assess functional coupling between astrocytes23,24. However, these methods do not provide information about the anatomical arrangement of astrocytic networks. A number of studies have already shown that &#34;dye- or tracer-coupling&#34;, as done here, occurs only in a fraction of coupled cells that are detected by electrophysiological methods25,26,</s...

Many studies have described how the neuron-astrocyte dialogue at a sub-cellular or synaptic level can have implications in neuronal functions and synaptic transmission. It is well established that astrocytes are sensitive to surrounding neuronal activity; in fact, they have receptors for many neurotransmitters including glutamate, GABA, acetylcholine, and ATP (see previously published reviews2,3,4). In return, astrocytic processes ensheath synaptic elements and influence neuronal activity both there and at extrasynaptic sites by regulating extracellular ionic homeostasis and releasing several factors or transmitters such as glutamate, D-serine, and ATP5,6,7.
The idea that astrocytes can also modulate neuronal function at the network level has emerged, with evidence that astrocytic coupling is spatially regulated and corresponds to neuronal segmentation in areas characterized by a clear anatomical compartmentalization (like areas with sensory representations), indicating that astrocytes will couple to other astrocytes serving the same function rather than just those that are close by. In the lateral superior olive, for instance, most astrocytic networks are oriented orthogonally to the tonotopic axis8, whereas in the barrel cortex or olfactoty glomeruli, communication between astrocytes is much stronger within barrels or glomeruli and weaker between adjacent ones9,10. In both cases, the astrocytic networks are oriented towards the center of the glomerule or barrel9,10.
We recently showed that astrocytic activity modulates neuronal firing by decreasing the concentration of extracellular Ca2+ ([Ca2+]e), presumably through the release of S100&#946;, a Ca2+-binding protein11. This effect, which was demonstrated in a population of trigeminal rhythmogenic neurons in the dorsal part of the trigeminal main sensory nucleus (NVsnpr, thought to play an important role in the generation of masticatory movements), results from the fact that rhythmic firing in these neurons depends on a persistent Na+ current that is promoted by decreases of [Ca2+]e11,12. Rhythmic firing in these neurons can be elicited &#34;physiologically&#34; by stimulation of their inputs or artificial decrease of [Ca2+]e. We further showed that astrocytic coupling was required for neuronal rhythmic firing1. This raised the possibility that astrocytic networks may form circumscribed functional domains where neuronal activity can be synchronized and coordinated. To assess this hypothesis, we first needed to develop a method to rigorously document the organization of these networks within NVsnpr.
Previous studies on astrocytic networks have mostly described the extent of coupling in terms of cell number and the density and area covered. Attempts to evaluate the shape of astrocytic networks and the direction of dye-coupling were mostly performed by comparing the size of networks along two axes (x and y) in the barrel cortex9, hippocampus13,14,15, barreloid fields of the thalamus16, lateral superior olive8, olfactory glomeruli10, and cortex14. The methods described here enable unbiased counts of labeled cells in a network and an estimation of the area they cover. We also developed tools to define the preferred orientation of coupling within a network and to assess whether the preferred orientation is towards the center of the nucleus or in a different direction. In comparison to previously used methods, this protocol provides a means to describe the organization and orientation of astrocytic networks in structures like the dorsal trigeminal main sensory nucleus that do not have a known clear anatomical compartmentalization. In the above studies, the network orientation is described as a relationship to the shape of the structure itself which is already documented (e.g., the barreloid in the thalamus, barrels in the cortex, layers in the hippocampus and cortex, glomeruli in the olfactory bulb, etc.). In addition, vectorial analysis allows for comparisons of coupling orientations revealed under different conditions. To analyze whether these parameters changed according to the position of the network within the nucleus, we also developed a method to replace each network in reference to the boundaries of the nucleus. These tools can be easily adapted to other areas for investigating networks of coupled cells.

<table>
	<tbody>
		<tr>
			<td>NaCl</td>
			<td>Fisher Chemicals</td>
			<td>S671-3</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Chemicals</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher Chemicals</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher Chemicals</td>
			<td>M65-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher Chemicals</td>
			<td>S233-500</td>
			<td></td>
		</tr>
		<tr>
			<td>C6H12O6 Dextrose anhydrous</td>
			<td>Fisher Chemicals</td>
			<td>D16-500</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 dihydrated</td>
			<td>Sigma</td>
			<td>C70-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr>
			<td>D-gluconic acid potassium salt</td>
			<td>Sigma</td>
			<td>G45001</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 anhydrous</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>ATPTris Salt</td>
			<td>Sigma</td>
			<td>A9062</td>
			<td></td>
		</tr>
		<tr>
			<td>GTPTris Salt</td>
			<td>Sigma</td>
			<td>G9002</td>
			<td></td>
		</tr>
		<tr>
			<td>Biocytin</td>
			<td>Sigma</td>
			<td>B4261</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenoxolone disodium salt</td>
			<td>Sigma</td>
			<td>C4790</td>
			<td></td>
		</tr>
		<tr>
			<td>avidin-biotin complex : ABC kit</td>
			<td>Vestor laboratories</td>
			<td>PK-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidine-alexa 594</td>
			<td>Molecular Probes</td>
			<td>S11227</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>Fisher Chemicals</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Chemicals</td>
			<td>X5-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqueous mounting medium 1 : Fluoromount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluen-based synthetic resin mounting medium : Permount</td>
			<td>Fisher Chemicals</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Drying Bench</td>
			<td>Fisherbrand</td>
			<td>11-474-470</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT 1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass</td>
			<td>Fisherbrand</td>
			<td>12-544A</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide ColorFrost</td>
			<td>Fisherbrand</td>
			<td>12-550-413</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Fisherchemicals</td>
			<td>04042-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus FluoView FV 1000 Confocal microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>40X water-immersion lens</td>
			<td>Olympus</td>
			<td>LUMPLFLN40XW</td>
			<td></td>
		</tr>
		<tr>
			<td>20X water-immersion lens</td>
			<td>Olympus</td>
			<td>XLUMPLFL20XW</td>
			<td></td>
		</tr>
		<tr>
			<td>4X water-immersion lens</td>
			<td>Olympus</td>
			<td>XLFLUOR4X/340</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P97</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instrument</td>
			<td>MP 225</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera CCD</td>
			<td>Sony</td>
			<td>CX-ST50</td>
			<td></td>
		</tr>
		<tr>
			<td>Black and white monitor</td>
			<td>Sony</td>
			<td>SSM-125</td>
			<td></td>
		</tr>
		<tr>
			<td>Digidata</td>
			<td>Molecular devices</td>
			<td>1322A</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch Clamp amplifier</td>
			<td>Axon instrument</td>
			<td>Mulitclamp 700A</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophysiology acquisition software</td>
			<td>Molecular devices</td>
			<td>pClamp 8</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophysiology analysis software</td>
			<td>Molecular devices</td>
			<td>Clampfit 8</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging analysis software</td>
			<td>ImageJFIJI</td>
			<td>Open source software. FIJI version including plug in package.</td>
			<td></td>
		</tr>
		<tr>
			<td>Vector image editor</td>
			<td>Adobe</td>
			<td>Illustrator CS4</td>
			<td></td>
		</tr>
		<tr>
			<td>Spreadsheet application</td>
			<td>Microsoft Office</td>
			<td>Excel 2010</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing the size, shape, and directionality of networks of coupled astrocytes

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network level. Astrocytes are strongly connected to each other through gap junctions and coupling through these junctions is dynamic and highly regulated. An emerging concept is that astrocytic functions are specialized and adapted to the functions of the neuronal circuit with which they are associated. Therefore, methods to measure various parameters of astrocytic networks are needed to better describe the rules governing their communication and coupling and to further understand their functions.
Here, using the image analysis software (e.g., ImageJFIJI), we describe a method to analyze confocal images of astrocytic networks revealed by dye-coupling. These methods allow for 1) an automated and unbiased detection of labeled cells, 2) calculation of the size of the network, 3) computation of the preferential orientation of dye spread within the network, and 4) repositioning of the network within the area of interest.
This analysis can be used to characterize astrocytic networks of a particular area, compare networks of different areas associated to different functions, or compare networks obtained under different conditions that have different effects on coupling. These observations may lead to important functional considerations. For instance, we analyze the astrocytic networks of a trigeminal nucleus, where we have previously shown that astrocytic coupling is essential for the ability of neurons to switch their firing patterns from tonic to rhythmic bursting1. By measuring the size, confinement, and preferential orientation of astrocytic networks in this nucleus, we can build hypotheses about functional domains that they circumscribe. Several studies suggest that several other brain areas, including the barrel cortex, lateral superior olive, olfactory glomeruli, and sensory nuclei in the thalamus and visual cortex, to name a few, may benefit from a similar analysis.

Coupling between cells in the brain is not static but rather dynamically regulated by many factors. The methods described were developed to analyze astrocytic networks revealed under different conditions and to understand their organization in NVsnpr. These results have been already published1. We performed biocytin filling of single astrocytes in the dorsal part of the NVsnpr in three different conditions: at rest (in control conditions in the absence of any stimu...

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Analyzing the Size, Shape, and Directionality of Networks of Coupled Astrocytes

Here we present a protocol to assess the organization of astrocytic networks. The described method minimizes bias to provide descriptive measures of these networks such as cell count, size, area, and position within a nucleus. Anisotropy is assessed with a vectorial analysis.

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network ...

This method can help answer key questions regarding the organization of astrocytic networks in brain structures. The main advantage of this technique is that it proposes an unbiased method to count label cells and document their anatomical organization within a defined structure. Open ImageJFIJI, select the desired file, and click OK in the bio-format's import options window.To redefine a Z stack that will contain only the optical slices needed for the final Z stack, first select STK and Z project, and then click on the stack knob in the tool bar. Select max intensity in the projection type setting. Save the file, and name it stack file.If the imaging file contains several channels, use image color and split channels to show only the channel with the image of the astrocytic network. Check the pixel settings of the image by selecting image, properties, pixel, with one for pixel dimension. Then use the subtract background tool by opening process and subtract background to remove background biocytin labeling.Use the preview function to set the rolling ball radius, which is generally set at 50 pixels. If further noise reduction is required following the subtract background step, select process, noise, and remove outliers. Select bright in the which outliers setting, and use the preview function to set the radius and threshold.Be careful using the remove outlier tool, since it may blur the data, as shown here. Adjust the threshold using image, adjust, threshold. Select default and B and W mode.Click on apply. Convert the image into a binary image with the binary process tool by selecting process, binary, and make binary. Save the file as a tiff file and name it binary file.To count cells, first set the measurement type by clicking analyze and set measurement. Then select the centroid option. Ensure that the binary file is open onscreen.Again, open the analyze menu, and this time select analyze particles. Next, select show and outlines. This generates a new file that shows the result of the detection.Optimize the parameters to refine the detection. To detect cells only, use a size value between 30 and 6000, and circularity between zero to one, where one defines a perfect circle and zero a random shape. Run the detection by clicking OK.Two tables will appear, a table titled summary that provides the number of detected cells, and a table titled results that provides the X and Y coordinates of each cell.Copy the values and paste them into a spreadsheet application. Save this table under the name detection table. A file with a plot of detected cells will also appear.Save this file as a tiff file under the name detection file. If a group of two or more labeled cells are detected as a single cell with the analyze particles tool, use the watershed tool on the binary image by selecting process, binary, and watershed before applying the analyze particles tool. To measure the surface area of the networks, use the detection file in ImageJ.Left click the mouse to select the polygon selection tool in the toolbar. Left click again to begin tracing a region of interest, or ROI, that connects all the cells located in the external periphery of the network. Right click to close the polygon.Open the set measurement window and select the area option. Then open the ROI manager and add the traced polygon in the ROI manager by clicking add. Run the measurement by clicking measure in the ROI manager.The area measurement will appear in a table and be expressed in pixels. Don't forget to convert this value with a conversion factor for the microscope used to obtain the value in square micrometers. Open the stack file in ImageJFIJI and identify the patched cell with its stronger labeling intensity.Then open the file named detection table in a spreadsheet application, and find the number associated with the patched cell and its corresponding coordinates. If unable to accurately determine the patched cell, use the polygon tool to surround the area where the deposit of biocytin is denser, and refer to this position as that of the patched cell. Use the ROI manager as before to draw an ROI at the patched cell location and add it to the ROI manager.Next, set the measurement and select the centroid option. In the RIO manager, click on measure to obtain the coordinates of the centroid of the traced area. Use these coordinates as the reference point for this specific network.Use this formula to calculate the coordinates of each cell in reference to the patched astrocyte. Expressing the coordinates of each cell in reference to the patched astrocyte is an important step to calculate vector from the patched astrocytes. Use this formula to calculate the coordinates of the main vector of preferential orientation.The main vector of preferential orientation of the network is the sum of all the previously calculated vectors for each cell comprised in the network. Divide the length of the main vector by the number of the cells in the network, minus one to exclude the patched cell, to normalize the values and enable comparisons between networks. To determine the position of each network in the nucleus of interest, first open the four x image with a vector image editor.Multiply the dimensions in the dimension window by five to increase the size of the four x image. Here, the image was sampled at 800 by 800 pixels, and so the sampling resolution is changed to 4000 by 4000 pixels. Export the file in tiff format and name it resized four x.Align the top left corner of the resized four x image with the bottom left corner of the Adobe Illustrator document. The software provides coordinates from a bottom left corner referential point. Open the 20 x image of the network.Use the binary file or detection file because they are easier to align. Then, play with the opacity tool on the binary file of the 20 x image to align it with the resized four x image. When the alignment is done, select and hold the pipette tool so the measure tool appears, and select it in the left toolbar.Use the measure tool to click on the top left corner of the 20 x image to get the coordinates of the 20 x referential point onto the resized four x image. This 20 x referential coordinates are useful for expressing the position of each network on a schematic drawing of the nucleus. To sum up the data, normalization of the nucleus as a rectangle is used.To do this, use the polygon tool to surround the nucleus in the resized four x image. Then open the ROI manager and add the drawn ROI. Use image with transparent lights if the borders of the nucleus cannot be seen, in which case remember to resize it first.In set measurement, select the bounding rectangle option to calculate the smallest rectangle within the drawn nucleus. Finally, click measure, then follow the steps in the written protocol to express the labeled cells coordinates in a normalized nucleus represented by the bounding rectangle. A single astrocyte in the dorsal part of the trigeminal main sensory nucleus, labeled by SR101, was targeted for whole-cell recording and filled with 0.2%biocytin.Electrical stimulation of the fifth tract increased biocytin labeling in the dorsal part of the trigeminal main sensory nucleus, representing an increase in the number of coupled cells. Astrocytic networks remain confined within the dorsal part of the trigeminal main sensory nucleus and their main orientation. This technique was developed to position astrocytic networks within a defined structure, but can be used to express the position of population of labeled cell in any structure.

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