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>
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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...

Watch this Scientific Journal Video about Analyzing the Size, Shape, and Directionality of Networks of Coupled Astrocytes at JoVE.com

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 ...

Research

JoVE Journal

Neuroscience

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