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Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Neuroscience

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

Published: October 4th, 2018

DOI:

10.3791/58116

1Groupe de Recherche sur le Système Nerveux Central, and Département de Neurosciences, Université de Montréal, 2Faculté de Médecine Dentaire, Université de Montréal

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 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.

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 ....

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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

  1. Prepare 1 L of a sucrose-based solution (Table 1) and 1 L of standard artificial cerebral-spinal fluid (aCSF) (Table 2).
  2. Bubble the sucrose-based solution with a mix of 95% O2 and 5% CO2 (carbogen) for 30 min before placing it in -80.......

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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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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 "dye- or tracer-coupling", as done here, occurs only in a fraction of coupled cells that are detected by electrophysiological methods25,26,

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

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

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