Published: October 4th, 2018
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 ....
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
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.......
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,
|C6H12O6 Dextrose anhydrous
|D-gluconic acid potassium salt
|Carbenoxolone disodium salt
|avidin-biotin complex : ABC kit
|Aqueous mounting medium 1 : Fluoromount-G
|Toluen-based synthetic resin mounting medium : Permount
|Slide Drying Bench
|Microscope cover glass
|Microscope slide ColorFrost
|Olympus FluoView FV 1000 Confocal microscope
|40X water-immersion lens
|20X water-immersion lens
|4X water-immersion lens
|Black and white monitor
|Patch Clamp amplifier
|Electrophysiology acquisition software
|Electrophysiology analysis software
|Imaging analysis software
|Open source software. FIJI version including plug in package.
|Vector image editor
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