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

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The authors have nothing to disclose.

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 methods25,26,27, suggesting that the number of cells detected with this method is underestimated. Nevertheless, this is still the best method for visualisation of coupling. Greater dye-coupling is obtained when using molecule tracers smaller than 1-1.2 KDa to permeate gap junctions20. Live visualization of coupling can be performed by monitoring diffusion of fluorescent glucose derivatives (2-NBDG) or a fluorescent marker (like some Alexa dyes or Lucifer Yellow)13, but due to its smaller size, biocytin yields better results and allows for quantification in fixed tissue. Thus, it should be noted that the number of cells detected with this method is largely underestimated. On the other hand, some of the cells may be labeled not because of their coupling, but because of the uptake of tracer that has leaked into the extracellular space. The extent of labeling resulting from leakage can be estimated in control experiments with bath applications of a gap junction blocker1. However, it can be assumed that any bias due to the method would apply to all conditions.
Finally, it should be emphasized that not all coupled cells are astrocytes. Panglial networks where astrocytes are coupled to oligodendrocytes have been described in several brain areas, including the lateral superior olive8, thalamus16, cortex, and hippocampus28. Our aim was to develop a method of comparing astrocytic networks revealed with dye-coupling under different conditions to assess the hypothesis that they form well-defined, distinctive functional domains revealed by different stimuli. Since in NVsnpr, the trigeminal brainstem nucleus in which this protocol was conducted, coupling between astrocytes is very limited under resting conditions, it was important to first develop a reliable and unbiased method to detect labeled cells. This was achieved with automated detection with ImageJFIJI. It is sometimes difficult to distinguish faintly labeled cells from background noise. Here, stack imaging and a method in ImageJFIJI are used to improve the signal, but background noise can still be too high and lead to data rejection. In future studies, clarification protocols may be used to improve the signal-to-noise ratio. A clearing protocol is an interesting method of clarification that preserves tissue morphology (no shrinkage), does not affect lipids, and is compatible with immunostaining29. Another limitation of the method described is the &#34;watershed&#34; tool used to discriminate cells that are too close to each other. Visual inspection of detected cells should be done when using this tool.
If astrocytic networks form functional domains, then they may define the boundaries of a nucleus or should at least be confined to the boundaries of a nucleus. Revealed networks may always be confined within a nucleus, more so if they are smaller in size, if the patched astrocyte is in the center (or within) the nucleus. To test if astrocytes sitting near the border of a nucleus preferably couple with other astrocytes outside the nucleus or with others within the nucleus, we needed to determine the preferential direction to which the dye spread from the patched astrocyte. Several other studies have addressed a similar issue in the barrel cortex, barreloid fields of the thalamus, olfactory glomeruli, hippocampus, and lateral superior olive, but they focused analyses mostly on the distance of diffusion of the tracer, electrophysiological characterization of different types of astrocytes in function of their localization, and spread of dye along orthogonal axes with respect to these well-defined structures8,9,10,13,14,16. Here, we used vectorial analysis to determine the dominant direction of tracer spread, which was given by the size and orientation of the normalized main vector. Small vectors indicate no clear &#34;dominant&#34; or preferential orientation. A critical step for vectorial analysis is the ability to accurately identify the patched astrocyte, which is not always possible. An interesting alternative to help locate the patched astrocyte is to add a large (non-gap junction permeant) and fixable tracer, like dextrans, to the recording solution. If it is not possible to determine the patched cell or patch location, it may be better to omit these experiments from the vector analysis.
Finally, to analyze whether properties of networks varied according to their location in the nucleus, we needed a method to express their position in a &#34;normalized&#34; nucleus. The only critical step in this procedure is the ability to clearly see the borders of the nucleus in the low magnification images (4X). A simple solution to this problem, if it occurs often, is to add a standard histological coloration to tissue processing before analysis.
Heterogeneity of astrocytes across brain regions is clearly established, and a growing body of evidence supports the concept that their specializations are particularly adapted for the function of the circuit that they are embedded in9,10,30,31. For this to be true, inter-astrocytic communication should be limited to astrocytes associated to the same circuit. Observations supporting this assumption are emerging in different brain areas but are more evident in areas with clustered representations, like sensory maps in the barrel cortex or olfactory bulb. However, most reports of overlap between neuronal and astrocytic maps are descriptive and qualitative. The methods reported here can be used to objectify&#160;these observations and develop tools to better analyze astrocytic networks according to their position in a given circuit.

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>KH&lt;sub&gt;2&lt;/sub&gt;PO4</td><td>Fisher Chemicals</td><td>P285-500</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Chemicals</td><td>M65-500</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Chemicals</td><td>S233-500</td><td></td></tr><tr><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt; Dextrose anhydrous</td><td>Fisher Chemicals</td><td>D16-500</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; 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>MgCl&lt;sub&gt;2 &lt;/sub&gt;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>ATP&lt;sub&gt;Tris Salt&lt;/sub&gt;</td><td>Sigma</td><td>A9062</td><td></td></tr><tr><td>GTP&lt;sub&gt;Tris Salt&lt;/sub&gt;</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.

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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 ...

analyzing-size-shape-directionality-networks-coupled

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.8K Views.  Université de Montréal. Deze methode kan helpen bij het beantwoorden van belangrijke vragen met betrekking tot de organisatie van astrocytische netwerken in hersenstructuren. Het belangrijkste voordeel van deze techniek is dat het een onbevooroordeelde methode voorstelt om labelcellen te tellen en hun anatomische organisatie binnen een gedefinieerde structuur te documenteren. Open ImageJFIJI, selecteer het gewenste bestand en klik op OK in het venster importopties van de bio-indeling.Als u een Z-stack wilt he...

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

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

Developmental Biology

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Bioengineering

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction of high-resolution imaged stacks to reveal ultrastructural patterns that could not be resolved using 2D images. Indeed, the latter might lead to a misinterpretation of morphologies, like in the case of mitochondria; the use of 3D models is, therefore, more and more common and applied to the formulation of morphology-based functional hypotheses. To date, the use of 3D models generated from light or electron image stacks makes qualitative, visual assessments, as well as quantification, more convenient to be performed directly in 3D. As these models are often extremely complex, a virtual reality environment is also important to be set up to overcome occlusion and to take full advantage of the 3D structure. Here, a step-by-step guide from image segmentation to reconstruction and analysis is described in detail.

The described pipeline is designed for the segmentation of electron microscopy datasets larger than gigabytes, to extract whole-cell morphologies. Once the cells are reconstructed in 3D, customized software designed around individual needs can be used to perform a qualitative and quantitative analysis directly in 3D, also using virtual reality to overcome view occlusion.

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction ...

Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, which include neurotransmitter clearance, ion regulation, synaptic modulation, metabolic support to neurons, and blood flow regulation. Astrocytes are complex cells that have a soma, several major branches, and numerous fine processes that contact diverse cellular elements within the neuropil. In order to assess the morphology of astrocytes, it is necessary to have a reliable and reproducible method to visualize their structure. We report a reliable&#160;protocol to perform intracellular iontophoresis of astrocytes using fluorescent Lucifer yellow (LY) dye in lightly fixed brain tissue from adult mice. This method has several features that are useful to characterize astrocyte morphology. It allows for three-dimensional reconstruction of individual astrocytes, which is useful to perform morphological analyses on different aspects of their structure. Immunohistochemistry together with LY iontophoresis can also be utilized to understand the interaction of astrocytes with different components of nervous system and to evaluate the expression of proteins within the labelled astrocytes. This protocol can be implemented in a variety of mouse models of CNS disorders to rigorously examine astrocyte morphology with light microscopy. LY iontophoresis provides an experimental approach to evaluate astrocyte structure, especially in the context of injury or disease where these cells are proposed to undergo significant morphological changes.

Astrocytes are morphologically complex cells, exemplified by their multiple processes and bushy territories. To analyze their elaborate morphology, we present a reliable protocol to perform intracellular Lucifer yellow iontophoresis in lightly fixed tissue.

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, ...

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.

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 ...

Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain cells and their interactions. Viral vector transduction enables the marking of diverse cell types in different fluorescence colors within the same tissue. Cells can be identified genetically by activity or projection. Using a modified CLARITY protocol, the potential sample size of astrocytes and neurons has grown by 2-3 orders of magnitude. The use of CLARITY allows the imaging of complete astrocytes, which are too large to fit in their entirety in slices, and the examination of the somata with all their processes. In addition, it provides the opportunity to investigate the spatial interaction between astrocytes and different neuronal cell types, namely, the number of pyramidal neurons in each astrocytic domain or the proximity between astrocytes and specific inhibitory neuron populations. This paper describes, in detail, how these methods are to be applied.

Combining viral vector transduction and brain clearing using the CLARITY method allows the investigation of a large number of neurons and astrocytes simultaneously.

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain ...

Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections

Astrocytes possess an astounding degree of morphological complexity that enables them to interact with nearly every type of cell and structure within the brain. Through these interactions, astrocytes actively regulate many critical brain functions, including synapse formation, neurotransmission, and ion homeostasis. In the rodent brain, astrocytes grow in size and complexity during the first three postnatal weeks and establish distinct, non-overlapping territories to tile the brain. This protocol provides an established method for analyzing astrocyte territory volume and astrocyte tiling using free-floating tissue sections from the mouse brain. First, this protocol describes the steps for tissue collection, cryosectioning, and immunostaining of free-floating tissue sections. Second, this protocol describes image acquisition and analysis of astrocyte territory volume and territory overlap volume, using commercially available image analysis software. Lastly, this manuscript discusses the advantages, important considerations, common pitfalls, and limitations of these methods. This protocol requires brain tissue with sparse or mosaic fluorescent labeling of astrocytes, and is designed to be used with common lab equipment, confocal microscopy, and commercially available image analysis software.

This protocol describes methods for sectioning, staining, and imaging free-floating tissue sections of the mouse brain, followed by a detailed description of the analysis of astrocyte territory volume and astrocyte territory overlap or tiling.

Astrocytes possess an astounding degree of morphological complexity that enables them to interact with nearly every type of cell and structure within the ...

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

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