The authors thank Ms. Soto, Dr. Yu, and Dr. Octeau for guidance as well as comments on the text. This work is supported by NS060677.

The animal experiments in this study were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the Chancellor&#39;s Animal Research Committee at the University of California, Los Angeles. Adult mice (6&#8722;8 weeks old) of mixed gender were used in all experiments.
1. Solution Preparation
<ol>
	<li>Artificial cerebrospinal fluid (ACSF) solution
	<ol>
		<li>Prepare fresh ACSF solution (135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 14.7 mM NaHCO3, 11 mM D-glucose, 1.25 mM Na2HPO4, and 2 mM CaCl2) before each experiment. Add MgCl2 and CaCl2 in the final step. Dissolve the components in high quality deionized water.</li>
		<li>Incubate the ACSF solution at 35 &#176;C in a water bath and bubble with 95% O2/ 5% CO2 for at least 30 min before the experiment.</li>
		<li>Add 2% lidocaine hydrochloride to reach a final concentration of 0.02% in ACSF (in 100 mL).</li>
	</ol>
	</li>
	<li>Fixative solution
	<ol>
		<li>Use 10% formalin buffered phosphate.</li>
	</ol>
	</li>
	<li>LY dye solution
	<ol>
		<li>Prepare 1.5% LY dye solution by dissolving LY CH dilithium salt or LY CH dipotassium salt in 5 mM KCl. Vortex thoroughly. A volume of 1 mL is sufficient for several experiments.</li>
		<li>Centrifuge for 10 min at 16,800 x g. Then, filter the supernatant with a 0.2 &#181;m syringe filter into a new tube. Aliquots can be kept at 4 &#176;C for up to 3 months. 
		NOTE: It is critical to centrifuge and filter the dye solution to prevent aggregation of the dye particles, which may clog the electrode.</li>
	</ol>
	</li>
</ol>
2. Mouse Transcardial Perfusion and Brain Dissection
<ol>
	<li>Preparation of equipment and mouse for transcardial perfusion 
	NOTE: C57/BL6 mice of either sex can be used. It has been empirically observed that for the method to work reliably, it is important that the mice are not older than 3 months (6&#8722;8 weeks old is ideal). The perfusion protocol is described below. An additional reference is provided for more details14.
	<ol>
		<li>Clear perfusion tubing with ACSF solution to remove any air bubbles in the line. Set up surgery tools in order of use (tweezers, curved, blunt scissors, iris scissors).</li>
		<li>Deeply anesthetize mouse by putting it into an isoflurane induction chamber, after adding 2&#8722;3 mL of isoflurane to the chamber. Allow 1&#8722;2 min for anesthetic to take effect. Ensure that breathing does not stop.</li>
		<li>Test for toe pinch reflex. Proceed when the mouse is unresponsive to pain stimulus and the reflex is absent. Secure the animal in the supine position with the head placed in the breathing cone with a supply of 5% isoflurane in oxygen and the limbs and tail taped down inside a chemical fume hood.</li>
	</ol>
	</li>
	<li>Transcardial perfusion
	<ol>
		<li>Using tweezers, lift up skin beneath rib cage. Make a 5&#8722;6 cm incision with the curved, blunt scissors through the skin to expose the abdominal cavity. Cut upwards through the abdominal wall until liver and diaphragm are visible.</li>
		<li>Gently move the liver away from the diagram. With iris scissors, make a 3&#8722;4 cm lateral incision in the diaphragm.</li>
		<li>Cut through the rib cage along the both sides of the body to expose the chest cavity. Be careful not to damage heart and avoid the lungs. Lift the sternum up to expose the heart.</li>
		<li>Once the heart is visible, inject 0.05 mL of heparin sodium solution (1,000 USP per mL) into the left ventricle as an anticoagulant. Then, insert perfusion needle carefully into the left ventricle. Make sure that the needle stays in the ventricle and does not pierce through to other heart chambers. Make a small incision in the right atrium with iris scissors.</li>
		<li>Perfuse with ACSF solution at a rate of approximately 10 mL/min until the fluid existing the body is cleared of blood (1&#8722;2 min).</li>
		<li>Switch from ACSF solution to fixative solution without introducing any air bubbles. Perfuse with fixative solution for 10 min at 10&#8722;20 mL/min. 
		CAUTION: Take care that no fixative solution is draining from the nose. This indicates that the needle reached the right ventricle and the fixative is traveling to the lungs rather than through the systemic circuit to the rest of the body.</li>
	</ol>
	</li>
	<li>Dissection of the brain
	<ol>
		<li>Remove the head of the mouse with scissors and carefully dissect out the mouse brain from the skull. Place in fixative solution for 1.5 h at room temperature for a short post-fixation period. 
		NOTE: A good perfusion is necessary for successful LY iontophoresis. Brain should be white-colored and absent of blood in brain vasculature. Body and limbs should appear stiff.</li>
	</ol>
	</li>
</ol>
3. Slice Preparation
<ol>
	<li>Preparation of hippocampal slices
	<ol>
		<li>Wash the brain with 0.1 M phosphate buffered saline (PBS) at room temperature for 5 min. Then, dry off the brain with filter paper and remove the olfactory bulb and cerebellum with a sharp razor blade.</li>
		<li>Mount the brain on the vibratome tray using cyanoacrylate glue and fill the tray with PBS at room temperature. Cut hippocampal coronal sections of 110 &#181;m thickness. 
		NOTE: Adjust the setting of the vibratome to make sure that slices have the same thickness and even surface. For this experiment, a speed setting of 4.5 and a frequency setting of 8 were used, which are arbitrary settings on the instrument (Table of Materials). Users may need to experiment with the settings on other devices.</li>
		<li>Collect the sections from the tray and place in a dish of PBS on ice.</li>
	</ol>
	</li>
</ol>
4. Electrode Preparation
<ol>
	<li>Prepare a sharp electrode with appropriate resistance.
	<ol>
		<li>Use a borosilicate glass single barrel electrode with filament (O.D. 1.0 mm, I.D. 0.58 mm). Pull an electrode on a micropipette puller (Table of Materials). Ideal electrodes filled with 1.5% LY in 5&#160;mM KCl should have a resistance of 200 M&#8486; when placed into a bath of PBS. 
		NOTE: Puller setting varies depending on the apparatus (type of machine used and puller filament). A higher heat setting usually gives longer and finer tips. For the micropipette puller used in this experiment, the settings were: heat: 317, pull: 90, velocity: 70, and delay: 70. A trough type filament was used.</li>
		<li>Store electrodes in a closed container to prevent dust from entering the tip. Keep electrodes elevated from the bottom of the box to prevent the tip from breaking.</li>
	</ol>
	</li>
	<li>Fill the electrode with LY dye solution.
	<ol>
		<li>Place an electrode in vertical position with the tip facing downward. Pipette 1&#8722;2 &#181;L of LY solution into the back of the electrode and wait for 5&#8722;10 min for the solution to move to the tip via capillary action.</li>
		<li>Gently secure the filled electrode in the electrode holder connected to a manipulator. 
		NOTE: The silver wire of the electrode holder needs to be in contact with the LY inside the electrode. Adjust volume of the LY solution if needed, depending on the wire length.</li>
	</ol>
	</li>
</ol>
5. Filling Astrocytes with Iontophoresis
<ol>
	<li>Test the electrode.
	<ol>
		<li>Place a brain slice gently into a glass bottom dish filled with 0.1 M PBS at room temperature. Hold the slice in place with a platinum harp with nylon strings.</li>
		<li>Ensure that the electrode is connected to a voltage source and place the ground electrode into the bath containing the brain slice.</li>
		<li>Move the objective to the brain region of interest.</li>
		<li>Lower the electrode into the solution. Under the bright field, move it to the center of the field of view and examine it carefully with the 40x water immersion lens to ensure that it appears clear and without debris or bubbles. If there is anything clogging the electrode tip, replace it with a new one. 
		NOTE: Clogging is a concern for the 200 M&#8486; electrode. With centrifugation and filtration of the dye solution before every experiment, this should not be a frequent problem. However, because the tip of the electrode is small, tissue may sometimes become stuck in the opening. The authors have not found a way to prevent this, but it can be easily dealt with by simply using a new electrode.</li>
		<li>Observe the tip of the electrode under the confocal laser-scanning microscope with the 488 nm laser. Then, test dye ejection by turning on the stimulator at 12 V. Note a large fluorescent dye cloud around the tip of the electrode while the stimulator is on. If no or little dye is ejected upon voltage stimulation, replace the electrode.</li>
		<li>Under the bright field, slowly lower the electrode toward the slice stopping just above the surface.</li>
	</ol>
	</li>
	<li>Fill astrocyte with iontophoresis.
	<ol>
		<li>Identify astrocytes 40&#8722;50 &#181;m below the slice surface with the infra-red differential interference contrast&#160;(IR-DIC). Look for cells with elongated, oval-shaped somata about 10 &#181;m in diameter. Once an astrocyte is chosen, move it to the center of the field of view. 
		NOTE: A good cell for iontophoresis has clear, defined borders around it. Do not choose cells too close to the surface of the slice because they cannot be completely filled. It takes some practice over a few days to be able to routinely identify astrocytes for dye filling. Depending on the brain area used for the experiment, the number of astrocytes may vary. This may affect the time needed to identify an astrocyte before filling.</li>
		<li>Slowly lower the electrode tip into the slice, navigating through the tissue, until it is on the same plane as the cell body. 
		NOTE: Move electrode slowly to avoid damaging the tissue.</li>
		<li>Once the cell body of the astrocyte is clearly visible and outlined, slowly and gently advance the electrode forward. Move electrode until the tip impales the soma of the cell. Move the focus of objective slowly up and down to note if the electrode is inside the soma. 
		NOTE: The electrode tip must be inside the cell body, and a small indentation on the soma should be observed. Do not move the electrode any further to avoid the tip going through the cell.</li>
		<li>Once the electrode tip is inside the cell, turn on the stimulator at ~0.5&#8722;1 V and continuously eject current into the cell. Using the confocal microscope, watch the cell fill. Increase the digital zoom to see the details of the cell and make sure that the tip of the electrode is visible inside the cell. 
		NOTE: Lower the voltage if it appears that dye is leaking out of the cell or filling other cells in the vicinity. If it appears that dye is still leaking out of the cell, pull electrode out slowly and find another cell. It is important that the dye does not leak to have a high signal/ background ratio in the final image.</li>
		<li>Wait for about 15 min until the finer branches and processes appear defined, turn off the voltage and gently withdraw the electrode tip from the cell.</li>
	</ol>
	</li>
	<li>Image the filled cell. 
	NOTE: Imaging can be performed immediately after filling or following staining with immunohistochemistry. An objective with a higher numerical aperture (NA) results in better resolution.
	<ol>
		<li>Wait until the cell returns to original form before imaging (15&#8722;20 min) with 40x objective. To image the cell, adjust the setting on the confocal to make sure that the finer branches and processes appear defined.</li>
		<li>Set up a z-stack with a step size of 0.3 &#181;m. While imaging, move the objective until there is no signal from the cell and set that as the top. Then, move the objective down (focusing through the cell) until there is no signal, set that as the bottom.</li>
		<li>After the imaging is complete, check the electrode for dye ejection. If a large dye cloud appears, it can be used for the next slice. Otherwise, replace it with a new electrode. 
		NOTE: Dye filling of a single astrocyte and imaging takes about 45 min to 1 h. For this specific experiment, it was possible to obtain about 3&#8722;6 cells per mouse. If needed, multiple cells can be labeled on the same slice. However, to distinguish individual astrocytes, be sure to keep a distance of about 200 &#181;m between cells.</li>
	</ol>
	</li>
</ol>
6. Staining with Immunohistochemistry (Optional)
<ol>
	<li>Once the imaging is done, immediately place the slice in 10% formalin on ice to preserve for immunohistochemistry. Keep brain slices in the dark and store overnight at 4 &#176;C.</li>
	<li>Wash brain sections 3x in 0.1 M PBS with 0.5% nonionic surfactant (i.e., Triton X 100) for 5 min each. Then incubate in a blocking solution of 0.1 M PBS with 0.5% nonionic surfactant and 10% normal goat serum (NGS) for 1 h at room temperature with gentle agitation.</li>
	<li>Incubate the sections with agitation in primary antibodies diluted in 0.1 M PBS with 0.5% nonionic surfactant and 5% NGS for 2 days at 4 &#176;C. 
	NOTE: Because of the thickness of the brain slices, the incubation period of the primary antibodies needs to be extended for better penetration and the antibody concentration may be increased. In this experiment, a 1:500 dilution was used for anti GFAP antibody and a 1:500 dilution was used for anti aquaporin-4 antibody.</li>
	<li>Wash the sections 3x in 0.1 M PBS with 0.5% nonionic surfactant for 10 min each and then incubate with secondary antibodies diluted in 0.1 M PBS with 0.5% nonionic surfactant and 10% NGS for 6 h at room temperature. 
	NOTE: Do not choose a secondary antibody excited by 488 nm, which is the wavelength used to visualize the LY dye. In this experiment, a 1:1,000 dilution was used for Alexa Fluor 546 goat anti-chicken IgG (H+L) and Alexa Fluor 647 goat anti-rabbit IgG (H+L).</li>
	<li>Rinse the sections 3x in 0.1 M PBS for 10 min each. Then mount the sections on glass microscope slides in mounting media suited for fluorescence. Seal slides. Image cells at a step size of 0.3 &#181;m.&#160;</li>
</ol>

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The authors have nothing to disclose.

The method outlined in this paper describes a way to visualize astrocyte morphology using intracellular iontophoresis of LY dye in lightly fixed brain slices. There are several critical factors highlighted in this protocol that contribute to successful LY iontophoresis and morphological reconstruction of the cells. One factor is the quality and reproducibility of the images, which is determined largely by the age of the mouse and the outcome of the perfusion. In this study, we used C57/BL6N mice 6&#8722;8 weeks old. A su...

Astrocytes are the most abundant glial cells in the central nervous system (CNS). They play roles in ion homeostasis, blood flow regulation, synapse formation as well as elimination, and neurotransmitter uptake1. The wide range of astrocyte functions is reflected in their complex morphological structure2,3. Astrocytes contain several primary and secondary branches which divide into thousands of finer branchlets and leaflets that directly interact with synapses, dendrites, axons, blood vessels, and other glial cells. Astrocyte morphology varies across different brain regions, which may hint at their ability to perform their functions differentially in neuronal circuits4. Moreover, astrocytes are known to alter their morphology during development, during physiological conditions, and in multiple disease states3,5,6.
A consistent, reproducible method is needed to accurately resolve the complexity of astrocyte morphology. Traditionally, immunohistochemistry has been used to visualize astrocytes with the use of astrocyte specific or astrocyte enriched protein markers. However, these methods reveal the pattern of protein expression rather than the structure of the astrocyte. The commonly used markers, such as glial fibrillary acidic protein (GFAP) and S100 calcium binding protein &#946; (S100&#946;), do not express in the entire cell volume and thus do not resolve complete morphology7. Genetic approaches to express fluorescent proteins ubiquitously in astrocytes (viral injections or transgenic mouse reporter lines) can identify the finer branches and overall territory. However, it is difficult to differentiate individual astrocytes, and analyses may be biased by the astrocyte population targeted by the specific promoter8. Serial section electron microscopy has been used to reveal a detailed picture of the interactions of astrocyte processes with synapses. Due to the thousands of astrocyte processes contacting synapses, it is currently not possible to reconstruct an entire cell with this technique9, although this is expected to change with the use of machine learning approaches for data analysis.
In this report, we focus on a procedure to characterize mouse astrocytes using intracellular iontophoresis with Lucifer yellow (LY) dye, using the CA1 stratum radiatum as an example. The method is based on pioneering past work by Eric Bushong and Mark Ellisman10,11. Astrocytes from lightly fixed brain slices are identified by their distinctive soma shape and filled with LY. The cells are then imaged with confocal microscopy. We demonstrate how LY iontophoresis can be used to reconstruct individual astrocytes and perform detailed morphological analyses of their processes and territory. Also, this method can be applied in conjunction with immunohistochemistry to identify spatial relationships and interactions between astrocytes and neurons, other glial cells, and brain vasculature. We consider LY iontophoresis to be a very suitable tool to analyze morphology in different brain regions and mouse models of healthy or disease conditions7,12,13.&#160;

<table><tbody><tr><td>10% Buffered Formalin Phosphate</td><td>Fisher</td><td>SF 100-20</td><td>An identical alternative can be used</td></tr><tr><td>Acrodisc Syringe Filters with Supor Membrane</td><td>Pall</td><td>4692</td><td>An identical alternative can be used</td></tr><tr><td>Ag/AgCl ground pellet</td><td>WPI</td><td>EP2</td><td>A similar alternative can be used</td></tr><tr><td>Alexa Fluor 546 goat anti-chicken IgG (H+L)</td><td>Thermo Scientific</td><td>A-11040</td><td>A similar alternative can be used</td></tr><tr><td>Alexa Fluor 647 goat anti-rabbit IgG (H+L)</td><td>Thermo Scientific</td><td>A27040</td><td>A similar alternative can be used</td></tr><tr><td>Anti Aquaporin-4 antibody</td><td>Novus Biologicals</td><td>NBP1-87679</td><td>A similar alternative can be used</td></tr><tr><td>Anti GFAP antibody</td><td>Abcam</td><td>ab4674</td><td>A similar alternative can be used</td></tr><tr><td>Borosilicate glass pipettes with filament</td><td>World precision instruments</td><td>1B150F-4</td><td></td></tr><tr><td>C57BL/6NTac mice</td><td>Taconic Stock</td><td>B6</td><td>A similar alternative can be used</td></tr><tr><td>Calcium Chloride</td><td>Sigma</td><td>21108</td><td>An identical alternative can be used</td></tr><tr><td>Confocal laser-scanning microscope</td><td>Olympus</td><td>FV1000MPE</td><td>A similar alternative can be used</td></tr><tr><td>D-glucose</td><td>Sigma</td><td>G7528</td><td>An identical alternative can be used</td></tr><tr><td>Disodium Phosphate</td><td>Sigma</td><td>255793</td><td>An identical alternative can be used</td></tr><tr><td>Electrode puller- Model P-97</td><td>Sutter</td><td>P-97</td><td>A similar alternative can be used</td></tr><tr><td>Fluoromount-G</td><td>Southern Biotech</td><td>0100-01</td><td>An identical alternative can be used</td></tr><tr><td>Heparin sodium injection (1,000 USP per mL)</td><td>Sagent Pharmaceuticals</td><td>400-10</td><td>An identical alternative can be used</td></tr><tr><td>Imaris software (Version 7.6.5)</td><td>Bitplane Inc.</td><td></td><td>A similar alternative can be used</td></tr><tr><td>Isofluorane</td><td>Henry Schein Animal Health</td><td>29404</td><td>An identical alternative can be used</td></tr><tr><td>Lidocaine Hydrochloride Injectable (2%)</td><td>Clipper</td><td>1050035</td><td>An identical alternative can be used</td></tr><tr><td>Lucifer Yellow CH dilithium salt</td><td>Sigma</td><td>L0259</td><td></td></tr><tr><td>Lucifer Yellow CH dipotassium salt</td><td>Sigma</td><td>L0144</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Sigma</td><td>M8266</td><td>An identical alternative can be used</td></tr><tr><td>Microscope Cover Glass</td><td>Thermo Scientific</td><td>24X60-1</td><td>An identical alternative can be used</td></tr><tr><td>Microscope Slides</td><td>Fisher</td><td>12-544-2</td><td>An identical alternative can be used</td></tr><tr><td>Normal Goat Serum</td><td>Vector Laboratories</td><td>S-1000</td><td>An identical alternative can be used</td></tr><tr><td>Objective lens (40x)</td><td>Olympus</td><td>LUMPLFLN 40XW</td><td>A similar alternative can be used</td></tr><tr><td>Objective lens (60x)</td><td>Olympus</td><td>PlanAPO 60X</td><td>A similar alternative can be used</td></tr><tr><td>PBS tablets, 100 mL</td><td>VWR</td><td>VWRVE404</td><td>An identical alternative can be used</td></tr><tr><td>Pipette micromanipulator- Model ROE-200</td><td>Sutter</td><td>MP-285 / ROE-200 / MPC-200</td><td>A similar alternative can be used</td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P3911</td><td>An identical alternative can be used</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S5761</td><td>An identical alternative can be used</td></tr><tr><td>Sodium Chloride</td><td>Sigma</td><td>S5886</td><td>An identical alternative can be used</td></tr><tr><td>Stimulator- Model Omnical 2010</td><td>World precision instruments</td><td>Omnical 2010</td><td>A similar alternative can be used</td></tr><tr><td>Triton X 100</td><td>Sigma</td><td>T8787</td><td>An identical alternative can be used</td></tr><tr><td>Vibratome- Model #3000</td><td>Pelco</td><td>100-S</td><td>A similar alternative can be used</td></tr></tbody></table>

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.

The data reported in this study are from 7&#8722;12 cells from 4 mice in each experiment. Average data are reported in the figure panels where appropriate.
To assess astrocyte morphology, we performed intracellular iontophoresis using LY dye to fill astrocytes in the CA1 stratum radiatum, which is summarized in Figure 1. Figure 2 depicts a representative astrocyte and its elaborate morphological structure. The cell was imaged post-fixation with th...

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Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

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-astrocyte-morphology-using-lucifer-yellow-iontophoresis

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JoVE Journal

Neuroscience

12.7K Views.  University of California Los Angeles. Deze methode kan worden gebruikt om de visualisatie van zeer fijne astrocytenprocessen te vergemakkelijken voor de evaluatie van astrocytenneuronale interacties bij de synaps tijdens ziekte en stabiele toestanden. Deze techniek kan ook worden toegepast in elk muismodel, celpopulatie of hersengebied. Om een scherpe elektrode met een geschikte weerstand voor te bereiden, trekt u een borosilicaatglas single barrel elektrode met een gloeidraad op een micropipet trekker.Bewaar de getrokken ...

Video: Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Biology

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

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 ...

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

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 ...

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.

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 ...

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 ...

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

Astrocytes tile the cerebral cortex uniformly, making the analysis of their complex morphology challenging at the cellular level. The protocol provided here uses multicolor labeling based on in utero electroporation to single out cortical astrocytes and analyze their volume and morphology with a user-friendly image analysis pipeline.

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

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 ...

Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

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