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;

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	<tbody>
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			<td height="21" style="height:21px;width:367px;">10% Buffered Formalin Phosphate</td>
			<td style="width:192px;">Fisher</td>
			<td style="width:196px;">SF 100-20</td>
			<td style="width:240px;">An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Acrodisc Syringe Filters with Supor Membrane</td>
			<td style="width:192px;">Pall</td>
			<td style="width:196px;">4692</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ag/AgCl ground pellet</td>
			<td>WPI</td>
			<td>EP2</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Alexa Fluor 546 goat anti-chicken IgG (H+L)</td>
			<td style="width:192px;">Thermo Scientific</td>
			<td>A-11040</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Alexa Fluor 647 goat anti-rabbit IgG (H+L)</td>
			<td style="width:192px;">Thermo Scientific</td>
			<td>A27040</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Anti Aquaporin-4 antibody</td>
			<td style="width:192px;">Novus Biologicals</td>
			<td>NBP1-87679</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Anti GFAP antibody</td>
			<td style="width:192px;">Abcam</td>
			<td style="width:196px;">ab4674</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate glass pipettes with filament</td>
			<td>World precision instruments</td>
			<td style="width:196px;">1B150F-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6NTac mice</td>
			<td>Taconic Stock</td>
			<td style="width:196px;">B6</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td style="width:192px;">Sigma</td>
			<td>21108</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal laser-scanning microscope</td>
			<td style="width:192px;">Olympus</td>
			<td style="width:196px;">FV1000MPE</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-glucose</td>
			<td style="width:192px;">Sigma</td>
			<td>G7528</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disodium Phosphate</td>
			<td style="width:192px;">Sigma</td>
			<td>255793</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electrode puller- Model P-97</td>
			<td>Sutter</td>
			<td style="width:196px;">P-97</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Fluoromount-G</td>
			<td style="width:192px;">Southern Biotech</td>
			<td style="width:196px;">0100-01</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Heparin sodium injection (1,000 USP per mL)</td>
			<td style="width:192px;">Sagent Pharmaceuticals</td>
			<td>400-10</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaris software (Version 7.6.5)</td>
			<td style="width:192px;">Bitplane Inc.</td>
			<td style="width:196px;"></td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Isofluorane</td>
			<td style="width:192px;">Henry Schein Animal Health</td>
			<td>29404</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lidocaine Hydrochloride Injectable (2%)</td>
			<td style="width:192px;">Clipper</td>
			<td>1050035</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Lucifer Yellow CH dilithium salt</td>
			<td style="width:192px;">Sigma</td>
			<td>L0259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Lucifer Yellow CH dipotassium salt</td>
			<td style="width:192px;">Sigma</td>
			<td>L0144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Chloride</td>
			<td style="width:192px;">Sigma</td>
			<td>M8266</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Microscope Cover Glass</td>
			<td style="width:192px;">Thermo Scientific</td>
			<td style="width:196px;">24X60-1</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Microscope Slides</td>
			<td style="width:192px;">Fisher</td>
			<td style="width:196px;">12-544-2</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Normal Goat Serum</td>
			<td style="width:192px;">Vector Laboratories</td>
			<td>S-1000</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Objective lens (40x)</td>
			<td>Olympus</td>
			<td style="width:196px;">LUMPLFLN 40XW</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Objective lens (60x)</td>
			<td>Olympus</td>
			<td>PlanAPO 60X</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">PBS tablets, 100 mL</td>
			<td style="width:192px;">VWR</td>
			<td>VWRVE404</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Pipette micromanipulator- Model ROE-200</td>
			<td style="width:192px;">Sutter</td>
			<td>MP-285 / ROE-200 / MPC-200</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Potassium Chloride</td>
			<td style="width:192px;">Sigma</td>
			<td>P3911</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Bicarbonate</td>
			<td style="width:192px;">Sigma</td>
			<td>S5761</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td style="width:192px;">Sigma</td>
			<td>S5886</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stimulator- Model Omnical 2010</td>
			<td>World precision instruments</td>
			<td style="width:196px;">Omnical 2010</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Triton X 100</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:196px;">T8787</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome- Model #3000</td>
			<td style="width:192px;">Pelco</td>
			<td style="width:196px;">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...

Watch this Scientific Journal Video about Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis at JoVE.com

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

Research

JoVE Journal

Neuroscience

12.5K Views.  University of California Los Angeles. 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.

Video: Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile stimulation is a positive early experience that mimics maternal licking and grooming in the rat. Exposing rat pups to this positive experience can be completed easily and cost-effectively by using highly accessible materials such as a household duster. Using a cross-litter design, pups are either stroked or left undisturbed, for 15 min, three times per day throughout the perinatal period. To measure the neuroplastic changes related to this positive early experience, Golgi-Cox staining of brain tissue is utilized. Owing to the fact that Golgi-Cox impregnation stains a discrete number of neurons rather than all of the cells, staining of the rodent brain with Golgi-Cox solution permits the visualization of entire neuronal elements, including the cell body, dendrites, axons, and dendritic spines. The staining procedure is carried out over several days and requires that the researcher pay close attention to detail. However, once staining is completed, the entire brain has been impregnated and can be preserved indefinitely for ongoing analysis. Therefore, Golgi-Cox staining is a valuable resource for studying experience-dependent plasticity.

This paper describes the procedures for tactile stimulation of rat pups and subsequent Golgi-Cox staining of neuronal morphology. Tactile stimulation is a positive experience that is administered in the perinatal period by stroking pups with a household duster. Golgi-cox staining is a reliable procedure permitting the visualization of entire neurons.

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

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 ...

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 ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer&#39;s disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia. This lack of information is mostly due to the lack of an assay system in which the kinetics of astrocyte- and microglia-mediated phagocytosis are easily comparable. To achieve this goal, we have developed a long-term live-imaging in vitro phagocytosis assay to evaluate the phagocytic capacity of purified astrocytes and microglia. In this assay, real-time detection of engulfment and degradation is possible using pH indicator-conjugated synaptosomes, which emit bright red fluorescence in acidic organelles, such as lysosomes. Our novel assay provides simple and effective detection of phagocytosis through live-imaging. In addition, this in vitro phagocytosis assay can be used as a screening platform to identify chemicals and compounds that can enhance or inhibit the phagocytic capacity of astrocytes. As synaptic pruning malfunction and pathogenic protein accumulation have been shown to cause mental disorders or neurodegenerative diseases, chemicals and compounds that modulate the phagocytic capacity of glial cells should be helpful in treating various neurological disorders.

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain.

Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape.

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As ...

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.