Name: visualizing astrocyte morphology using lucifer yellow iontophoresis
Uploaded: 2019-09-14T15:00:00
Description: Watch this Scientific Journal Video about Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis at JoVE.com

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
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	<li>Artificial cerebrospinal fluid (ACSF) solution
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		<li>Prepare fresh ACSF solution (135 mM NaCl...

<ol>
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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherin-9+regulates+synapse-specific+differentiation+in+the+developing+hippocampus.">Cadherin-9 regulates synapse-specific differentiation in the developing hippocampus.</a> Neuron. 71 (4), 640-655 (2011).</li><li>Ogata, K., Kosaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+quantitative+analysis+of+astrocytes+in+the+mouse+hippocampus.">Structural and quantitative analysis of astrocytes in the mouse hippocampus.</a> Neuroscience. 113 (1), 221-233 (2002).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), e3564 (2012).</li><li>Hubbard, J. A., Hsu, M. S., Seldin, M. M., Binder, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+Astrocyte+Water+Channel+Aquaporin-4+in+the+Mouse+Brain.">Expression of the Astrocyte Water Channel Aquaporin-4 in the Mouse Brain.</a> ASN Neuro. 7 (5), (2015).</li><li>Benediktsson, A. 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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 border="0" cellpadding="0" cellspacing="0" style="width:995px;" width="994">
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	<tbody>
		<tr height="21">
			<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, ...

This method can be used to facilitate the visualization of very fine astrocyte processes for the evaluation of astrocyte neuronal interactions at the synapse during disease and steady states. This technique can also be applied in any mouse model, cell population, or brain region. To prepare a sharp electrode with an appropriate resistance, pull a borosilicate glass single barrel electrode with a filament on a micropipette puller.Store the pulled electrodes in a closed container to prevent dust from entering the tips and keep the electrodes elevated from the bottom of the box to prevent the tips from breaking. To fill an electrode with lucifer yellow dye solution, place an electrode in the vertical position with the tip facing downward, and pipette one to two microliters of lucifer yellow solution into the back of the electrode. Wait five to 10 minutes for the solution to move to the tip via capillary action before gently securing the filled electrode in an electrode holder connected to a manipulator with the silver wire of the electrode holder in contact with the lucifer yellow inside the electrode.For the dye ejection test, gently place a lightly fixed brain slice into a glass bottom dish filled with 0.1 molar PBS at room temperature and hold the slice in place with a platinum harp with nylon strings. Connect the electrode to a voltage source and place the ground electrode into the bath containing the brain slice. Move the objective of a confocal microscope to the brain region of interest and lower the electrode into the solution using the 10X water immersion lens, moving the electrode to the center of the field of view.Under bright-field illumination, examine the electrode carefully with the 40X water immersion lens to ensure that the electrode appears clear and without debris or bubbles. Switch to confocal laser scanning microscopy with a 488-nanometer laser. Turn on the stimulator at 12 volts to test the dye ejection.A large fluorescent dye cloud should be visible around the tip of the electrode. Then, under bright-field slowly lower the electrode toward the slice, stopping just above the surface. To fill an astrocyte of interest with iontophoresis, use the infrared differential interference contrast to identify astrocytes with elongated oval-shaped somata about 10 micrometers in diameter, 40 to 50 micrometers below the slice surface.This is the stratum radiatum of the hippocampus directly below the pyramidal cell layer. As we move through the tissue, we can see blood vessels, neurons, astrocytes, and other cell types. Once an astrocyte has been selected, move the cell to the center of the field of view and slowly lower the electrode tip into the slice, navigating through the tissue until the electrode is on the same plain as the cell body.Once the cell body of the astrocyte is clearly visible and outlined, slowly and gently advance the electrode forward, moving the electrode until the tip impales the soma of the cell. Move the focus of the objective slowly up and down to note if the electrode is inside the soma. Once the electrode tip is inside the cell, turn on the stimulator at 0.5 to one volts to continuously eject current into the cell.Using the confocal microscope, watch the cell film, increasing the digital zoom to visualize the details of the cell and to make sure that the tip of the electrode is visible inside the cell as necessary. Wait for about 15 minutes until the finer branches and processes appear defined before turning off the voltage and gently withdrawing the electrode tip from the cell. To image the filled cell, wait 15 to 20 minutes for the cell to return to its original form before adjusting the setting on the confocal microscope to make sure that the finer branches and processes appear defined under the 40X objective.Set up a Z stack with a step size of 0.3 micrometers, moving the objective while imaging until there is no signal from the cell and set that step as the top. Then move the objective down, focusing through the cell until there is no signal and set that step as the bottom. After the imaging is complete, check the electrode for dye ejection.If a large dye cloud appears, the electrode can be used for the next slice. Otherwise, replace the electrode. By generating a maximum intensity projection, a detailed view of the structure of an astrocyte in its domain can be observed.A four X magnitude zoom into the astrocyte reveals a maximum intensity projection of one major branch, several secondary branches, and the distribution of the surrounding branchlets and leaflets. Here, the original image of a CA1 astrocyte is shown. The cell body, major branches, processes, and territory volume enclosed by the astrocyte are reconstructed in these subsequent images.After the reconstructions were created, the volumes of the soma, entire cell, and territory were quantified. And the number of major branches were counted. As cytoskeletal protein that labels intermediate astrocyte filaments is expressed in the cell soma, major branches, and some secondary astrocyte branches, but not in the finer branches and processes.In this representative analysis, no significant differences were found in the number of primary branches labeled by glial fibrillary acidic protein and those visualize by lucifer yellow. Further, the cell area and volume of the astrocyte labeled by glial fibrillary acidic protein were significantly smaller than the area and volume visualized with lucifer yellow, demonstrating that glial fibrillary acidic protein is a reliable marker for labeling major branches but is not useful for determining the overall area or volume of the cell. The amount of dye released from the electrode tip is dependent on the electrode resistance which must be high enough to allow a steady stream of dye to be ejected.Future studies can examine the different components of astrocyte structure by super resolution light microscopy or by immunohistochemical analysis of the expression of specific proteins within the astrocyte structure.

Research

JoVE Journal

Neuroscience

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