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, ...

Research

JoVE Journal

Neuroscience

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 ...

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

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 ...

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

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 ...

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 ...

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 ...

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 ...

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

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 ...

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 ...