The authors would like to thank Drs. Rick Ryan, Umesh Patel, Jeff Till, Matthew Hsu, Jehangir Mistry and Michele Hatler for support during the development of these projects and protocols.

Cell Preparation:
<ol>
	<li>Prior to cell seeding for assay, culture neurons/astrocytes in growth media until ~70-80% confluent (unless cell seeding directly from thaw or isolation).</li>
	<li>Detach cells from culture flasks/plates via method appropriate for cell type of interest. If necessary, coat assay plate wells with poly-D-lysine or extracellular matrix protein to enhance cell adhesion. Co-cultures of astrocytes and neurons may be seeded simultaneously or sequentially. For sequential seedings, plating of astrocytes first as a &#34;basal&#34; layer is recommended, followed by neuronal plating and differentiation, if necessary. Adjust cell density as appropriate for cell type, subsequent culture time, parameters of interest, etc. Depending on culture age, cell source and seeding density, primary cultures may vary greatly in rate of proliferation, GFAP expression or neurite outgrowth - it is important to characterize and optimize your cell system to provide the most biological relevance for your experimental model, as well as to provide for effective imaging, segmentation and analysis using HCS (see Figure 1). After adding cells to plate (cells may be seeded in 90 &#956;L media, to facilitate toxin treatment as described in Step 3 below), allow plate to sit on a level surface at room temperature for 15-30 min, enabling even cell distribution. Following this period, incubate cells in growth media (37&#176;C/5% CO2) for at least 24 hours, then switch to differentiation culture conditions (e.g., low serum/NGF for PC12 cells), as appropriate. Continue culture until cells reach desired level of confluence or differentiation, changing media at intervals appropriate for cell type.</li>
	<li>Cell treatments (control compounds, test compounds, etc.) can be introduced at any point during this culture period, as appropriate for time-course of treatment of interest. Acrylamide, hydrogen peroxide and K-252a are provided as neurotoxic control compounds. Sufficient reagents are provided for duplicate 12-point dose response curves (including one dH2O or DMSO-control set within the dose response) for all five 96-well plates. The compounds are provided at 250X concentration (for K-252a, assuming a maximum treatment of 1 &#181;M) or 10X (for acrylamide and hydrogen peroxide, assuming maximum treatments of 100 mM and 10 mM, respectively). Recommended treatment preparation involves half-log (1:&#8730;10) serial dilution of the 250X compound in DMSO, followed by dilution in Compound Dilution Buffer to 10X (10X control compounds originating in dH2O may be serially diluted directly in sterile dH2O or Compound Dilution Buffer). 10 &#181;L of each treatment may then be added to the 90 &#181;L of culture media already present in each well, for a final 1X concentration (0.4% DMSO or 10% dH2O). Sample data is provided for 24 or 96 hours of compound treatment at 37&#176;C prior to fixation.</li>
</ol>
Cell Fixation and Immunofluorescent Staining:
Note: Staining time is ~2.5 hours post-fixation. Do not allow wells to dry out between staining steps. Aspiration and dispensation of reagents should be conducted at low flow rates to diminish any cell loss due to fluid shear. All recommended &#8216;per well&#8217; volumes refer to a single well of a 96-well microplate. All recommended &#8216;per 96-well plate&#8217; volumes include 25% excess for liquid handling volume loss. All staining steps are performed at room temperature (RT). All buffers and antibody solutions are stable for at least 24 hours at RT.
<ol start="4">
	<li>At end of culture period, pre-warm HCS Fixation Solution (2X) to room temperature (RT) or 37&#186;C if desired (12 mL/96-well plate). In a chemical fume hood, add 100 &#181;L/well directly to culture media and allow to fix for 30 min at RT. Remove fixative/toxin-containing media and dispose of in compliance with regulations for hazardous waste (see MSDS). If proceeding immediately to staining, rinse each well twice with 200 &#181;L of HCS Immunofluorescence Buffer. Alternatively, if plates are to be stained at a later time, rinse twice with 200 &#181;L of Wash Buffer, then leave second rinse volume in wells and store plates tightly sealed at 4&#186;C until staining.</li>
	<li>If fixed samples have been stored at 4&#186;C prior to staining, rinse twice with 200 &#181;L HCS Immunofluorescence Buffer before proceeding with staining protocol.</li>
	<li>Prepare working solution of Rabbit Anti-&#946;III Tubulin/Mouse Anti-GFAP HCS Primary Antibodies (6 mL/96-well plate) as follows: Add 60 &#181;L of each thawed primary antibody to 5.88 mL of HCS Immunofluorescence Buffer. Mix well. Remove previous Immunofluorescence Buffer rinse. Add 50 &#181;L of Primary Antibody solution to each well and incubate for 1 hour at RT.</li>
	<li>Remove Primary Antibody solution. Rinse three times with 200 &#181;L HCS Immunofluorescence Buffer.</li>
	<li>Prepare working solution of HCS Secondary Antibody/Hoechst HCS Nuclear Stain (6 mL/96-well plate) as follows: Add 30 &#181;L of each thawed secondary antibody and 30 &#181;L of thawed Hoechst HCS Nuclear Stain to 5.91 mL of HCS Immunofluorescence Buffer. Mix well, protecting solution from light. Remove previous HCS Immunofluorescence Buffer rinse. Add 50 &#181;L of HCS Secondary Antibody/Hoechst HCS Nuclear Stain solution and incubate for 1 hour at RT, protected from light.</li>
	<li>Remove HCS Secondary Antibody/Hoechst HCS Nuclear Stain solution. Rinse twice with 200 &#181;L HCS Immunofluorescence Buffer.</li>
	<li>Remove previous HCS Immunofluorescence Buffer rinse. Rinse twice with 200 &#181;L of HCS Wash Buffer, leaving second rinse volume in wells.</li>
	<li>Seal plate and image immediately, or store plate at 4&#186;C protected from light until ready for imaging.</li>
</ol>
Image Acquisition and Analysis:
<ol start="12">
	<li>Imaging and analysis of stained plates may be performed upon a variety of available HCS platforms, including the IN Cell Analyzer (GE Healthcare), ArrayScan (ThermoFisher Scientific) or Opera (Perkin Elmer). Some guidelines for imaging and analysis are provided in Table 1:</li>
</ol>
<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="4" style="background:#000000; color:#FFFFFF;">HCS222 Image Acquisition Guidelines</td>
		</tr>
		<tr>
			<td>Detection Reagent</td>
			<td>Objective Lens</td>
			<td>Excitation Filter Range [peak/bandwidth (nm)]</td>
			<td>Emission Filter Range [peak/bandwidth (nm)]</td>
		</tr>
		<tr>
			<td>Hoechst HCS Nuclear Stain</td>
			<td>20X</td>
			<td>360/40</td>
			<td>460/40 (or 535/50 if necessary)</td>
		</tr>
		<tr>
			<td>HCS Secondary Antibody, FITC-Donkey anti-Rabbit IgG</td>
			<td>20X</td>
			<td>480/40</td>
			<td>535/50</td>
		</tr>
		<tr>
			<td>HCS Secondary Antibody, Cy3-Donkey anti-Mouse IgG</td>
			<td>20X</td>
			<td>535/50</td>
			<td>600/50</td>
		</tr>
	</tbody>
</table>

<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="4" style="background:#000000; color:#FFFFFF;" width="545"> 
			HCS222 Image Analysis Guidelines</td>
		</tr>
		<tr>
			<td>Cell Parameter</td>
			<td>Detection</td>
			<td>Segmentation/ Measurement</td>
			<td>Rationale </td>
		</tr>
		<tr>
			<td>Cell Number, Nuclear Characteristics</td>
			<td>Hoechst HCS Nuclear Stain</td>
			<td>Nuclear region (460 nm emission channel).&#160; Count number of nuclei.&#160; DNA content (nuclear intensity) or nuclear area analyses are also possible.</td>
			<td>Use cell number, nuclear characteristics to determine cell loss, toxicity phenotypes, etc.&#160; Can &#8220;filter&#8221; nuclei for those associated with &#946;III-tubulin or GFAP expression to obtain separate neuronal and astrocytic cell counts/characterizations (strongly recommended).</td>
		</tr>
		<tr>
			<td>&#946;III-Tubulin Expression, Neurite Outgrowth</td>
			<td>HCS Secondary Antibody, FITC-conjugated</td>
			<td>Cytoplasmic region (535 nm emission channel).&#160; FITC signal may be used to distinguish neuronal cell bodies from neurites (e.g., via minimum/average cell body areas, minimum/maximum neurite lengths and widths).&#160; Determine parameters such as total neurite length, neurite counts/cell, etc.</td>
			<td>Neurite outgrowth measurements may be modulated during neuronal differentiation or as a result of chemical injury, disease states, etc.&#160; Can &#8220;filter&#8221; cell bodies for those associated with &#946;III-tubulin expression to obtain distinct neuronal characterization (strongly recommended).</td>
		</tr>
		<tr>
			<td>GFAP Expression, Astrocyte Area 
			&#160;</td>
			<td>HCS Secondary Antibody, Cy3-conjugated 
			&#160;</td>
			<td>Cytoplasmic region (600 nm emission channel).&#160; Cy3 signal may be used to define astrocytic cytoplasmic segmentation.&#160; Determine parameters such as average cytoplasmic signal intensity, cell area, etc.</td>
			<td>GFAP expression and astrocyte cell area may be modulated during astrocyte development or as a result of chemical injury, disease states, etc.&#160; Can &#8220;filter&#8221; cell bodies for those associated with GFAP expression to obtain distinct astrocytic characterization (strongly recommended).</td>
		</tr>
	</tbody>
</table>
Table 1. Image Acquisition and Analysis Guidelines &#8211; HCS222 &#946;III-Tubulin/GFAP (Co-Culture) Assay
Representative Results:
<img alt="Figure 1" src="/files/ftp_upload/1173/figure1.jpg" /> 
Figure 1. &#946;III-tubulin and GFAP immunofluorescence of mixed rat neural cell cultures. 
 
Co-cultures of primary rat hippocampal astrocytes with either primary rat hippocampal neurons or rat PC12 cells were generated by pre-plating astrocytes for several days in culture, followed by neuronal seeding. Primary neurons were cultured for an additional 11 days, while PC12s were cultured for an additional 2 days under differentiation conditions (low serum/NGF). Cell handling, fixation and immunostaining were performed according to HCS222 assay protocols. Cells were imaged on the GE IN Cell Analyzer 1000 (3.3) at 20X (primary neurons) or 10X (PC12) objective magnification and analyzed using the GE IN Cell Analyzer 1000 Workstation (3.4) Neurite Outgrowth and Multi Target Analysis algorithms. Top panel: Fused images of Hoechst HCS Nuclear Stain (blue), &#946;III-tubulin (green) and GFAP (red) fluorescence. Separate analysis of the &#946;III-tubulin fluorescence channel allows for neurite outgrowth segmentation (middle panel: cell bodies outlined in blue (primary neurons) or yellow (PC12), neurites in green (primary neurons) or light blue (PC12)). Analysis of the GFAP channel allows for astrocyte segmentation (bottom panel: nuclei outlined in blue, cytoplasm in green). GFAP segmentation for the primary rat hippocampal neuron/astrocyte co-culture demonstrates the ability to distinguish between nuclei/cell bodies that are GFAP (+) (green outlines) and those that are GFAP (-) (red), enabling separate cell counts for neurons and astrocytes in a mixed culture setting. 
 
<img alt="Figure 2" src="/files/ftp_upload/1173/figure2.jpg" /> 
Figure 2. Dose responses of primary rat hippocampal neuron/astrocyte co-cultures to toxic stresses. 
 
Primary rat hippocampal astrocytes (P6) were plated on 96-well plates in growth media and cultured for 6 days. Primary rat hippocampal neurons (direct from thaw) were then seeded on top of pre-plated astrocytes and cultured in growth media for 11 days. Cells were treated with serial dilutions of acrylamide or hydrogen peroxide (max. concentrations = 100mM and 10mM, respectively) for the last 24 hours of culture. Cell handling, fixation and immunostaining were performed according to HCS222 assay protocols. Cells were imaged on the GE IN Cell Analyzer 1000 (3.3) at 20X (10 fields/well) and analyzed (nuclear/cytoplasmic/neurite segmentation) using the GE IN Cell Analyzer 1000 Workstation (3.4) Neurite Outgrowth and Multi Target Analysis algorithms. Data presented are mean &#177; SEM, n = 3. Multiple parameters (neurite outgrowth, GFAP intensity, astrocyte area and neuronal or astrocytic cell counts) were investigated for dose-dependent trends.

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The authors are all employees of Millipore Corporation.

To date, in vitro experiments designed to study neurotoxicity risk assessment using mixed cultures of neurons and astrocytes have been limited. It is well-accepted that glial cells are integral in providing spatial and metabolic support to neurons, and play a critical role in modulating several neuronal functions, including neuronal migration, differentiation and synaptic activity. Glial astrocytes also release cytokines and other soluble factors which are capable of both inducing adverse responses in surrounding neuronal tissue, as well as promoting neuronal tolerance of many toxins. Demonstrating the depth of neuronal-glial interactions, in co-culture studies, astrocytes have been shown to protect neurons against the toxicity of oxidative stress, whereas impairment of astrocytic functions, such as maintenance of antioxidant defense and cellular energy levels, has been shown to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test-system may prove more relevant to human central nervous system structure and function than neuronal cells alone. Despite the growing awareness of the physiological significance of neuronal-astrocytic interactions, it has previously been difficult to perform quantitative analyses of these interactions in intact cells. The advent of High Content Screening enables the development of powerful new assays to address this.
In this study, we have demonstrated that quantitative analyses of multiple neuronal and astrocytic phenotypes within a single assay are made possible by HCA. In primary neurons we have performed quantitative measurements of neurotoxicity markers such as neuronal number, neurite count and neurite length. In astrocytes, reactive gliosis is a known biomarker of neurotoxicity, characterized by alterations in GFAP expression, astrocyte number and astrocyte morphology. We have demonstrated that each of these endpoints may readily be assessed via HCA. These studies support the concept that HCA of neural-specific biomarkers could be used as part of a battery of in vitro tests to rapidly screen large numbers of chemicals for neurotoxic endpoints.
Millipore&rsquo;s HCS222 High Content Analysis kit for co-culture of neurons and astrocytes is comprised of high-quality detection reagents and protocols for simultaneously profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP) in a wide variety of cellular models of neurotoxicity. The highly-validated reagents provided with this kit allow the user to standardize assays, minimize assay-to-assay variability, and to reproducibly generate images with a high signal-to-background ratio.

<table><tbody><tr><td>Rabbit Anti-&amp;#946;III-Tubulin HCS Primary Antibody, 100X</td><td></td><td>Part No. CS201672</td><td>1 vial containing 300 &amp;#x03BC;L&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>HCS Secondary Antibody (donkey anti-rabbit IgG, FITC conjugate), 200X</td><td></td><td>Part No. CS201649</td><td>1 vial containing 150 &amp;#x03BC;L&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>Mouse Anti-GFAP HCS Primary Antibody, 100X</td><td></td><td>Part No. CS201671</td><td>1 vial containing 300 &amp;#x03BC;L&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>HCS Secondary Antibody (donkey anti-mouse IgG, Cy3 conjugate), 200X</td><td></td><td>Part No. CS200437</td><td>1 vial containing 150 &amp;#x03BC;L&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>H&amp;#8211;chst HCS Nuclear Stain, 200X</td><td></td><td>Part No. CS200438</td><td>1 vial containing 150 &amp;#x03BC;L&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>HCS Fixation Solution with Phenol Red, 2X</td><td></td><td>Part No. CS200434</td><td>1 bottle containing 100 mL&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>HCS Immunofluorescence Buffer, 1X</td><td></td><td>Part No. CS200435</td><td>1 bottle containing 1000 mL&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>HCS Wash Buffer, 1X</td><td></td><td>Part No. 2007643</td><td>1 bottle containing 500 mL&lt;br/&gt;Millipore HCS222 Kit Components</td></tr><tr><td>Acrylamide (Control Compound), 10X</td><td></td><td>Part No. CS201679</td><td>1 vial containing 500 &amp;#x03BC;L of 1M acrylamide in dH&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>Hydrogen Peroxide (Control Compound), 10X</td><td></td><td>Part No. CS201730</td><td>1 vial containing 500 &amp;#x03BC;L of 100 mM hydrogen peroxide in dH&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt;Millipore HCS222 Kit Components</td></tr><tr><td>K-252a (Control Compound), 250X</td><td></td><td>Part No. CS201741</td><td>1 vial containing 100 &amp;#x03BC;L of 250 &amp;#x03BC;M K-252a in DMSO&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>DMSO for Compound Serial Dilution</td><td></td><td>Part No. CS200441</td><td>1 bottle containing 10 mL&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>Compound Dilution Buffer</td><td></td><td>Part No. CS200442</td><td>1 bottle containing 25 mL&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>Plate Sealers</td><td></td><td>Part No. CS200443</td><td>10 each&lt;br/&gt;Millipore HCS222 Kit Components</td></tr><tr><td>tissue culture-treated black/clear bottom microplates </td><td></td><td></td><td></td></tr><tr><td>HCS imaging/analysis system</td><td></td><td></td><td></td></tr></tbody></table>

a neuronal and astrocyte co-culture assay for high content analysis of neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

Watch this Scientific Journal Video about A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity at JoVE.com

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

a-neuronal-astrocyte-co-culture-assay-for-high-content-analysis

Research

JoVE Journal

Biology

Video: A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is very poor 1. The failure of axon regeneration is a combined result of several factors including the hostile glial cell environment, inhibitory myelin related molecules and decreased intrinsic neuron regenerative capacity 2. Astrocytes are the most predominant glial cell type in central nervous system and play important role in axon functions under physiology and pathology conditions 3. Contrast to the homologous oligodendrocytes, astrocytes are a heterogeneous cell population composed by different astrocyte subpopulations with diverse morphologies and gene expression 4. The functional significance of this heterogeneity, such as their influences on axon growth, is largely unknown.
To study the glial cell, especially the function of astrocyte heterogeneity in neuron behavior, we established a new method by co-culturing high purified dorsal root ganglia neurons with glial cells obtained from the rat cortex. By this technique, we were able to directly compare neuron adhesion and axon growth on different astrocytes subpopulations under the same condition.
In this report, we give the detailed protocol of this method for astrocytes isolation and culture, dorsal root ganglia neurons isolation and purification, and the co-culture of DRG neurons with astrocytes. This method could also be extended to other brain regions to study cellular or regional specific interaction between neurons and glial cells.

In this protocol, we described a new method to study the influence of glial cell heterogeneity on axon growth with an in vitro co-culture system. Rat cortical glial cells were cultured to confluence and cocultured with highly purified rat dorsal root ganglia neurons. Different glial cell influence on neurons adhesion and axon growth was compared directly in the same culture. This method provides a new way to directly study the glial cell heterogeneity influence on neuron adhesion and axon growth.

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

Neuroscience

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect.
Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.&#34;

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides ...

Developmental Biology

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 ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

A Scalable Method to Study Neuronal Survival in Primary Neuronal Culture with Single-cell and Real-Time Resolution

Neuronal loss is at the core of many neuropathologies, including stroke, Alzheimer's disease, and Parkinson's disease. Different methods were developed to study the process of neuronal survival upon cytotoxic stress. Most methods are based on biochemical approaches that do not allow single-cell resolution or involve complex and costly methodologies. Presented here is a versatile, inexpensive, and effective experimental paradigm to study neuronal survival. This method takes advantage of sparse fluorescent labeling of the neurons followed by live imaging and automated quantification. To this aim, the neurons are electroporated to express fluorescent markers and co-cultured with non-electroporated neurons to easily regulate cell density and increase survival. 
Sparse labeling by electroporation allows a simple and robust automated quantification. In addition, fluorescent labeling can be combined with the co-expression of a gene of interest to study specific molecular pathways. Here, we present a model of stroke as a neurotoxic model, namely, the oxygen-glucose deprivation (OGD) assay, which was performed in an affordable and robust homemade hypoxic chamber. Finally, two different workflows are described using IN Cell Analyzer 2200 or the open-source ImageJ for image analysis for semi-automatic data processing. This workflow can be easily adapted to different experimental models of toxicity and scaled up for high-throughput screening. In conclusion, the described protocol provides an approachable, affordable, and effective in vitro model of neurotoxicity, which can be suitable for testing the roles of specific genes and pathways in live imaging and for high-throughput drug screening.

Here, we present a simple, potent, and versatile methodology to investigate neuronal survival upon cytotoxic stress in primary cortical neurons with cellular resolution in real time or in fixed material.

Neuronal loss is at the core of many neuropathologies, including stroke, Alzheimer's disease, and Parkinson's disease. Different methods were developed to ...

 Primary Microglia and Neuron Co-Culture Protocol for CNS Interaction Studies

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

Author Spotlight: In Vitro Co-Culture Model for Studying Microglia-Neuronal Interactions in Disease Conditions

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS ...

An Indirect Neuron-Astrocyte Co-Culture Technique for Studying Neuron-Glia Interactions

Source: Gottschling, C. et al., <a href="https://app.jove.com/v/54757">The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions.</a>&#160;J. Vis. Exp.&#160;(2016)
This video illustrates a method for establishing an indirect neuron-astrocyte co-culture using a compartmentalized approach. In this setup, neurons and astrocytes are spatially separated but share a conditioned medium. Neurotrophic factors released by astrocytes promote neuron differentiation and the formation of synaptic connections, suggesting neuron-glia interactions.

Technika pośredniej kohodowli neuronów i astrocytów do badania interakcji neuron-glej

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Co-culture and Interaction Studies

Recording Electrophysiological Activity of the Neuronal Network in a Neuron-Astrocyte Co-culture

Source: Taga, A., et al. <a href="https://app.jove.com/v/62726">Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons.</a> J. Vis. Exp. (2021).
This video demonstrates establishing a neuron-astrocyte co-culture for recording neuronal network activity. Upon establishing the co-culture of motor neurons and astrocytes on a multi-electrode array (MEA) plate, the synchronous rhythmic electrical activity of the neuronal network was recorded extracellularly using the MEA.

Rejestrowanie aktywności elektrofizjologicznej sieci neuronalnej w kokulturze neuron-astrocyt

1. Co-culturing MN and spinal cord astrocytes in multi-electrode array plates

	Differentiate and plate the motor neurons and astrocytes when they are ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

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Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

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

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

A Scalable Method to Study Neuronal Survival in Primary Neuronal Culture with Single-cell and Real-Time Resolution

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Technika pośredniej kohodowli neuronów i astrocytów do badania interakcji neuron-glej

Rejestrowanie aktywności elektrofizjologicznej sieci neuronalnej w kokulturze neuron-astrocyt