We would like to thank Dr. Jeffrey Rothstein for providing BAC GLT1 eGFP mice and GLT1 antibody; Tufts Center for Neuroscience Research (NIH P30 NS047243; PI, Rob Jackson) for providing valuable core facilities; New faculty recruitment grant (NIH P30 5P30NS069254-02; PI, Phil Haydon) in Tufts Neuroscience Department.

1. Assembly of the Microfluidic Culture Chamber (MCP)
<ol>
 <li>MCP (Figure 1) are open chambers designed for compartmented cultures of different types of cells 4. It typically has two compartments that are connected through the central channels (3 &mu;m in diameter). Assembly of MCP with glass-bottomed dishes is necessary for preparing cultures and subsequent imaging analysis.</li>
 <li>First, coat sterile glass-bottomed dishes with Polyornithine (Sigma-Aldrich, 1 mg/ml) dissolved in sodium tetraborate (Sigma-Aldrich, 10 mM pH 8.5) and were incubated overnight at 37 &deg;C.</li>
 <li>On the following day, the coated glass-bottomed dishes were washed three times with ddH2O and dried under the sterile fume hood. The glass-bottomed dishes were then further coated with laminin (Sigma-Aldrich, 1 mg/ml) and dried under UV light for 1 hour. Coated dishes are ready to use or can be stored at -20 &deg;C until use. These coating are necessary for plating neurons and astrocytes in the co-culture.</li>
 <li>To assemble the culture platform, microfluidic culture platforms were placed on top of the coated glass-bottomed dishes with central connecting channels on its bottom side to form a tight seal. Regular neuron or astrocyte culture mediums were added (from the cell plating areas) on both sides (Figure 1) into the assembled MCP and incubated for 2-4 hours at 37 &deg;C, to ensure there is no leakage between MCP and the glass-bottomed dish. We have tested with medium before plating cells to ensure there is no leak. The assembly of MCP on the glass-bottomed dish should be freshly prepared before use.</li>
</ol>
2. Preparation of Neuronal Culture and Induction of Neuronal Axons in Assembled MCP
<ol>
 <li>Cortical neuronal cell cultures were freshly prepared from embryonic 14-16 day-old mouse brains. The neuron culture medium is composed of neurobasal medium, 2% B27 neurobasal supplement, 2 mM glutamine by adding 1% of 100x GlutaMAX, and 1% Penicillin-streptomysin. Following dissection of the mouse brain, the meninges was removed from the cortex under the dissecting microscope. The tissue was then minced with a razor blade to make small tissue blocks in order to increase the surface area exposed to trypsin. Tissue blocks were trypsinized (Sigma-Aldrich, 0.05% Trypsin) for 10 min in a 37 &deg;C water bath, and then dissociated gently by trituration with a fire-polished Pasteur pipette. Dissociated cells were filtered through a 70 &mu;m strainer to collect clear neuronal cell suspension.</li>
 <li>Neurons (2-3 x 106/ml, 150 &mu;l) were plated on the cell plating areas on right side (Figure 1) of the assembled MCP so that neurons can attach in the cell retention area (Figure 1). Freshly prepared neurons were plated with neuron plating medium which is supplemented with 5% fetal bovine serum (Hyclone) into the regular neuron culture medium. Because only the neurons that attach in the cell retention area are able to grow axons into the other side of the assembled MCP through central channels, it is important to plate high density of neurons in the cell plating area to ensure enough neurons are attached to the cell retention area. A representative image of high density of neuronal axons is shown in Figure 2A.</li>
 <li>On the following day, the neuron plating medium was replaced by the regular neuron culture medium. Changing medium was accomplished by carefully aspirating the medium from the cell plating area of the chamber, but not from the cell retention area of the assembled MCP, in order to avoid air bubbles in the cell retention area and the central connecting channels. Air bubbles will severely inhibit the axon outgrowth and subsequent entry into the central channels in the MCP. Glial-cell derived neurotrophic factor (GDNF, 10-20 ng/ml) was also added on the other (left) side of the chamber on the same day to facilitate the induction of axon outgrowth5 from the neuronal side and cross through central channels of the assembled MCP (Figure 2A). Fair amount of spontaneous neurite outgrowth without GDNF is also observed from the neuronal side and cross through central channels of the assembled MCP. Axons that enter into the central channel are often observed 2-3 days after plating. Once the axons enter into the central channel, they usually enter the other side within one day. Axons are normally intact for at least one week after entering the other side.</li>
</ol>
3. Addition of Cultured Astrocytes to MCP to Establish a Compartmentalized Co-culture System
Primary astrocyte cultures were prepared from the P1-3 mouse pup brain. The brain dissociation procedure is similar to the neuronal cell isolation procedure described above. Astrocytes were first plated into 10 cm dish that were pre-coated with Polyornithine (Sigma-Aldrich, 1 mg/ml). The astrocyte culture medium (DMEM, 10% fetal bovine serum, 1% Penicillin-streptomysin) was changed every day for the next two days to remove the debris. After that, the medium was changed every three days.
<ol>
 <li>Astrocytes become 90% confluent and form a monolayer after 7 days. Confluent astrocytes were trypsinized and 150 &mu;l of re-suspended astrocytes (1x106 /ml) were re-plated into the cell plating area of the left side of the assembled MCP when axons are about to enter or have just entered into the left side of the assembled MCP. Astrocytes were usually plated 4-5 days after the neurons were plated. GDNF was first removed before the re-plating of the astrocytes into the left side of the MCP. Re-plated astrocytes were attached in the cell retention area, as shown by the GFAP immunostaining (Figure 1). Only minimal flow of medium between both sides of the chambers (determined by fluorescence dyes) was found in the MCP, as previously shown4,6 .</li>
 <li>Astrocytes become 90% confluent and form a monolayer after 7 days. Confluent astrocytes were trypsinized and 150 &mu;l of re-suspended astrocytes (1 x 106 / ml) were re-plated into the cell plating area of the left side of the assembled MCP when axons are about to enter or have just entered into the left side of the assembled MCP. Astrocytes were usually plated 4-5 days after the neurons were plated. GDNF was first removed before the re-plating of the astrocytes into the left side of the MCP. Re-plated astrocytes were attached in the cell retention area, as shown by the GFAP immunostaining (Figure 1). Only minimal flow of medium between both sides of the chambers (determined by fluorescence dyes) was found in the MCP, as previously shown4,6 .</li>
 <li>After the re-plating of the astrocytes, axons that entered the left side of the assembled MCP directly interact with the astrocytes by either direct axonal contact or release of soluble factors. Immunostaining of astroglial plasma membrane glutamate transporter GLT1 and neuronal &beta;III-tubulin was performed to visualize the interaction between the axons and the astrocytes, as shown in Figure 2D-F.</li>
</ol>

<ol>
	<li>Stevens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-astrocyte+signaling+in+the+development+and+plasticity+of+neural+circuits.">Neuron-astrocyte signaling in the development and plasticity of neural circuits.</a> Neuro-Signals. 16, 278-288 (2008).</li><li>Paixao, S., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-astrocyte+communication+and+synaptic+plasticity.">Neuron-astrocyte communication and synaptic plasticity.</a> Current opinion in neurobiology. 20, 466-473 (2010).</li><li>Fields, R. D., Stevens-Graham, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+neuron-glia+communication.">New insights into neuron-glia communication.</a> Science. 298, 556-562 (2002).</li><li>Park, J. W., Vahidi, B., Taylor, A. M., Rhee, S. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nature. 1, 2128-2136 (2006).</li><li>Wang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+neuromuscular+synapse+development+by+glial+cell+line-derived+neurotrophic+factor+and+neurturin.">Regulation of neuromuscular synapse development by glial cell line-derived neurotrophic factor and neurturin.</a> The Journal of biological chemistry. 277, 10614-10625 (2002).</li><li>Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+regulation+of+astroglial+excitatory+neurotransmitter+transporter+GLT1.">Presynaptic regulation of astroglial excitatory neurotransmitter transporter GLT1.</a> Neuron. 61, 880-894 (2009).</li><li>Regan, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variations+in+promoter+activity+reveal+a+differential+expression+and+physiology+of+glutamate+transporters+by+glia+in+the+developing+and+mature+CNS.">Variations in promoter activity reveal a differential expression and physiology of glutamate transporters by glia in the developing and mature CNS.</a> The Journal of neuroscience. 27, 6607-6619 (2007).</li><li>Swanson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+regulation+of+glutamate+transporter+subtype+expression+in+astrocytes.">Neuronal regulation of glutamate transporter subtype expression in astrocytes.</a> The Journal of neuroscience. 17, 932-940 (1997).</li><li>Schlag, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+glial+Na+-dependent+glutamate+transporters+by+cyclic+AMP+analogs+and+neurons.">Regulation of the glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons.</a> Molecular pharmacology. 53, 355-369 (1998).</li><li>Ponomarev, E. D., Novikova, M., Maresz, K., Shriver, L. P., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+culture+system+that+supports+adult+microglial+cell+proliferation+and+maintenance+in+the+resting+state.">Development of a culture system that supports adult microglial cell proliferation and maintenance in the resting state.</a> Journal of immunological. 300, 32-46 (2005).</li><li>Espinosa-Jeffrey, A., Wakeman, D. R., Kim, S. U., Snyder, E. Y., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+system+for+rodent+and+human+oligodendrocyte+specification,+lineage+progression,+and+maturation.">Culture system for rodent and human oligodendrocyte specification, lineage progression, and maturation.</a> Current protocols in stem cell biology. Chapter 2, (2009).</li><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mystery+and+magic+of+glia:+a+perspective+on+their+roles+in+health+and+disease.">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60, 430-440 (2008).</li><li>Debanne, D., Rama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+shape+axonal+signaling.">Astrocytes shape axonal signaling.</a> Science signaling. 4, pe11 (2011).</li></ol>

No conflicts of interest declared.

The MCP based neuron and astrocytes co-culture system allows dissection of detailed neuron to astroglia signaling pathways by allowing only the axons pass the central channels and interacting with the astroglial cells. This co-culture system can be conveniently set up with conventional neuron and astrocyte culture procedures. We also described a practical application of this co-culture system by employing an eGFP based reporter for demonstrating axon-dependent GLT1 promoter activation in astrocytes.
	<p class="...

<table border="1">
 <tbody>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Hyclone</td>
 <td>SH30070.03</td>
 <td>for plating neuron for neuron cutlure medium</td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Sigma-Aldrich</td>
 <td>F4135</td>
 <td>for astrocyte culture medium</td>
 </tr>
 <tr>
 <td>Glial derived nerve factor</td>
 <td>R&amp;D systems</td>
 <td>212-GD</td>
 <td>Apply 10-20 ng/ml to neuron side of chamber</td>
 </tr>
 <tr>
 <td>Dulbecco modified eagle medium high glucose</td>
 <td>Sigma-Aldrich</td>
 <td>11995</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>70 mm cell strainer</td>
 <td>BD Falcon</td>
 <td>352350</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sterile glass bottom dish</td>
 <td>MatTek Corporation</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microfluidic culture platforms</td>
 <td>Xona Microfluidics LLC</td>
 <td>SND150</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>6 wells of the culture plate</td>
 <td>Cellstar</td>
 <td>657 160</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Neuron culture medium
 <ul>
 <li>Neurobasal medium</li>
 <li>2% B27 Neurobasal supplement</li>
 <li>2 mM glutamate by adding 1% 100x GlutaMAX</li>
 <li>1% Penicillin-streptomysin</li>
 </ul>
 </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>
 Neuron culture medium for plating cell
 <ul>
 <li>Neurobasal medium</li>
 <li>2% B27 Neurobasal supplement</li>
 <li>2 mM glutamate by adding 1% 100x GlutaMAX</li>
 <li>1% Penicillin-streptomysin</li>
 <li>5% Fetal bovine serum SH30070.03</li>
 </ul>
 </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>
 Astrocyte culture medium
 <ul>
 <li>Dulbecco modified eagle medium high glucose</li>
 <li>10% Fetal bovine serum F4135</li>
 <li>1% Penicillin-streptomysin</li>
 </ul>
 </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>
 Table 1. Materials used in the microfluidic culture platform-based neuronal axon and glia co-culture system.</td>
 </tr>
 </tbody>
</table>

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.

Time-lapse imaging analysis of axon-induced GLT1 promoter activation in astrocytes
The compartmented neuron and astrocyte co-culture system allows only the neuronal processes, especially the axons, to selectively interact with astrocytes. Following the successful establishment of axon and astrocyte (or other glial cells) co-culture in the assembled MCP, different types of axon-glia interactions can be studied such as; axon-induced activation of astroglial gene promoter activation, astrocyte-induc...

Watch this Scientific Journal Video about Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System at JoVE.com

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.

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 ...

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14.9K Views.  Tufts University. 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.

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

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

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 ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly useful for disorders in which identifiable neuronal circuitry plays a key role, such as epilepsy and movement disorders. Currently available surgical modalities, while effective, generally involve an invasive surgical procedure, which can result in surgical injury to non-target tissues. Consequently, it would be of value to expand the range of surgical approaches to include a technique that is both non-invasive and neurotoxic.
Here, a method is presented for producing focal, neuronal lesions in the brain in a non-invasive manner. This approach utilizes low-intensity focused ultrasound together with intravenous microbubbles to transiently and focally open the Blood Brain Barrier (BBB). The period of transient BBB opening is then exploited to focally deliver a systemically administered neurotoxin to a targeted brain area. The neurotoxin quinolinic acid (QA) is normally BBB-impermeable, and is well-tolerated when administered intraperitoneally or intravenously. However, when QA gains direct access to brain tissue, it is toxic to the neurons. This method has been used in rats and mice to target specific brain regions. Immediately after MRgFUS, successful opening of the BBB is confirmed using contrast enhanced T1-weighted imaging. After the procedure, T2 imaging shows injury restricted to the targeted area of the brain and the loss of neurons in the targeted area can be confirmed post-mortem utilizing histological techniques. Notably, animals injected with saline rather than QA do demonstrate opening of the BBB, but dot not exhibit injury or neuronal loss. This method, termed Precise Intracerebral Non-invasive Guided surgery (PING) could provide a non-invasive approach for treating neurological disorders associated with disturbances in neural circuitry.

The goal of the protocol is to provide a method for producing non-invasive neuronal lesions in the brain. The method utilizes Magnetic Resonance-guided Focused Ultrasound (MRgFUS) to open the Blood Brain Barrier in a transient and focal manner, in order to deliver a circulating neurotoxin to the brain parenchyma.

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

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 ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral (dorsal root ganglia) and central tissues (cortex, subventricular zone, organoid) that are dynamically studied in terms of calcium shifts. The model chosen to illustrate the results is the retina, a simple tissue with complex cellular interactions. Calcium is a universal messenger involved in most of the important cellular roles. We explain in a step-by-step protocol how retinal neuron-glial cells in culture can be prepared and evaluated, envisioning calcium shifts. In this model, we differentiate neurons from glia based on their selective response to KCl and ATP. Calcium permeable receptors and channels are selectively expressed in different compartments. To analyze calcium responses, we use ratiometric fluorescent dies such as Fura-2. This probe quantifies free Ca2+ concentration based on Ca2+-free and Ca2+-bound forms, presenting two different peaks, founded on the fluorescence intensity perceived on two wavelengths.

Cell calcium imaging is a versatile methodology to study dynamic signaling of individual cells, on mixed populations in culture or even on awakened animals, based on the expression of calcium-permeable channels/receptors that gives unique functional signatures.

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral ...

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain ...