The present work was supported by the German research foundation (Deutsche Forschungsgemeinschaft DFG: GRK 736, Fa 159/22-1; the research school of the Ruhr University Bochum (GSC98/1) and the priority program SSP 1172 "Glia and Synapse", Fa 159/11-1,2,3).

The experiments with mice were in accordance with the German Law and the German Society for Neuroscience guidelines of animal husbandry. The animal care and utilization committees of the Ruhr-University Bochum have granted the appropriate permits.
1. Preparation and Cultivation of Cortical Astrocytes
Note: Complete these steps of the protocol at least 7 d before proceeding to the next steps, as the astrocyte cultures should develop into confluent monolayers before the neurons are prepared. Primary astrocytes are derived from mixed glial cultures obtained from mouse pups around postnatal day (P) 0-3. Three brains (6 cortices) per T75 flask are to be used.
<ol>
	<li>Prepare the astrocyte medium by supplementing Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% v/v horse serum and 0.1% v/v gentamycin under sterile conditions. Store the medium at 4 &#176;C for up to 2 weeks.</li>
	<li>Coat 3x T75 flasks with 10 &#181;g/mL&#160;Poly-D-Lysine (PDL; in cell-culture-grade water) for at least 1 h&#160;at 37 &#176;C in the presence of 6% v/v CO2. After coating, wash the flasks twice with Phosphate-Buffered Saline (PBS) to remove unbound cations, and add 9 mL&#160;astrocyte medium.</li>
	<li>For the preparation of the brains, add 10 mL&#160;Hank&#39;s Balanced Salt Solution (HBSS) to 1x 10 cm Petri dish and 1 mL&#160;HBSS to 1x 15 mL tube (regardless of the number of brains that will be used). Keep a pair of surgical scissors, 2 fine forceps and a binocular within reach for the procedure.</li>
	<li>Decapitate 3 pups quickly with the surgical scissors and collect the heads in an empty 10 cm dish.
	<ol>
		<li>Perform the preparation of the cortices under the binocular. Using two fine forceps, remove the dorsal skin from the skull at the midline, starting from the neck up towards the level of the eyes. Thereafter, the brain with its blood vessels is visible under the skull.</li>
		<li>Fix the head at the rostral end with a pair of forceps and incise the midline of the skull, starting at the posterior end. Subsequently, clip off the right and the left halves of the skull to expose the brain.</li>
		<li>Close the tip of the forceps and position it ventrally under the brain. Lift the brain out of the skull and transfer it into the HBSS-filled 10 cm Petri dish. Proceed in the same manner with the remaining specimen until all brains are collected in the dish.</li>
	</ol>
	</li>
	<li>After collecting the brains, start with the separation of the cortices by pinching off the hindbrain using a pair of forceps like a pair of scissors. Perform a midline incision between the two hemispheres with one pair of forceps. Carefully fix the brain with a forceps and cut off the midbrain and the olfactory bulbs using a second forceps to end up with the cortical halves.</li>
	<li>Fix one cortical half with one pair of forceps and peel the meninges from the hemispheres by detaching them from the lateral edge and subsequently pulling them from the cortical surface using a second forceps.</li>
	<li>Flip the cortical half with its surface oriented down towards the dish; carefully dissect and remove the crescent-shaped hippocampus using one pair of forceps. Transfer the single cortices into the conical 15 mL&#160;tube using one closed forceps to carry an individual cortex.</li>
	<li>After collecting all cortices, transit to the sterile laminar flow hood and begin with the preparations for the dissociation of the cortical tissue.</li>
	<li>Add 1 mL&#160;DMEM to a 2 mL&#160;reaction tube and add 0.1% w/v papain (30 units). Incubate the suspension in a water bath at 37 &#176;C until a clarified solution develops (about 3 min). Add 0.24 mg/mL&#160;L-cysteine and 20 &#181;g/mL&#160;DNAse and shake gently. 
	NOTE: For digestion, approximately 1 mL&#160;of enzyme solution is needed.</li>
	<li>Filter (0.22 &#181;m pore size) to obtain 1 mL&#160;sterile solution before adding it to the cortical tissue. Incubate the tubes for 30 min - 1 h&#160;at 37 &#176;C (i.e., in a water bath). 
	NOTE: Steps 1.11 - 1.14 are critical and should be completed within 15 min.</li>
	<li>To terminate the digestion reaction, add 1 mL&#160;astrocyte medium and carefully triturate the digested tissue using a 1 mL&#160;pipette. For trituration, gently move the pipette plunger, thereby aspirating and extruding the cortical tissue suspension.</li>
	<li>Once the tissue has been dissociated into a single-cell suspension, add 5 mL&#160;astrocyte medium and centrifuge for 5 min at 216 x g.</li>
	<li>Aspirate the supernatant carefully from the resulting pellet and resuspend the cells in 1 mL&#160;astrocyte medium.</li>
	<li>Add the cell suspensions to T75 flasks replenished with 9 mL astrocyte medium (see 1.2).</li>
	<li>Incubate the cells at 37 &#176;C in the incubator in the presence of 6% v/v CO2. After 4 d, perform the first complete medium change (10 mL). Thereafter, change the culture medium every 2 - 3 d.</li>
	<li>After 7 d, check if the cells have formed a confluent monolayer. If not, wait for another 2 - 3 days until complete confluence of the culture is attained. 
	NOTE: Astrocytes display large flattened cell bodies, localize at the bottom of the flask and do not develop notable cellular processes. They differ from the small phase-contrast bright progenitor cells and microglia residing on top&#160;of the monolayer17.</li>
	<li>In order to get rid of the progenitor cells and obtain a pure astrocyte culture, place the T75 flasks on an orbital shaker and shake the cultures O/N at 250 rpm. Ensure that the temperature is adjusted to 37 &#176;C and that the filter of the flask is sealed with laboratory film to prevent evaporation of the CO2.</li>
	<li>The next day, aspirate the medium and add 10 mL fresh culture medium replenished with 20 &#181;M cytosine-1-&#946;-D-arabinofuranoside (AraC) to eliminate residual dividing cells. 
	NOTE: incubation with AraC for 2 - 3 d in the incubator at 37 &#176;C,&#160;6% CO2&#160;will result in a pure astrocyte culture.</li>
	<li>Monitor the purity of the astrocyte cultures by visual inspection under the light microscope. If residual phase-contrast bright progenitor cells and microglia still reside on top of the astrocytic monolayer17 (see step 1.16), repeat steps 1.18 and 1.19. 
	NOTE: The confluent and pure astrocyte monolayers can be maintained for up to 14 d in the incubator (37 &#176;C, 6% v/v CO2) before proceeding to the next step. Change half of the medium every 3 d. Do not collect, freeze and thaw the astrocytes, as these cells will lose their neuronal supportive properties through the storage procedure.</li>
</ol>
2. Preparing Astrocytes for the Indirect Coculture
Note: 48 - 72 h&#160;before preparing the neurons, transfer astrocytes to the cell-culture inserts. Generally, a single&#160;T75 flask delivers a sufficient number of cells to support the neuronal cultures obtained with one preparation.
<ol>
	<li>Place as many inserts as needed into 24-well-plates. Coat each insert with 10 &#181;g/mL PDL (dissolved in cell-culture-grade water) for about 1 h at 37 &#176;C, 6% v/v CO2. After the incubation, wash the inserts twice with PBS. In the meantime, proceed with the preparation of astrocytes. 
	NOTE: The inserts filled with PBS can be kept until the astrocyte cell suspension is ready for use.</li>
	<li>Aspirate the astrocyte medium (with AraC) and wash the culture one time with 10 mL PBS to ensure any residual serum is removed.</li>
	<li>Add 3 mL of 0.05% trypsin-EDTA (Ethylenediaminetetraacetic acid) to the flasks and incubate the cells for trypsinization at 37 &#176;C, 6% v/v CO2 for approximately 10 min.</li>
	<li>Control the cell detachment by visual inspection using a microscope and facilitate the detachment of the cells by tapping the side of the flasks against the desktop. 
	NOTE: Steps 2.5 - 2.8 are critical and should be finished within 15 - 20 min.</li>
	<li>Following the trypsinization, gently re-suspend the cells in 7 mL astrocyte medium and transfer the cell suspension to a 15 mL tube. Centrifuge for 5 min at 216 x g.</li>
	<li>Carefully aspirate the supernatant and resuspend the cell pellet in 1 mL astrocyte medium.</li>
	<li>Count the cells using a counting chamber.</li>
	<li>Fill the bottom of the wells of the 24-well-plate with 500 &#181;L astrocyte medium and transfer 25,000 cells in 500 &#181;L astrocyte medium into each individual insert. Incubate the culture at 37 &#176;C, 6% v/v CO2. 
	NOTE: After 42 - 72 h&#160;the cultures will reach approximately 100% confluence. At this stage the purity of the culture can be verified by immunocytochemistry11. The confluent cultures can be maintained for up to 7 d until proceeding to the next step. Change half of the medium every 3 d.</li>
</ol>
3. Preparation of Primary Hippocampal Neurons
Note: Primary mouse hippocampal neurons should be derived from E15.5 - E16 embryos of timed pregnant mice.
<ol>
	<li>Prepare neuron medium (MEM with 10 mM sodium pyruvate, 0.1% w/v ovalbumin,&#160;2% v/v B27 medium supplement, and 0.1% v/v gentamicin (sterile filtered)), and preparation medium (HBSS with 0.6% w/v glucose and 10mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid)). Pre-warm and pre-equilibrate the neuron medium in filter flasks at 37 &#176;C, 6% v/v CO2.</li>
	<li>Place the glass-coverslips (12 mm diameter) into the wells of 24-well-plates (use the special coverslips, which are notched for the use with cell culture inserts).
	<ol>
		<li>Coat for at least 1 h with 15 &#181;g/mL&#160;Poly-DL-Ornithine in water (cell-culture-grade) at 37 &#176;C. Use at least 500 &#181;L per well to ensure that the coverslips are completely covered. Wash the wells twice with PBS. Leave the PBS in the wells until plating the cells. Ensure that the wells do not dry out.</li>
	</ol>
	</li>
	<li>To dissect the hippocampi, prepare 3x 10 cm dishes filled with preparation medium and 1x 2 mL tube with 1 mL preparation medium. Prepare scissors and 2 fine forceps.</li>
	<li>Sacrifice the pregnant mouse by cervical dislocation, sterilize the abdomen by washing with 70% v/v ethanol, and open the abdomen using sharp scissors. Excise the beaded uterus carefully with scissors and transfer the organ into 1x 10 cm dish filled with preparation medium.</li>
	<li>Next, remove the embryos from the uterus. Carefully incise the uterus and remove the amnion without harming the embryos by using 2 forceps. Thereafter, decapitate each embryo with a pair of scissors and transfer the heads into a second 10 cm dish supplied with preparation medium.</li>
	<li>Proceed with the preparation of the heads (similar to the preparation described in 1.4 - 1.7).
	<ol>
		<li>Immobilize the head with a pair of forceps and incise the skull and skin in the neck region close to the hindbrain, perpendicular to the neuraxis. Use a second pair of forceps to lift both skull and&#160;covering skin, and bend the soft skull cap towards the rostral end of the head, thereby exposing the brain.</li>
		<li>With the help of 2 forceps, detach the brain from the cranial cavity. To do this, close 1 pair of forceps and separate the brain from the skull starting at the olfactory bulbs and moving&#160;towards the hindbrain. Collect the brains in another 10 cm dish filled with preparation medium.</li>
	</ol>
	</li>
	<li>Dissect the hippocampi from the cortex moieties as described above for the astrocyte preparation (step 1.6). Remove the hippocampi from each hemisphere and collect them in the 2 mL tube filled with preparation medium.</li>
	<li>After completing the dissection, transfer the tube to the sterile laminar flow bench and digest the hippocampal tissue with 1 mL digestion solution containing papain. Prepare the digestion solution as described in step 1.9 - 1.10, but use MEM instead DMEM. 
	NOTE: Steps 3.9 - 3.12 are critical and should be completed within 10 - 15 min.</li>
	<li>After completion of digestion, carefully withdraw the digestion solution by gentle suction with a pipette.</li>
	<li>Wash the hippocampi 3 times with neuron medium by sequentially adding and thereafter carefully aspirating 1 mL fresh culture medium per wash cycle. Do not centrifuge the cell suspension.</li>
	<li>After the final washing step, carefully triturate the tissue in 1 mL neuron medium.</li>
	<li>Count the cells using a counting chamber and plate 35,000 cells in 500 &#181;L neuron medium per a single well of a 24-well-plate (the plate mentioned in 3.2). 
	NOTE: Optionally, a cell aliquot can be counterstained with trypan blue to assess the vitality of the cell preparation. Control the plating density by visual inspection using the microscope (typically 90 - 110 cells per visual field of a standard 10X objective).</li>
	<li>Place neurons in the incubator at 37 &#176;C, 6% v/v CO2 for 1 h.</li>
	<li>Post incubation, take the prepared inserts (seeded with confluent astrocyte monolayers) out of the incubator (see step 2.8). Exchange the medium by aspirating the astrocyte medium and replacing it with 500 &#181;l fresh neuron medium. 
	NOTE: The inserts should harbor confluent astrocyte monolayers after 2 - 3 DIV (Days In Vitro).</li>
	<li>Place the inserts with astrocytes carefully into the wells that contain the neuron cultures (step 3.13) using sterile forceps.</li>
	<li>Place the resulting indirect neuron-astrocyte coculture back into the incubator at 37 &#176;C, 6% v/v CO2. 
	NOTE: Under these conditions, neurons can be cultivated for up to 4 weeks in completely defined medium. No medium change is necessary. For long-time cultures, it is recommended to fill empty wells with sterile water in order to reduce the evaporation from the 24-well-plate.</li>
	<li>Perform in vitro cell analyses after desired culture periods (e.g., for&#160;immunocytochemistry, PCR, Western blot, electrophysiological recordings (patch clamp or multielectrode array))7,15,18. 
	NOTE: The culture system is also suitable for acute and chronic pharmacological treatment of the cultures11.</li>
</ol>

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The authors have nothing to disclose.

The main goal of the current protocol is to completely separate neuronal and astrocytic cultures, while maintaining them in shared medium. For this reason, the purity of the cultures obtained should be verified at the beginning of the procedure. We recommend the use of neuron-specific tubulin, neurofilaments or NeuN protein as neuronal markers, GFAP as astrocytic marker, O4 antigen as oligodendrocyte precursor marker and Iba1 protein to identify microglia.
Pay special attention when performing a critical step of the protocol, as specified by a &#39;NOTE&#39; before the step description. Take into consideration that both primary cultures of neurons and astrocytes are rather sensitive. Therefore, several problems may arise at the beginning of the procedure. If the astrocytes do not reach a confluent monolayer in T75 flasks after 10 DIV (protocol section 1), check the components of the astrocyte medium (for expiration date). In addition, increase the amount of cortical tissue prepared to initiate the culture (up to 10 cortices per flask). If the astrocytes do not reach a confluent monolayer on cell culture inserts after 72 h&#160;(protocol section 2), check the components of the astrocyte medium and PDL (for expiration date).Try to plate out freshly prepared cultures, determine the proportion of viable cells prior to plating and adjust cell numbers accordingly (e.g., use trypan blue counterstaining). If low survival of neurons is detected after 24 h, hippocampus tissue was not triturated gently enough (protocol step 3.11).Pay attention to avoid the formation of bubbles when dissociating the tissue. If survival of neurons is significantly lowered after 7 - 14 DIV and if neurons form aggregates of 3 - 5 cells after 7 DIV, use freshly prepared inserts with astrocytes, check for astrocyte purity by immunocytochemistry, and check the neuron medium components. If big aggregates of neurons (more than 10 cells) are visible, it is possible that the hippocampus tissue was not sufficiently triturated, resulting in incomplete dissociation (protocol step 3.11). Pay attention to suspension homogeneity. If neuronal culture is contaminated with non-neuronal cells (microglia, astrocytes, oligodendrocyte precursor cells, etc.), completely remove the neighboring cortical tissue adjacent to the hippocampus (step 3.7) and also check the neuron medium components.
The proposed indirect coculture of primary neurons and astrocytes provides a versatile tool for the long-term investigation of neuron-glia interactions. Importantly, this protocol is optimized for mouse cells, which opens perspective for the implementation of mouse genetic models. Because of the complete physical separation of the 2 cell types, neurons and astrocytes of distinct genotypes can be combined in the desired way.
Among a variety of applications, this approach can be used to investigate neural networks development, synaptogenesis, network activity and astrocyte-neuron signaling. Although the indirect cocultivation of neurons and astrocytes opens vast opportunities for their segregated analysis and long-range signaling, the lack of direct contact may compromise&#160;the subtle regulation of synaptic plasticity22. Thus, the lack of interaction between astrocytic protrusions and neuronal synapses has to be treated carefully when using this model.
The assay is based on two previous protocols for astrocyte and neuronal preparations14,17 and has been improved in the course of the past years. The approach originally has been established for mouse7 and rat11 cells. Previous publications of the laboratory provide a detailed characterization of the assays concerning cell survival, synapse formation and electrophysiological characterization of the resulting cells7,11,15,18. Furthermore, the assay can be adapted to other settings, such as the micro-electrode-arrays (MEAs)7. For future applications, the reader should consider other protocols for the detailed investigation of synapse formation23, as well as for MEA analysis in vitro. In conclusion, the assay provides a versatile tool for laboratories focusing on various aspects of neuron-glia interactions.

Throughout the past decades, the general interpretation of neuroglia function has evolved&#160;from the attribution of a merely supportive towards an active regulatory role concerning neuronal function1. Because of their prominent impact on brain homeostasis in health and disease2, astrocytes are of special interest for the scientific community. In the past few years, a diversity of studies have focused on neuron-glia interactions in vivo and in vitro3. However, most of the culture systems do not allow for the separate analysis of both cell types and of their respective secretomes.
Several approaches exploit the direct cocultivation of neurons and glia to achieve long lasting survival and physiologically relevant neuronal network development4-6. The present protocol reaches the same goals while keeping both cell types physically separated7. Compared to conditioned medium approaches8,9, our system allows to study the bidirectional communication between neurons and astrocytes. The expression of secreted signaling molecules can be monitored while the cells maturate in the shared medium. This opportunity is especially relevant, as astrocytes release soluble factors, such as cytokines, growth factors and extracellular matrix molecules10,11, thereby regulating neuronal growth and function7,12. Thus, it has been demonstrated that the addition of thrombospondin to retinal ganglion cells in vitro induces the formation of synapses13. However, other yet unknown factors are necessary to render synapses functional13. Furthermore, molecules released by astrocytes have to be identified in order to understand the basis of neuron-glia interactions.
The cultivation of primary neurons and astrocytes from mouse and rat has been described previously14-16. Here we present an elegant and versatile tool to combine both cell types in an indirect coculture approach. Since the two cultures are physically separated&#160;yet sharing the same medium, the impact of neurons, astrocytes and soluble molecules, can be separately analyzed, thus creating a powerful tool for neuron-glia interaction studies.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>B27</td><td>Gibco (Life Technologies)</td><td>17504-044</td><td></td></tr><tr><td>Cell-culture-grade water</td><td>MilliQ</td><td></td><td></td></tr><tr><td>Cytosine-&amp;#946;-D-arabinofuranoside (AraC)</td><td>Sigma-Aldrich</td><td>C1768</td><td>CAUTION: H317, H361</td></tr><tr><td>DMEM</td><td>Gibco (Life Technologies)&amp;nbsp;</td><td>41966-029</td><td></td></tr><tr><td>DNAse</td><td>Worthington</td><td>LS002007</td><td></td></tr><tr><td>Gentamycin</td><td>Sigma-Aldrich</td><td>G1397</td><td>CAUTION: H317-334</td></tr><tr><td>Glucose</td><td>Serva</td><td>22700</td><td></td></tr><tr><td>HBSS</td><td>Gibco (Life Technologies)</td><td>14170-088</td><td></td></tr><tr><td>HEPES</td><td>Gibco (Life Technologies)</td><td>15630-056</td><td></td></tr><tr><td>Horse serum</td><td>Biochrom AG</td><td>S9135</td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma-Aldrich</td><td>C-2529</td><td></td></tr><tr><td>MEM</td><td>Gibco (Life Technologies)</td><td>31095-029</td><td></td></tr><tr><td>Ovalbumin</td><td>Sigma-Aldrich</td><td>A7641</td><td>CAUTION: H334</td></tr><tr><td>Papain</td><td>Worthington</td><td>3126</td><td></td></tr><tr><td>PBS</td><td>self-made</td><td></td><td></td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>P0899</td><td></td></tr><tr><td>Poly-L-ornithine</td><td>Sigma-Aldrich</td><td>P3655</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>S8636</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco (Life Technologies)</td><td>25300054</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>24-well-plates</td><td>Thermoscientific/Nunc</td><td>142475</td><td></td></tr><tr><td>24-wells-plate (for the&amp;nbsp; indirect co-culture)</td><td>BD Falcon</td><td>353504</td><td></td></tr><tr><td>Binocular</td><td>Leica</td><td>MZ6</td><td></td></tr><tr><td>Cell-culture inserts</td><td>BD Falcon</td><td>353095</td><td></td></tr><tr><td>Centrifuge</td><td>Heraeus</td><td>Multifuge 3S-R</td><td></td></tr><tr><td>Counting Chamber</td><td>Marienfeld</td><td>650010</td><td></td></tr><tr><td>Forceps</td><td>FST Dumont (#5)</td><td>11254-20</td><td></td></tr><tr><td>glass cover slips (12&amp;nbsp;mm)</td><td>Carl Roth (Menzel- Gl&amp;auml;ser)</td><td>P231.1</td><td></td></tr><tr><td>Incubator</td><td>Thermo Scientific</td><td>Heracell 240i</td><td></td></tr><tr><td>Micro-tube (2 mL)</td><td>Sarstedt</td><td>72,691</td><td></td></tr><tr><td>Microscope</td><td>Leica</td><td>DMIL</td><td></td></tr><tr><td>Millex Syringe-driven filter unit</td><td>Millipore</td><td>SLGV013SL</td><td></td></tr><tr><td>Orbital shaker</td><td>New Brunswick Scientific</td><td>Innova 4000</td><td></td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM-996</td><td></td></tr><tr><td>Petri dishes (10 cm)</td><td>Sarstedt</td><td>833,902</td><td></td></tr><tr><td>pipette (1 mL)</td><td>Gilson</td><td>Pipetman 1000</td><td></td></tr><tr><td>Sterile work bench</td><td>The Baker Company</td><td>Laminar Flow SterilGARD III</td><td></td></tr><tr><td>Surgical scissors</td><td>FST Dumont</td><td>14094-11</td><td></td></tr><tr><td>Syringe</td><td>Henry Schein</td><td>9003016</td><td></td></tr><tr><td>T75 flask</td><td>Sarstedt</td><td>833,911,002</td><td></td></tr><tr><td>tube (15 mL)</td><td>Sarstedt</td><td>64,554,502</td><td></td></tr><tr><td>Water bath</td><td>GFL</td><td>Water bath type 1004</td><td></td></tr></tbody></table>

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 analysis of the neuronal cultures via the indirect coculture system is multifarious and can be performed at different stages of culture maturation. Due to the fact that the cells can be maintained for up to 4 weeks, long-term investigations of the cultures are possible.
The schematic in the middle left panel of Figure 1 demonstrates the cocultivation setup. With the use of this system, live cell imaging of both cell types can be performed, as exemplified by phase contrast pictures of the astrocyte monolayer (top left panel) and the neuronal network (bottom left panel). After fixation, immunocytochemical assessment is possible, as shown in top right and bottom right panels.
Neurons begin to establish synaptic connections starting at approximately 7 DIV in our model (data not shown). By 14 DIV multiple synapses are formed within neuronal networks, as identified by the combined immunocytochemical detection of pre&#160;and postsynaptic markers (Figure 1, bottom right panel). Importantly, not only the amount of synaptic marker puncta can be visualized and quantified using this approach, but also the structurally completed synapses, which are most likely to contribute to network connectivity.
The astrocyte cultures not only provide trophic support to neurons11, but also synthesize several secreted factors19,20 involved in neural plasticity. The expression of one of these factors, namely matrix metalloproteinase 2 (MMP2), is demonstrated in the top right panel of Figure 1. Interestingly, 2 distinct MMP2-isoforms were detected in the coculture medium using Western Blot (Figure 1, middle right panel). Potentially astrocytes secrete MMP2 into the shared medium, thus affecting the plasticity of neurons. Also multiple isoforms of Tenascin-C, a known regulator of axon outgrowth, cell migration and differentiation21 were identified.
Taken together, these results demonstrate two examples and thereby provide proof of principle for the variety of investigations of astrocytes, neurons and their reciprocal communication that can be realized using the indirect cocultivation method described herein.
<img alt="Figure 1" src="/files/ftp_upload/54757/54757fig1.jpg" /> 
Figure 1: The Indirect Astrocyte-Neuron Coculture System Allows for Separate Analysis of Astrocytes, Neurons and Secreted Molecular Mediators. Top panel, left: the astrocytes form monolayers on the cell culture insert membrane, phase contrast; scale bar:&#160;250 microns. Top panel, right: astrocytes are immunostained for Glial Acidic Fibrillar Protein (GFAP) (mouse clone GA5) and Matrix Metalloprotease 2 (MMP2) (rabbit polyclonal); scale bar: 250 microns. Middle panel, left: the scheme illustrates the indirect coculture setup. Although two cultures are physically separated, they share the same medium. Middle panel, right: two secreted molecular mediators of neuron-glia interactions are revealed in the co-cultivation medium using Western Blot. Multiple isoforms of Tenascin C (TNC), a neurite outgrowth regulator, and MMP2, an extracellular matrix modifier, are documented. Bottom panel, left: primary neurons develop highly interconnected networks by the 14th day of cultivation, phase contrast; scale bar: 250 microns. Bottom panel, right: starting with 14 d&#160;in vitro, multiple synaptic connections are established between neurons, as detected by immunocytochemical labeling; scale bar: 50 microns. The co-localization of the presynaptic marker Bassoon (rabbit polyclonal) with the postsynaptic PSD95 scaffolding protein (mouse clone 6G6-1C9) documents the structurally completed synapses formation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54757/54757fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions at JoVE.com

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.

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 ...

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Nanyang Technological University

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Neuroscience

14.6K Views.  Ruhr-University.  Dit protocol beschrijft de indirecte neuron-astrocyten co-cultuur voor verzuilde analyse van neuron-glia interacties. 

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

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 ...

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 ...

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.

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 ...

Biology

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 ...

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

Developmental Biology

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 ...

 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.remotexs.ntu.edu.sg/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.

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

Co-Culturing Glioblastoma Cells on Patterned Neurons: A Technique to Monitor the Migration of Glioblastoma Cells on Rat Hippocampal Neurons In Vitro

Source: Guyon, J. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/62213" target="_blank">Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions.</a> J. Vis. Exp. (2021)
This video describes a co-culture assay of glioblastoma cells on micropatterned neurons, which mimic the myelinated neuronal tracts invasion by the glioblastoma stem-like cells as they migrate on neurons in the peritumoral spaces. This assay can help analyze the effects of pharmacological drugs that inhibit glioblastoma cells&#39; migration on neurons in a glioma.

Source: Guyon, J. et al. Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions. J. Vis. Exp. 
...

Cancer Research

Cancers of the Nervous System

In vitro 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.remotexs.ntu.edu.sg/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.

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

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

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

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