Name: The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions (Video) | JoVE Journal
Uploaded: 2016-11-14T14:00:00.000+00:00
Duration: 11 min 8 s
Description: Learn the indirect neuron-astrocyte coculture assay for studying neuron-glia interactions using primary cells in vitro. This method enables longitudinal monitoring of synapse formation and analysis of cellular exchanges.

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5 minutes

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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<li>Christopherson, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thrombospondins+are+astrocyte-secreted+proteins+that+promote+CNS+synaptogenesis%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis.</a> Cell. 120, 421-433 (2005).</li>
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</ol>

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...

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

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 b...

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 ...

The overall goal of this Indirect Coculture Assay, is to investigate the impact of astrocytes on the development of neurons. Our laboratory is interested in the role of neuron-glia interactions. It is believed that astrocytes contribute to the formation of the synapses, the connecting structures of the central nervous system.In order to study their role, we have designed an in vitro model where we can combine primary astrocytes and primary embryonic neurons, in separate compartments. The compartments are connected by a permeable membrane, which allows for the analysis of exchanges between both cell types. Using this approach, we have been able to monitor synapse formation over periods of up to four weeks.In addition, it is possible to investigate the individual roles of astrocytes on the one hand, and neurons on the other hand, using this assay. And finally, we can also investigate in more detail the secretome of our cell compartments which contains molecules that mediate the reciprocal influence of both cell compartments in that system. This method can provide insights into the interactions between neurons and astrocytes.Also, it can be applied to other model organisms, such as the cells from rat. Generally individuals new to this method would struggle because experience is required to obtain the optimal maturation operation of hippocampal tissue after dissection. To dissect the cortices, remove the skin from the skull along the midline starting from the neck up towards the level of the eyes.Next, hold the head by the rostral end with a pair of forceps and make an incision along the midline of the skull. Then, clip off the left and the right halves of the skull to expose the brain. After that, lift the brain up from the skull and transfer it to a 10 cm petri dish filled with HBSS.Proceed in the same manner with remaining specimens until all the brains are collected in the dish. To separate the cortices, pinch off the hindrind using a pair of forceps. Make a midline incision between the two hemispheres.Carefully fix the brain and cut off the midbrain and the olfactory bulb using a second pair of forceps, which results in two cortical halves. Following this, fix one cortical half with one pair of forceps and peel the meninges off by detaching it from the lateral edge. And subsequently pulling it from the cortical surface using a second pair of forceps.Orient the cortical half with its surface facing down. Carefully dissect and remove the crescent shaped hippocampus. Then transfer the cortices into a 15 ml conical tube.Add one ml of denem and 0.1%wv papain to a 2ml reaction tube. Incubate the suspension in a water bath at 37 degrees Celsius until the solution is clear. Add L-cysteine and DNAse to the mixture and shake gently.Afterward filter the mixture to obtain one ml of sterile solution, then add it to the cortical tissue and incubate for 30 minutes to one hour at 37 degrees Celsius. To terminate the digestion reaction, add one ml of astrocyte medium and carefully titrate the digested tissue. Afterwards, add five ml of astrocyte medium to the cell suspension.Once the tissue has been dissociated into a single cell suspension, centrifuge it for five minutes. After that, aspirate the supernatant carefully from the resulting pellet and re-suspend the cells in one ml of astrocyte medium. Add the cell suspension to the T75 flasks replenished with nine ml of astrocyte medium.After seven days of culture, check if the cells have formed a confluent monolayer. Ensure that the temperature is adjusted to 37 degress Celsius and the filter of the flask is sealed with laboratory film to prevent evaporation of the carbon dioxide. 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 overnight at 250 rotations per minute.The next day aspirate the medium and add 10 ml of fresh culture medium replenished with 20 micromolar RSE to eliminate residual dividing cells. In this procedure place as many inserts as needed in the 24 well plate. Coat each insert with 10 micrograms per ml of poly d'lysine and incubate for one hour.After one hour wash the inserts twice with PBS. In the meantime, aspirate the astrocyte medium and wash the culture once with 10 ml of PBS to ensure that residual serum is removed. Next, add three ml of 0.05%trypsin edta to the flasks and incubate the cells for trypsinization for approximately 10 minutes.Afterward, gently re-suspend the cells in seven ml of astrocyte medium. Transfer the cell suspension to a 15 ml tube and centrifuge for five minutes at 216 x g. Then carefully aspirate the supernatant and re-suspend the cell pellet in one ml of astrocyte medium.Count the cells using a counting chamber. Subsequently, fill the 24 well plate with 500 microliters of astrocyte medium per well. Aspirate the PBS and transfer 25, 000 cells and 500 microliters of astrocyte medium into each individual insert and incubate the culture at 37 degrees Celsius.To dissect the hippocampi, prepare three 10 cm dishes filled with preparation medium and one 2 ml tube with one ml of preparation medium. Dissect the hippocampi from the cortex noyadees and collect them in a two ml tube filled with preparation medium. It is critical for the hippocampi to be isolated without an adjacent tissue from other regions of the CNS.Therefore, after removing the hippocampus the foreign contaminating tissues have to be additionally removed. After dissection, transfer the tube to a sterile laminar flow bench. Remove the preparation medium carefully and digest the hippocampal tissue of one ml of digestion solution containing papain.After 15 minutes of digestion, carefully withdraw the digestion solution by gentle suction with the pipette. Due to the neuron's high sensitivity, it is critical to titrate the hippocampal carefully in order to avoid bubbles and to obtain the optimal titration intensity. Wash the hippocampi three times with neuron medium by adding and carefully aspirating one ml of fresh culture medium per wash cycle.After the final washing step, carefully titrate the tissue in one ml of neuron medium. Then count the cells using a counting chamber. Plate 35, 000 cells in 500 microliters of neuron medium per well of the 24 well plate and incubate the neurons at 37 degrees Celsius for one hour.Now take the prepared inserts, which have been seeded with confluent astrocyte monolayers out of the incubator. Exchange the medium by aspirating the astrocyte medium and replacing it with 500 microliters of fresh neuron medium. Then place the inserts with astrocytes carefully into the wells that contain the neuron cultures using sterile forceps.Subsequently, place the resulting indirect neuron astrocyte coculture back into the incubator until the experiments. This image shows the monolayers formed by astrocytes on the cell culture insert membrane. The astrocytes are immunostained for GFAP and MMP2.The scheme here illustrates the indirect coculture setup. Although two cultures are physically separated, they share the same medium. Two secreted molecular mediators of neuron glia interactions are revealed in the co cultivation medium using western blot with multiple isoforms of TNC, an outright growth regulator and MMP2, an extra cellular matrix modifier.This image shows that the primary neurons develop highly interconnected networks by day 14 of cultivation. The co localization of the presynaptic marker bassoon with the postsynaptic PSD95 scaffolding documents the structurally completed synaptic formation. In conclusion, using our assay system we have shown that it possible to combine primary astrocytes and primary neurons in the coculture model.Both surtypes are separated but share the same medium. Hence, we can also investigate the secretome of both surtypes in our model. In this model synapses develop and maturate.And furthermore, we can also show the emergence of additional specific structures such as perineuronal nets. Thus, our model system is well suited to investigate the influence of astrocytes on synapse formation, plasticity and function.

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

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