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...

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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.

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			<td height="41" style="height:41px;width:115px;">Reagents</td>
			<td style="width:152px;"></td>
			<td style="width:151px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">B27</td>
			<td style="width:152px;">Gibco (Life Technologies)</td>
			<td style="width:151px;">17504-044</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:115px;">Cell culture grade water</td>
			<td style="width:152px;">MilliQ</td>
			<td style="width:151px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:115px;">Cell culture grade water</td>
			<td style="width:152px;">MilliQ</td>
			<td style="width:151px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:115px;">Cytosine-&#223;-D arabinofuranoside (AraC)</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">C1768</td>
			<td style="width:191px;">CAUTION: H317, H361</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">DMEM</td>
			<td style="width:152px;">Gibco (Life Technologies)&#160;</td>
			<td style="width:151px;">41966-029</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">DNAse</td>
			<td style="width:152px;">Worthington</td>
			<td style="width:151px;">LS002007</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Gentamycin</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">G1397</td>
			<td style="width:191px;">CAUTION: H317-334</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Glucose</td>
			<td style="width:152px;">Serva</td>
			<td style="width:151px;">22700</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">HBSS</td>
			<td style="width:152px;">Gibco (Life Technologies)</td>
			<td style="width:151px;">14170-088</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">HEPES</td>
			<td style="width:152px;">Gibco (Life Technologies)</td>
			<td style="width:151px;">15630-056</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Horse serum</td>
			<td style="width:152px;">Biochrom AG</td>
			<td style="width:151px;">S9135</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">L-Cysteine</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">C-2529</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">MEM</td>
			<td style="width:152px;">Gibco (Life Technologies)</td>
			<td style="width:151px;">31095-029</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Ovalbumin</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">A7641</td>
			<td style="width:191px;">CAUTION: H334</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Papain</td>
			<td style="width:152px;">Worthington</td>
			<td style="width:151px;">3126</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">PBS</td>
			<td style="width:152px;">self-made&#160;</td>
			<td style="width:151px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Poly-D-lysine</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">P0899</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Poly-L-ornithine</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">P3655</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Sodium pyruvate</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">S8636</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">Trypsin-EDTA</td>
			<td style="width:152px;">Gibco (Life Technologies)</td>
			<td style="width:151px;">25300054</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Equipment</td>
			<td style="width:152px;"></td>
			<td style="width:151px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">24 well plates</td>
			<td style="width:152px;">Thermoscientific/Nunc</td>
			<td style="width:151px;">142475</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">24-wells plate (for the&#160; indirect co-culture)</td>
			<td style="width:152px;">BD Falcon</td>
			<td style="width:151px;">353504</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Binocular</td>
			<td style="width:152px;">Leica</td>
			<td style="width:151px;">MZ6</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Cell-culture inserts</td>
			<td style="width:152px;">BD Falcon</td>
			<td style="width:151px;">353095</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Centrifuge</td>
			<td style="width:152px;">Heraeus</td>
			<td style="width:151px;">Multifuge 3S-R</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Counting Chamber</td>
			<td style="width:152px;">Marienfeld</td>
			<td style="width:151px;">650010</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Forceps</td>
			<td style="width:152px;">FST Dumont (#5)</td>
			<td style="width:151px;">11254-20</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">glass cover slips (12 mm)</td>
			<td style="width:152px;">Carl Roth (Menzel- Gl&#228;ser)</td>
			<td style="width:151px;">P231.1</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Incubator</td>
			<td style="width:152px;">Thermo Scientific</td>
			<td style="width:151px;">Heracell 240i</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Micro tube (2 ml)</td>
			<td style="width:152px;">Sarstedt</td>
			<td style="width:151px;">72,691</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Microscope</td>
			<td style="width:152px;">Leica</td>
			<td style="width:151px;">DMIL</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Millex Syringe-driven filter unit</td>
			<td style="width:152px;">Millipore</td>
			<td style="width:151px;">SLGV013SL</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Orbital shaker</td>
			<td style="width:152px;">New Brunswick Scientific</td>
			<td style="width:151px;">Innova 4000</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Parafilm</td>
			<td style="width:152px;">Bemis</td>
			<td style="width:151px;">PM-996</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Petri dishes (10 cm)</td>
			<td style="width:152px;">Sarstedt</td>
			<td style="width:151px;">833,902</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">pipette (1 ml)</td>
			<td style="width:152px;">Gilson</td>
			<td style="width:151px;">Pipetman 1000</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Sterile work bench</td>
			<td style="width:152px;">The Baker Company</td>
			<td style="width:151px;">Laminar Flow SterilGARD III</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Surgical scissors</td>
			<td style="width:152px;">FST Dumont</td>
			<td style="width:151px;">14094-11</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Syringe</td>
			<td style="width:152px;">Henry Schein</td>
			<td style="width:151px;">9003016</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">T75 flask</td>
			<td style="width:152px;">Sarstedt</td>
			<td style="width:151px;">833,911,002</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">tube (15 ml)</td>
			<td style="width:152px;">Sarstedt</td>
			<td style="width:151px;">64,554,502</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Water bath</td>
			<td style="width:152px;">GFL</td>
			<td style="width:151px;">Water bath type 1004</td>
			<td style="width:191px;"></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 b...

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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 ...

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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14.4K Views.  Ruhr-University. 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. T...