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...

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...

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 ...

the-indirect-neuron-astrocyte-coculture-assay-an-vitro-set-up-for

Research

JoVE Journal

Neuroscience

14.6K Views.  Ruhr-University. This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

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

Brain Imaging Investigation of the Neural Correlates of Observing Virtual Social Interactions

The ability to gauge social interactions is crucial in the assessment of others&#x2019; intentions. Factors such as facial expressions and body language affect our decisions in personal and professional life alike 1. These "friend or foe" judgements are often based on first impressions, which in turn may affect our decisions to "approach or avoid". Previous studies investigating the neural correlates of social cognition tended to use static facial stimuli 2. Here, we illustrate an experimental design in which whole-body animated characters were used in conjunction with functional magnetic resonance imaging (fMRI) recordings. Fifteen participants were presented with short movie-clips of guest-host interactions in a business setting, while fMRI data were recorded; at the end of each movie, participants also provided ratings of the host behaviour. This design mimics more closely real-life situations, and hence may contribute to better understanding of the neural mechanisms of social interactions in healthy behaviour, and to gaining insight into possible causes of deficits in social behaviour in such clinical conditions as social anxiety and autism 3.

This article demonstrates an experimental design in which whole-body animated characters are used in conjunction with functional magnetic resonance imaging (fMRI) to investigate the neural correlates of observing virtual social interactions.

The ability to gauge social interactions is crucial in the assessment of others&#x2019; intentions. Factors such as facial expressions and body language ...

Brain Imaging Investigation of the Memory-Enhancing Effect of Emotion

Emotional events tend to be better remembered than non-emotional events1,2. One goal of cognitive and affective neuroscientists is 
to understand the neural mechanisms underlying this enhancing effect of emotion on memory. A method that has proven particularly influential in the 
investigation of the memory-enhancing effect of emotion is the so-called subsequent memory paradigm (SMP). This method was originally used to investigate the 
neural correlates of non-emotional memories3, and more recently we and others also applied it successfully to studies of emotional memory (reviewed in4, 5-7). 
Here, we describe a protocol that allows investigation of the neural correlates of the memory-enhancing effect of emotion using the SMP in conjunction with 
event-related functional magnetic resonance imaging (fMRI). An important feature of the SMP is that it allows separation of brain activity specifically 
associated with memory from more general activity associated with perception. Moreover, in the context of investigating the impact of emotional stimuli, 
SMP allows identification of brain regions whose activity is susceptible to emotional modulation of both general/perceptual and memory-specific processing. 
This protocol can be used in healthy subjects8-15, as well as in clinical patients where there are alterations in the neural correlates of emotion perception 
and biases in remembering emotional events, such as those suffering from depression and post-traumatic stress disorder (PTSD)16, 17.

We present a protocol that uses functional magnetic resonance imaging to investigate the neural correlates of the memory-enhancing effect of emotion. This protocol allows identification of brain activity specifically linked to memory-related processing, contrary to more general perceptual processing, and can be used with healthy and clinical populations.

Emotional events tend to be better remembered than non-emotional events1,2. One goal of cognitive and affective neuroscientists is 
to understand the ...

Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional distractibility) effects (reviewed in 1). Complementing our recent protocol 2 describing a method that allows investigation of the neural correlates of the memory-enhancing effect of emotion (see also 1, 3-5), here we present a protocol that allows investigation of the neural correlates of the detrimental impact of emotion on cognition. The main feature of this method is that it allows identification of reciprocal modulations between activity in a ventral neural system, involved in 'hot' emotion processing (HotEmo system), and a dorsal system, involved in higher-level 'cold' cognitive/executive processing (ColdEx system), which are linked to cognitive performance and to individual variations in behavior (reviewed in 1). Since its initial introduction 6, this design has proven particularly versatile and influential in the elucidation of various aspects concerning the neural correlates of the detrimental impact of emotional distraction on cognition, with a focus on working memory (WM), and of coping with such distraction 7,11, in both healthy 8-11 and clinical participants 12-14.

We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2. CSF is a protein rich fluid that is essential for normal brain development and function3-6.
In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8, while claudin 5a is necessary for tight junction formation 9. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.
To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2).
This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell leukodystrophy (GLD) is unclear. This gap in knowledge has been hindered by the lack of an appropriate in vitro model for study. Herein, we describe a primary murine glial culture system in which treatment with psychosine results in multinucleation of microglia resembling the characteristic globoid cells found in GLD. Using this novel system, we defined the conditions and modes of analysis for study of globoid cells. The potential use of this model system was validated in our previous study, which identified a potential role for matrix metalloproteinase (MMP)-3 in GLD. This novel in vitro system may be a useful model in which to study the formation and function, but also the potential therapeutic manipulation, of these unique cells.

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

Globoid cells are a defining pathological feature of Krabbe disease, a leukodystrophy currently lacking an effective long-term therapy. We have developed a cell culture model to study the innate biology and pathogenic potential of activated microglia and their transformation into globoid cells.

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

The tau aggregation assay described in this manuscript mimics the anticipated features of in vivo tau misfolding and aggregation.

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other ...