Name: the indirect neuron-astrocyte coculture assay: an in vitro set-up for the detailed investigation of neuron-glia interactions
Uploaded: 2016-11-14T14:00:00
Description: 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 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>
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			<td height="63" style="height:63px;width:115px;">B27</td>
			<td style="width:152px;">Gibco (Life Technologies)</td>
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		<tr height="84">
			<td height="84" style="height:84px;width:115px;">Cell culture grade water</td>
			<td style="width:152px;">MilliQ</td>
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		<tr height="84">
			<td height="84" style="height:84px;width:115px;">Cell culture grade water</td>
			<td style="width:152px;">MilliQ</td>
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			<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>
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		<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>
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			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">G1397</td>
			<td style="width:191px;">CAUTION: H317-334</td>
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		<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>
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			<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>
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			<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>
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			<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>
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			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">C-2529</td>
			<td style="width:191px;"></td>
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			<td height="63" style="height:63px;width:115px;">MEM</td>
			<td style="width:152px;">Gibco (Life Technologies)</td>
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			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">A7641</td>
			<td style="width:191px;">CAUTION: H334</td>
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			<td height="42" style="height:42px;width:115px;">Papain</td>
			<td style="width:152px;">Worthington</td>
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			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">P0899</td>
			<td style="width:191px;"></td>
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			<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>
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		<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>
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			<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>
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		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Equipment</td>
			<td style="width:152px;"></td>
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			<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>
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		<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>
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			<td height="42" style="height:42px;width:115px;">Binocular</td>
			<td style="width:152px;">Leica</td>
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			<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>
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			<td height="21" style="height:21px;width:115px;">Centrifuge</td>
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			<td style="width:152px;">Marienfeld</td>
			<td style="width:151px;">650010</td>
			<td style="width:191px;"></td>
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			<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>
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			<td height="42" style="height:42px;width:115px;">glass cover slips (12 mm)</td>
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			<td style="width:151px;">P231.1</td>
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			<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>
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			<td style="width:152px;">Millipore</td>
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			<td style="width:191px;"></td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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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Research

JoVE Journal

Neuroscience

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 ...

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 ...

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 ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

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 ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

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

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

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

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