The authors acknowledge the funding support by Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia through Projects UIDB/00709/2020, POCI-01-0145-FEDER-029311 and the fellowship SFRH/BD/135936/2018 to JP, by &#8216;&#8216;Programa Operacional do Centro, Centro 2020&#8221; through the project CENTRO-01-0145-FEDER-000013 and funding to the PPBI-Portuguese Platform of BioImaging through the Project POCI-01-0145-FEDER-022122.

All animals used were bred at the CICS-UBI Health Science Research Centre in accordance with the national ethical requirements for animal research and with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Directive 2010/63/EU).
1. Rat embryo cortex primary cell culture
<ol>
	<li>Culture medium preparation
	<ol>
		<li>Prepare the Neurobasal Medium (NBM) by adding the following supplements: 2% B27, 0.5 mM glutamine, 25 &#181;M glutamate and 120 &#181;g/mL gentamicin. Homogenize, adjust the pH to 7.2 and sterilize the medium with a vacuum filtration step, using a 0.22 &#181;m filter. For the neuron-glia cell culture, supplement the NBM cell culture medium with 10% of Heat-Inactivated Fetal Bovine Serum (HI-FBS).</li>
		<li>Prepare the Minimum Essential Medium Eagle (MEM) medium with the following supplements: sodium hydrogen carbonate 2.2 g/L, insulin from bovine pancreas 5 mg/L, D-glucose anhydrous 3.4 g/L, penicillin (12 U/mL) /streptomycin (12 &#181;g/mL) and 10% HI-FBS. Homogenize, adjust the pH to 7.2 and sterilize the medium with a filtration step.</li>
	</ol>
	</li>
	<li>Preparation of materials and equipment
	<ol>
		<li>Puncture the terminal part of a 1 mL plastic micropipette tip from side to side, using a needle with a specific diameter (0.5 mm for S; 0.6 mm for M; 0.8 mm for L) and seal the opening of the tip&#160;using a flame.</li>
		<li>Sterilize all the glassware. Throughout the dissection keep the tools used (e.g., scissors, tweezers, scalpel) immersed in 70% ethanol. Spray the materials with 70% ethanol before entering them in the laminar flow chamber.</li>
		<li>Set the water bath temperature to 37 &#176;C and place the cell culture medium in the water bath before starting the procedure. 
		NOTE: For immunocytochemistry assays, poly-D-lysine coating should be performed in multiwell plates containing coverslips.</li>
	</ol>
	</li>
	<li>Rat embryonic cortex culture
	<ol>
		<li>Remove the rat embryos from a female Wistar rat with 15-16 days of gestation (the end of the mating, which should last 24 h, is considered the 1st day of the embryonic development). For that purpose, anaesthetize the female with ketamine (87.5 mg/kg) and xylazine (12 mg/kg) and remove the embryos. Then euthanize the female rat by cervical dislocation, following standard protocol.</li>
		<li>Place the embryos in a 50 mL sterile tube, add phosphate buffer saline (PBS) until it covers the embryos and quickly take them to the culture room.</li>
		<li>Still inside the yolk sac, place the embryos in a Petri dish containing 25 mL of cold PBS. With the help of scissors and tweezers, break the yolk sac, remove the embryo and transfer it to another Petri dish containing also cold PBS. The PBS in the Petri dish should be enough to cover the entire embryo. 
		NOTE: Be careful when opening the yolk sac to avoid damaging the embryo. In 1.3.3 and 1.3.4 we used 90 mm diameter Petri dishes placed on top of ice packs covered with absorbent paper to keep the PBS at low temperature.</li>
		<li>For dissection of the embryo, transfer it to another Petri dish containing 30 mL of cold PBS. Place the embryo under a dissecting microscope and immobilize it using a tweezer. Make the initial incision parallel to the cortex, going from the ocular cavity to the end of the muzzle and be careful not to decapitate the animal.</li>
		<li>Carefully remove the scalp and the meninges using tweezers, in order not to damage the cortical brain tissue. Make the next incision to separate the cortex. Transfer the cortical tissue to a 15 mL tube containing 5 mL of PBS using a Pasteur pipette.</li>
		<li>Perform the mechanical digestion of the cortical brain tissue using the 1 mL plastic tips prepared in 1.2.1. Triturate 10 times with a regular pipette and repeat the process using pipettes with progressively smaller holes (L, M and S), until the chunks have fallen apart.</li>
		<li>After the mechanical digestion, centrifuge the suspension at 400 x g for 3 min. Discard the supernatant and resuspend the sediment with the appropriate cell culture medium previously warmed at 37 &#176;C.</li>
		<li>Determine the total number of cells present in the cell suspension (cell density) using a Neubauer chamber, make the appropriate dilutions and plate the cells. The initial cell density was defined based on a previous study5.
		<ol>
			<li>For a neuron-enriched culture, use 0.21 x 106 cells/cm2 as the initial cell density and maintain the cells in NBM culture medium without HI-FBS.</li>
			<li>For the neuron-glia cultures use 0.14 x 106 cells/cm2 as the initial cell density and maintain the cells in NBM culture medium supplemented with 10% HI-FBS.</li>
			<li>For an astrocyte-enriched culture, use 0.26 x 106 cells/cm2 as the initial cell density and maintain the cells in MEM supplemented as previously indicated.</li>
			<li>Place the cells in an incubator set at 37 &#176;C, 95% O2 and 5% CO2. 
			NOTE: For longer culture periods, an anti-mitotic such as 27 &#956;M 5-fluoro-2&#8242;-deoxyuridine with 68 &#956;M uridine should be added to suppress cell growth.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Co-culture system
<ol>
	<li>Preparation of materials
	<ol>
		<li>Heat the paraffin at 150 &#176;C in a heating block for approximately 7 min. Keep at 150 &#176;C until finishing the procedure. Then, with the help of a 1 mm diameter sterile glass Pasteur pipette, add a small drop over sterilized and PDL-coated coverslips. The paraffin spheres are irregular, but they have approximately 2 mm diameter. The spheres will allow the two cultures to be separated by approximately 1.25 mm.</li>
		<li>As the paraffin is not sterile, place the multiwell with the paraffin spheres under ultraviolet radiation for 15 min.</li>
		<li>To establish the co-culture, transfer the coverslip with the paraffin spheres using a tweezer previously immersed in 70% ethanol for 15 min.</li>
	</ol>
	</li>
	<li>Co-culture
	<ol>
		<li>When the two cultures are ready to use (i.e., after 7 days in culture under the conditions mentioned in step 1.3.8), transfer the neurons seeded in the coverslips with paraffin spheres to the wells containing the astrocytes.</li>
		<li>24 h before the two cultures are brought in contact, change the culture medium of neurons and astrocytes to NBM supplemented, or not, with HI-FBS, depending on the purpose of the experiment.</li>
		<li>After placing both cell types in contact, wait 8-12 h before starting the different stimuli and procedures. 
		NOTE: The schematic representation of the co-culture system is shown in Figure 1.</li>
	</ol>
	</li>
</ol>
3. Oxygen and glucose deprivation
<ol>
	<li>Culture Medium Preparation
	<ol>
		<li>For the OGD experiments, use Hank&#39;s Balanced Salt Solution (HBSS). Prepare the HBSS medium with the following reagents: 1.26 mM CaCl2, 5.36 mM KCl, 0.44 mM KH2PO4, 0.49 mM MgCl2, 139.9 mM NaCl, 4.17 mM NaHCO3, 3.38 mM Na2HPO4. Homogenize and adjust the pH to 7.2. Sterilize the medium by filtration. 
		NOTE: If appropriate, supplement the HBSS solution with glucose (HBSSglu+), by adding 5.56 mM glucose. For cultures submitted to OGD do not supplement the HBSS medium with glucose (HBSSglu-).</li>
	</ol>
	</li>
	<li>OGD procedure
	<ol>
		<li>7 days after seeding the cells, remove the culture medium and wash two times with HBSSglu-. After washing add the HBSSglu- cell culture medium and place the multiwell in the hypoxia chamber.</li>
		<li>Seal the hypoxia chamber and add a gas mix containing 95% N2/5% CO2 for 4 min with a flow of 20 L/min to remove the oxygen present inside the chamber. After this, stop the flow and place the hypoxia chamber in an incubator at 37 &#176;C for 4 h or 6 h, depending on the extent of the intended ischemia.</li>
		<li>After the period of OGD, replace the HBSSglu- medium with the appropriate culture medium for the remaining procedures. 
		NOTE: The OGD experiment aims to simulate the in vitro conditions that the cells suffer during an ischemic event, so it is important to certify that all the media used previously is removed.</li>
	</ol>
	</li>
</ol>
4. Immunocytochemistry assay
NOTE: Perform the immunocytochemistry assay as previously described5.
<ol>
	<li>Briefly, to characterize the different cortical cultures, incubate the cells overnight at 4 &#176;C with rabbit anti-GFAP (1:2000) and mouse anti-microtubule-associated protein 2 (MAP2; 1:500); and then 1 h at room temperature with the following secondary antibodies: anti-rabbit conjugated to Alexa Fluor 546 and anti-mouse conjugated to Alexa Fluor 488, both at 1:1000 dilution.</li>
	<li>Label the cell nuclei by incubation with 2 &#181;M Hoechst 33342 for 10 min at room temperature.</li>
	<li>Mount coverslips in fluorescence mounting medium and acquire images on an epifluorescence microscope with a 63x magnification.</li>
</ol>
5. Statistical analysis
<ol>
	<li>Express data as percentage of the&#160;total number of cells or as a percentage of control and presented as the mean &#177; standard error of the mean (SEM) of at least 3 independent experiments performed in triplicate.</li>
	<li>Perform statistical analysis with software (GraphPad Software Inc., San Diego, CA), using the unpaired Student&#8217;s t test. The results were considered significant when values of p &#60; 0.05.</li>
</ol>

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D., Mohr, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+metabolism+and+signaling+during+brain+ischemia.">Astrocyte metabolism and signaling during brain ischemia.</a> Nature Neuroscience. 10 (11), 1377-1386 (2007).</li></ol>

The authors declare that they have no conflict of interests.

The method here described consists of the astrocyte and neuron isolation from rat embryonic cortical tissue, allowing the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures. It was adapted from a previous study of our group5, where the cortical neuron&#8211;glia and neuron-enriched embryonic cultures isolation were described and the two cultures characterized. Using these cultures, Roque et al. found that astrocytes play a key role in responding to an ischemic damage a...

According to data from the World Health Organization, about 5.5 million people die every year from ischemic stroke1. This condition is characterized by the interruption of blood flow to a certain brain region, resulting in a reversible or irreversible loss in the supply of oxygen and nutrients to the tissue, which alters tissue function and leads to mitochondrial dysfunction, calcium dysregulation, glutamate excitotoxicity, inflammation and cell loss2,3.
Apart from vascular cells, neuronal and glial cells are involved in the pathophysiology of the ischemic stroke4. In particular, astrocytes are essential to the maintenance of neurons and recently were shown to play a critical role in the response to the ischemic lesion5. This type of glial cell performs functions of structural support, defence against oxidative stress, synthesis of neurotransmitters, stabilization of cell-cell communication, among others6. Along with neurons, astrocytes play a direct role in synaptic transmission, regulating the release of molecules such as adenosine triphosphate, gamma-aminobutyric acid and glutamate7. Part of the injury induced by ischemia is caused by the excessive release of glutamate and its accumulation at the synaptic cleft, leading to the overactivation of N-methyl-D-aspartate receptors, activating downstream signalling cascades, ultimately resulting in excitotoxicity8. Since astrocytes are able to remove glutamate from the synaptic cleft and convert it into glutamine, they are crucial in defending against excitotoxicity, thereby exerting a neuroprotective effect on ischemia. These cells also play a role in ischemia-induced neuroinflammation. After the ischemic insult, activated astrocytes undergo morphologic changes (hypertrophy), proliferate, and show an increase in glial fibrillary acidic protein (GFAP) expression. They can become reactive (astrogliosis), releasing pro-inflammatory cytokines such as tumour necrosis factor-&#945;, interleukin-1&#945; and interleukin-1&#946;, and producing free radicals, including nitric oxide and superoxide, which in turn can induce neuronal death9,10. In contrast, reactive astrocytes may also play a neuroprotective effect, since they release anti-inflammatory cytokines, such as transforming growth factor-&#946;, that is upregulated after stroke11. Moreover, they can generate a glial scar, which can limit tissue regeneration by inhibiting axonal sprouting; however, this glial scar can isolate the injury site from viable tissue, thus preventing a cascading wave of uncontrolled tissue damage12,13.
Thus, it is imperative to establish models that allow studying neuron-glia interactions under an ischemic injury in order to find therapeutic strategies that limit or reverse the effects of ischemic injury. Compared to other models used to study ischemic injury, namely in vivo models14,15,16, organotypic cultures17,18,19 and acute brain slices20,21,22, primary cell cultures are less complex, which makes possible the study of individual contributions of each cell type in the pathophysiology of ischemic stroke and how each cell type responds to possible therapeutic targets. Typically, in order to study the interactions between neuron-enriched cultures and astrocyte-enriched cultures, neurons and glial cells of postnatal origin are used23,24, or postnatal glial cells and embryonic neurons25,26. Herein is proposed a simple approach to establish neuron- or astrocyte-enriched cultures and neuron-glia cultures from the same tissue. These primary cells are obtained from rat embryonic cortex, a region frequently affected by stroke27,28. Moreover, the dissociation of the tissue is performed only by a mechanical procedure. Therefore, this protocol allows isolating cells in the same stage of development, in a fast and inexpensive way, and with high performance and reproducibility.
The crosstalk between astrocytes and neurons can be explored using a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes seeded in multiwell plates. Small paraffin spheres can be used to ensure the separation of the two cell cultures. This approach allows independent manipulation of each cell type before they are brought into contact. For example, it is possible to silence a specific gene in astrocytes and see how it can influence the neuronal vulnerability or protection against ischemic-induced damage. An established method to induce ischemic-like conditions in vitro is oxygen and glucose deprivation (OGD)3, which consists in replacing the regular atmosphere (95% air and 5% CO2) by an anoxic atmosphere (95% N2 and 5% CO2), associated with the omission of glucose.
The method described is suitable for studying the interactions between neurons and astrocytes in the context of ischemic stroke, in a simple, fast, reproducible and inexpensive way.

<table>
	<tbody>
		<tr>
			<td>24 -well culture plates</td>
			<td>Thermo Fischer Scientific</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>95% N2/5% CO2 gas cylinder</td>
			<td>ArL&#237;quido</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse conjugated to Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td>1/1000 dilution; incubation period - 1 h at room temperature</td>
		</tr>
		<tr>
			<td>Anti-rabbit conjugated to Alexa Fluor 546</td>
			<td>Invitrogen</td>
			<td>A11010</td>
			<td>1/1000 dilution; incubation period - 1 h at room temperature</td>
		</tr>
		<tr>
			<td>B27 supplement (50x)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako Fluorescence Mounting Medium</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose anhydrous</td>
			<td>Fisher Scientific</td>
			<td>G/0450/60</td>
			<td>3.4 g/L</td>
		</tr>
		<tr>
			<td>Epifluorescence microscope</td>
			<td>Zeiss</td>
			<td>AxioObserver Z1x</td>
			<td>63x objective</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Biochrom</td>
			<td>S0615</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G1272</td>
			<td>120 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G8415</td>
			<td>25&#181;M</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G3126</td>
			<td>0.5 mM</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H1399</td>
			<td>2 &#181;M; incubation period - 10 min at room temperature</td>
		</tr>
		<tr>
			<td>Hypoxia incubation chamber</td>
			<td>Stemcell Technologies</td>
			<td>27310</td>
			<td>Chamber used for OGD induction</td>
		</tr>
		<tr>
			<td>Insulin from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>I5500</td>
			<td>5 mg/L</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Sigma-Aldrich</td>
			<td>K-002</td>
			<td>87.5 mg/Kg</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium Eagle medium</td>
			<td>Sigma-Aldrich</td>
			<td>M0268</td>
			<td>warm up to 37 &#176;C before use</td>
		</tr>
		<tr>
			<td>Mouse Anti-MAP2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>Sc-74421</td>
			<td>1/500 dilution; incubation period overnight at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>warm up to 37 &#176;C before use</td>
		</tr>
		<tr>
			<td>Paraffin pastilles for histology</td>
			<td>Sigma-Aldrich</td>
			<td>1.07164</td>
			<td>Solidification point 56-58&#176;C</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma -Aldrich</td>
			<td>P6148</td>
			<td>4% in PBS</td>
		</tr>
		<tr>
			<td>Penicilin/Streptomycin</td>
			<td>Biochrom</td>
			<td>A 2213</td>
			<td>penicillin (12U/mL) /streptomycin (12&#181;g/mL)</td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P1024</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti-GFAP</td>
			<td>DAKO</td>
			<td>Z0334</td>
			<td>1/2000 dilution; incubation period overnight at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate</td>
			<td>Fisher Scientific</td>
			<td>S/4240/60</td>
			<td>2.2g/L</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Sigma-Aldrich</td>
			<td>X1126</td>
			<td>12 mg/Kg</td>
		</tr>
	</tbody>
</table>

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.

To characterize the cultures, immunocytochemistry to assess the number of cells that expressed GFAP or MAP2, widely used markers of astrocytes and neurons (Figure 2), was performed in each type of cortical culture. This analysis revealed that astrocyte-enriched cultures presented 97% of the cells expressing GFAP (Figure 2A). Regarding the neuron-enriched culture 78% of the cells expressed MAP2, 4% of the cells expressed GFAP, and 18% of the cells were both GFAP ...

Watch this Scientific Journal Video about A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia at JoVE.com

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

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 ...

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9.2K Views.  Universidade da Beira Interior. 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.

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

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats

The most insidious aspect of drug addiction is the high propensity for relapse. Animal models of relapse, known as reinstatement procedures, have been used extensively to study the neurobiology and phenomenology of relapse to drug use. Although procedural variations have emerged over the past several decades, the most conventional reinstatement procedures are based on the drug self-administration (SA) model. In this model, an animal is trained to perform an operant response to obtain drug. Subsequently, the behavior is extinguished by withholding response-contingent reinforcement. Reinstatement of drug seeking is then triggered by a discrete event, such as an injection of the training drug, re-exposure to drug-associated cues, or exposure to a stressor 1.
Reinstatement procedures were originally developed to study the ability of acute non-contingent exposure to the training drug to reinstate drug seeking in rats and monkeys 1, 2. Reinstatement procedures have since been modified to study the role of environmental stimuli, including drug-associated cues and exposure to various forms of stress, in relapse to drug seeking 1, 3, 4.
Over the past 15 years, a major focus of the reinstatement literature has been on the role of stress in drug relapse. One of the most commonly used forms of stress for studying this relationship is acute exposures to mild, intermittent, electric footshocks. The ability of footshock stress to induce reinstatement of drug seeking was originally demonstrated by Shaham and colleagues (1995) in rats with a history of intravenous heroin SA5. Subsequently, the effect was generalized to rats with histories of intravenous cocaine, methamphetamine, and nicotine SA, as well as oral ethanol SA 3, 6.
Although footshock-induced reinstatement of drug seeking can be achieved reliably and robustly, it is an effect that tends to be sensitive to certain parametrical variables. These include the arrangement of extinction and reinstatement test sessions, the intensity and duration of footshock stress, and the presence of drug-associated cues during extinction and testing for reinstatement. Here we present a protocol for footshock-induced reinstatement of cocaine seeking that we have used with consistent success to study the relationship between stress and cocaine seeking.

Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.

The most insidious aspect of drug addiction is the high propensity for relapse.  Animal models of relapse, known as reinstatement procedures, have been ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

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 ...

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

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 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.

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with neurodegenerative diseases or injuries, but also in the normal brain. Here, we describe a newly-established micromechanical system to rapidly, reliably, and reversibly induce axonal varicosities, allowing us to understand the mechanisms governing varicosity formation and heterogeneous protein composition. This system represents a novel means to evaluate the effects of compression and shear stress on different subcellular compartments of neurons, different from other in vitro systems that mainly focus on the effect of stretching. Importantly, owing to the unique features of our system, we recently made a novel discovery showing that the application of pressurized fluid can rapidly and reversibly induce axonal varicosities through the activation of a transient receptor potential channel. Our biomechanical system can be utilized conveniently in combination with drug perfusion, live cell imaging, calcium imaging, and patch clamp recording. Therefore, this method can be adopted for studying mechanosensitive ion channels, axonal transport regulation, axonal cytoskeleton dynamics, calcium signaling, and morphological changes related to traumatic brain injury.

This protocol describes a physiologically relevant, pressurized fluid approach for rapid and reversible induction of varicosities in neurons.

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...