Name: rapid genotyping of animals followed by establishing primary cultures of brain neurons
Uploaded: 2015-01-29T13:00:00.000+00:00
Duration: 9 min 51 s
Description: Watch this Scientific Journal Video about Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons at JoVE.com

The authors thank researchers at the University of Iowa, Drs. Luis Tecedor, Ines Martins and Beverly Davidson for instructions and helpful comments regarding striatal cultures, and Drs. Kara Gordon, Nicole Bode and Pedro Gonzalez-Alegre for genotyping assistance and discussions. We also thank Dr. Eric Weyand (Animal Identification and Marking Systems) for helpful comments regarding tattooing, and Dr. Shutaro Katsurabayashi (Fukuoka University) for helpful comments regarding the mouse culture. This work was supported by grants from the American Heart Association, the Department of Defense (Peer Reviewed Medical Research Program award W81XWH-14-1-0301), the Dystonia Medical Research Foundation, the Edward Mallinckrodt, Jr. Foundation, the National Science Foundation, and the Whitehall Foundation (N.C.H.).

NOTE: All animal procedures performed in this study were approved by the Institutional Animal Care and Use Committee of the University of Iowa.
1. Long-term Identification of Mice Using Tattooing the Paw Pads
<ol>
	<li>Immobilize a paw with the paw pad (plantar surface) facing the experimenter. Hold the paw with the thumb and the index finger. Be careful not to pinch the paw. 
	NOTE: Stable immobilization is important to ensure that the tattoo pigment is placed into the dermis of the paw pad, and thus is permanent 7.</li>
	<li>Swab the paw pad with 70% ethanol on a gauze sponge or swab.</li>
	<li>Apply skin oil to a cotton-tipped applicator, and gently press the tip against the surface of the paw pad several times. Use only a small amount of skin oil; when present in large amounts, it will prevent the tattoo pigment from reaching the skin.</li>
	<li>Dip the tips of the tattoo needles into the tattooing ink just prior to tattooing. Use clean, aseptic and sharp tattooing needles, to decrease the pain and the possibility of infection, and also to increase the tattooing efficiency.</li>
	<li>While the paw pad surface is covered with the skin oil, press the tattoo needle tips vertically and lightly against the skin in the center of the paw pad, and inject the ink multiple times. See Table of Materials/Equipment for information about electric tattooing system.</li>
	<li>Spray 70% ethanol on a gauze sponge, gently press it against the paw to remove extra tattoo pigment on the skin surface, and inspect the quality of tattoo. Check that the middle of paw pad has a dark, round spot that differs from normal skin pigmentation. If the tattoo is not dark or large enough for easy viewing, repeat the prior tattooing steps.</li>
</ol>
2. Genotyping Newborn Mice Using a Fast PCR Genotyping Kit
<ol>
	<li>Disinfect the distal end of a mouse tail with 70% ethanol, cut 5 mm or less of the tail tip and transfer it to a tube of an 8-tube strip of the type used for PCR. Use an un-used razor blade for each pup to avoid cross contamination between specimens. Alternatively, use a pair of scissors, but in this case, carefully and thoroughly remove the remaining tissue on the blades using 70% ethanol. Check for bleeding. If bleeding occurs, apply pressure to the cut portion of the tail with a gauze sponge until bleeding has stopped.</li>
	<li>Add 200 &#181;l of DNA Extraction Solution to each PCR tube containing a specimen. See Table of Materials/Equipment for its composition and information about the kit.</li>
	<li>Place the tube strip into a PCR thermal cycler, and start the DNA extraction using the following program: 1 cycle at 55 &#176;C for 10 min, 1 cycle at 95 &#176;C for 10 min, and holding at 4 &#176;C. 
	NOTE: This is the same PCR thermal cycler that is later used for PCR.</li>
	<li>After the DNA extraction is finished, remove the tube strip from the thermal cycler and invert 5 times.</li>
	<li>Transfer 4 &#181;l of the solution (DNA extract) of each specimen to an un-used tube of an 8-tube strip, and mix with: 10 &#181;l of 2X PCR Ready Mix II, 2 &#181;l of mixed forward &#38; reverse primers (recommended at 0.5 &#181;M; see Table of Materials/Equipment for sequences), and 4 &#181;l of nuclease-free H2O, for each specimen. Spin briefly (e.g., 3 sec) using a table-top centrifuge. Keep the tubes on ice at all times except when handling.</li>
	<li>Perform thermal cycling. See Table of Materials/Equipment for the thermal program.</li>
	<li>Detect the amplified DNA products. Load the total reaction solution from the PCR (20 &#181;l) directly into a well of an agarose gel. Load the molecular weight markers into a separate well. Apply an electric field.</li>
	<li>Acquire fluorescence images of the bands under ultraviolet light.</li>
</ol>
3. Primary Culture of Mouse Brain Neurons on Glial Feeder Layer
NOTE: The procedures for brain dissection and cell dissociation (3.1) are common to all the subsequent procedures. The procedures for mouse glial cultures (3.2), rat glial cultures (3.3), and mouse neuronal cultures (3.4) are described separately afterwards.
<ol>
	<li>Brain Dissection and Cellular Dissocaiation
	<ol>
		<li>Sacrifice one mouse or rat pup by decapitation, rapidly remove the brain, and place it into Hanks&#39; solution (see Table 1 for composition) + 20% fetal bovine serum (FBS) in a 35 mm dish (kept on ice).</li>
		<li>Cut the brain through the midline into two hemispheres. Remove the region of interest(e.g.,&#160;cerebral cortex, striatum and hippocampus) from each hemisphere of the brain. Remove the meninges and major blood vessels from the surface. Cut the brain region into 4-10 thin slices using a surgical blade.</li>
		<li>Rinse the brain slices in a 15 ml centrifugation tube, once with Hanks&#39; solution + 20% FBS, then 3 times with the Hanks&#39; solution (i.e., without serum) (all at 4 &#176;C). To rinse, add 5-10 ml solution, let the brain slices settle at the bottom of the tube, aspirate the solution from the top, and add a fresh solution. Finish the rinsing procedure by aspirating the solution.</li>
		<li>Filter 2 ml of the trypsin-containing digestion solution (see Table 1 for composition) using a 3 ml syringe and a 0.2 &#181;m syringe filter, and add the filtrate directly into the tube containing the brain slices. Let the trypsinization proceed for 13 min at RT.</li>
		<li>Neutralize the trypsin solution first by aspirating most of it and then by adding 7-10 ml of Hanks&#39; solution + 20% FBS (4 &#176;C).</li>
		<li>Rinse the brain slices, twice with Hanks&#39; solution + 20% FBS, and then three times with the Hanks&#39; solution (i.e. without serum) (all at 4 &#176;C). Finish the rinsing procedure by aspirating the solution.</li>
		<li>Filter 2 ml of the dissociation solution (see Table 1 for composition) using a 3 ml syringe and a 0.2 &#956;m syringe filter, and add the filtrate directly to the tube with the brain slices.</li>
		<li>Mechanically dissociate the cells by gently triturating 10-20 times, until visible tissue pieces disappear. Use a cotton-plugged, fire-polished Pasteur pipette and avoid making bubbles during trituration.</li>
		<li>Wait 3 min for small pieces to settle down.</li>
		<li>Transfer the majority of the solution (~1.5 ml, leaving some solution at the bottom) to a 15 ml centrifugation tube that contains 3 ml Hanks&#39; solution + 20% FBS solution (4 &#176;C), using the cotton-plugged, fire-polished Pasteur pipette. Do not transfer all the solution because the inclusion of any sediment at the bottom typically results in deterioration of the culture.</li>
		<li>Centrifuge for 13 min at ~185 g (~1,100 rpm) at 4 &#176;C.</li>
		<li>Aspirate the supernatant gently, add 1 ml of pre-warmed plating medium (37 &#176;C) to the pellet, and resuspend it by gently pipetting several times using the cotton-plugged, fire-polished Pasteur pipette. 
		NOTE: Three different types of plating media are used for different culture purposes: plating medium-1 for mouse glial cells, plating medium-2 for mouse neurons, and plating medium-3 for rat neurons and glial cells (see Table 1 for compositions).</li>
		<li>Take out 10 &#956;l of the cell suspension, mix it with 10 &#956;l of 0.4% trypan blue solution, and measure the density of live cells, using either a hemocytometer or an automated cell counter.</li>
	</ol>
	</li>
	<li>Mouse Glial Cultures
	<ol>
		<li>Wash the coverslips to help establish healthy cultures of mouse cells.
		<ol>
			<li>Immerse glass coverslips (round, 12 mm diameter) in 70% nitric acid in a glass Petri dish. Protect from light using aluminum foil, and place it on an orbital shaker O/N.</li>
			<li>Rinse the glass coverslips in the Petri dish at least three times with distilled water. Immerse them in distilled water. Place the dish on an orbital shaker O/N.</li>
			<li>Dry the coverslips on a Whatman 150 mm filter paper in the biological safety cabinet.</li>
			<li>Autoclave the coverslips.</li>
			<li>Place the coverslips into 24-well culture dish.</li>
		</ol>
		</li>
		<li>Label and genotype newborn mice according to steps 1 and 2.</li>
		<li>Obtain the brain cells from the mouse pups, according to step 3.1. 
		NOTE: Use of the cerebral cortex is typical for preparing the mouse glial feeder layer. However, other brain regions, such as hippocampus and striatum, will work after proper adjustment of cell density due to different numbers and yields of cells from different regions.</li>
		<li>Add ~4 ml of pre-warmed plating medium-1 to the final cell suspension (~1 ml). For pre-warming culture media, place the solution in the culture incubator (5% CO2-95% O2, 37 &#176;C) O/N, to allow the temperature and pH values to stabilize.</li>
		<li>Transfer the cell suspension to an uncoated T25 culture flask, using the cotton-plugged, fire-polished Pasteur pipette. Place the flask in the culture incubator.</li>
		<li>At 1 day in vitro (DIV), rinse the cultured cells in the T25 flask twice with plating medium-1 (4 &#176;C). Place the flask back into the incubator. Perform rinsing by aspirating the medium inside the flask completely with a Pasteur pipette, adding ~5 ml of fresh medium and gently tilting the flask several times in a swirling motion.</li>
		<li>At 6-9 DIV, (i.e.,&#160;one day before trypsinization and plating on coverslips, in steps 3.2.8-3.2.13), place 100 &#181;l of the coating material with extracellular matrix proteins (see Table of Materials/Equipment for comments about the coating material) on the glass coverslips in a culture dish, and place the culture dish in the culture incubator.</li>
		<li>At 7-10 DIV, when the cells are 20-40% confluent (spatially continuous), trypsinize them.
		<ol>
			<li>Rinse the flask once, by aspirating all the solution within the flask and adding ~13 ml of Hanks&#39; solution (4 &#176;C), gently tilt the flask several times in a swirling motion, and aspirate the solution completely.</li>
			<li>Add 40 &#181;l of DNase solution (final concentration, 750 units/ml) to 4 ml Trypsin-EDTA solution, pass the solution through a 0.2 &#181;m syringe filter, and add the filtrate directly to the cells in the flask.</li>
			<li>Let the trypsinization proceed for 13 min at 37 &#176;C in the incubator.</li>
			<li>Neutralize the trypsin solution by adding 2 ml of 100% FBS (4 &#176;C) to the flask.</li>
			<li>Transfer the trypsinized cells to a 15 ml centrifugation tube using a 5- to 10-ml pipette, add ~4 ml of Hanks&#39; solution + 20% FBS (4 &#176;C), centrifuge at ~185 g and 4 &#176;C for 13 min, and aspirate the supernatant.</li>
		</ol>
		</li>
		<li>Resuspend the pellet in 1 ml of pre-warmed plating medium-1.</li>
		<li>Measure the density of the cells according to step 3.1.13.</li>
		<li>Aspirate the coating material completely from the glass coverslips, and plate ~50 &#956;l of the resuspended glial cells on coverslips. 
		NOTE: These cells will establish the glial feeder layer.</li>
		<li>Place the culture dish with coverslips in the incubator.</li>
		<li>20-60 min later, add 1 ml of pre-warmed plating medium-1 to each well, and place the dish back in the incubator. Note: While the plating medium is added, it will be helpful to use another pipette to press down the periphery of the coverslip (where there are no plated cells), so that the coverslip will not float in the medium.</li>
		<li>At 1 DIV of the glial feeder layer (i.e., on the glass coverslip), replace the medium with 1 ml of pre-warmed plating medium-1, by aspirating all of the solution in a well and then filling it with fresh medium.</li>
		<li>At 2-3 DIV of the glial feeder layer when the cells are 80-100% confluent, add mitotic inhibitor (10 &#181;l of a mixture of 5-fluoro-2&#39;-deoxyuridine + uridine; final concentrations of 81.2 and 204.8 &#181;M, respectively) to each well, to inhibit DNA replication and therefore to suppress glial-cell proliferation.</li>
		<li>At 7-9 DIV, when the cells are 90-100% confluent (1-2 hr before plating neurons on the glial feeder layer), replace the culture medium with pre-warmed plating medium-2 (37 &#176;C). 
		NOTE: Neurons will be plated on the same day, using step 3.4.</li>
	</ol>
	</li>
	<li>Rat Glial Cultures
	<ol>
		<li>Coat a T25 culture flask with the same coating material. 
		NOTE: This is necessary for later removal of non-adherent cells in step 3.3.5.
		<ol>
			<li>Add 2.0 ml of the coating material to the flask, and place the flask in the culture incubator.</li>
			<li>After 2-3 hr, aspirate the coating material completely, such that the floor of the flask becomes dry.</li>
		</ol>
		</li>
		<li>Obtain the cells from the rat pups, according to step 3.1. 
		NOTE: The CA3-CA1 region of the hippocampus is used for preparing the rat glial feeder layer. However, other brain regions, such as the cerebral cortex and striatum, will work after adjusting for differences in cell density due to different numbers and yields of cells from different regions.</li>
		<li>Add ~4 ml of pre-warmed plating medium-3 to the final cell suspension (~1 ml).</li>
		<li>Transfer the cell suspension into the coated T25 culture flask, using the cotton-plugged, fire-polished Pasteur pipette. Place the flask in the culture incubator (5% CO2-95% O2, 37&#176;C).</li>
		<li>At 2-3 DIV, when cells are 20-40% confluent, remove non-adherent or weakly adherent cells, by closing the lid tightly, shaking the flask vigorously ~10 times, aspirating the solution, and adding 4-5 ml of plating medium-3 (at 4 &#176;C). Repeat this procedure once. Examine the cultured cells across the entire flask floor (e.g.,&#160;using phase-contrast microscope) to confirm that those that appear neuronal (i.e., those for which the outer rim of the cell body appears phase-bright) are completely removed. Repeat the procedure as often as necessary to remove these cells, and then return the flask to the incubator. 
		NOTE: The strength and the total number of &#39;shakes&#39; required may differ among individual experimenters. The important thing is not to keep the total number of shakes to a set number, but to confirm that neuron-like cells are eliminated.</li>
		<li>At 6-8 DIV, when cells are 90-100% confluent, passage the glial cells by trypsinizing them as in step 3.2.8.</li>
		<li>After centrifugation, resuspend the pellet in 1 ml of plating medium-3 (4 &#176;C), add 10 ml of plating medium-3 (4 &#176;C), transfer the trypsinized cells to an un-coated T75 flask, and culture them in the incubator. 
		NOTE: Rat glial cells will be passaged twice; in contrast the mouse glial cells are passaged only once because they become unhealthy after multiple passages. Rat glial cells will be passaged in T75 flasks that are not coated with any coating material. The coating allows rat glial cells to grow too quickly and yield many ciliated cells (putative ependymal cells), which will deteriorate the neuronal culture condition later.</li>
		<li>At 17-19 DIV of glial culture in a flask (i.e., 11 days after passaging and one day before trypsinization and plating onto coverslips by steps 3.3.9-3.3.14), place 100 &#181;l of the coating material on unwashed glass coverslips (round, 12 mm diameter) in a culture dish, and place the culture dish in the culture incubator. 
		NOTE: For rat glial cultures, a difference was not noticed in the culture results with or without washing the coverslips.</li>
		<li>At 18-20 DIV of glial culture in a flask (i.e.,&#160;12 days after passaging), prepare the glial feeder layer by trypsinizing the cultured cells, according to step 3.2.8.</li>
		<li>During centrifugation, aspirate the coating material completely from the glass coverslips.</li>
		<li>After centrifugation, resuspend the pellet in 1 ml of plating medium-3 (4 &#176;C), and measure the cell density, according to step 3.1.13.</li>
		<li>Adjust the glial density to 104 live cells/ml, and plate 100 &#181;l of the glial cell suspension on the coated glass coverslips. 
		NOTE: These cells will establish the glial feeder layer.</li>
		<li>Place the culture dish with coverslips into the incubator for 20-60 min.</li>
		<li>Add 1 ml of plating medium-3 (4 &#176;C) to each well, and place the dish back in the incubator.</li>
		<li>At 3-4 DIV of the glial feeder layer, when the cells on the coverslip are 40-80% confluent, add 1 ml of the pre-warmed growth medium (see Table 1 for composition) that contains cytosine &#946;-D-arabinofuranoside (AraC, final concentration of 4 &#181;M) to stop glial-cell proliferation. Plate neurons at 7-9 DIV of the glial feeder layer when the cells are 60-80% confluent, using step 3.4.</li>
	</ol>
	</li>
	<li>Mouse Neuronal Cultures
	<ol>
		<li>Label and genotype newborn mice according to steps 1 and 2.</li>
		<li>Use the mouse pups for step 3.1.</li>
		<li>Adjust the density of live-cell suspension to ~2.0 x 105 cells/ml using pre-warmed plating medium-2 for plating on mouse glial cells, or using plating medium-3 for plating on rat glial cells.</li>
		<li>Plate the cells on the glial feeder layer, by gently adding the cell suspension to the culture medium of each well. Note: The volume of the cell suspension to be added will be determined by the target number of cells in a culture well. For example, plate ~60 &#181;l of cell suspension to achieve 12,000 cells/well.</li>
		<li>Note that no solution changes are necessary after the neurons are plated. Be careful to avoid evaporation of the solution from the wells over the course of culture, by humidifying the inside of the culture incubator.</li>
	</ol>
	</li>
</ol>

The author (Zhengmin Huang) is the president of EZ BioResearch LLC that produces reagents described in this article.

The protocol presented here includes procedures for tattooing to label/identify mice, for genotyping mice from tail tips, and for culturing mouse brain neurons at low density. In one round of experiments using 6-8 pups, these procedures typically require ~0.5 hr, ~4 hr and ~2 hr, respectively, at a total of 6-7 hr. This makes it practical for a single experimenter to complete all the procedures necessary from the time of the pups&#39; birth to the plating of neuronal cultures &#8211; in less than a single working day (wi...

Rodent models of genetic diseases have proven useful in establishing the physiological functions of normal proteins and nucleic acids, as well as the pathophysiological consequences of defects in these. Examples include mice deficient for proteins involved in key cellular functions, as well as mouse models of disorders such as Alzheimer&#39;s disease. However, certain genetic manipulations can lead to neonatal lethality shortly or a few days after birth. In these cases, primary cell cultures are an important tool because live cells can be obtained from the embryonic or neonatal pups before death, they can be maintained for at least a few weeks in vitro, and during this time early neuronal development can be followed by biochemical, functional and morphological experiments. For the primary cultures, it can be beneficial to plate the neurons at low density; this makes it possible to visualize the individual somata, dendrites, axonal shafts and nerve terminals at high spatial resolution. However, the survival and differentiation of neurons at low density typically requires that they are plated on a glial feeder layer, co-cultured with glial cells in the absence of physical contact with them, or cultured in medium conditioned by glia 1.
The establishment of low-density neuronal cultures on glial feeder layers can be dependent on fast and reliable genotyping beforehand &#8211; within a few hr in contrast to a few days. Speed is especially important when the neuronal genotype needs to be matched to that of a glial feeder layer prepared beforehand. As a more practical example, it may be necessary to decide which pups of which genotype to use in generating cultures, to optimize the efficiency of an experiment.
Here we demonstrate the working protocol that has been used for fast, simplified and reliable mouse genotyping in previous publications 2-6. Mouse tails and a commercially available kit are used. This protocol includes single-step extraction of nucleic acids from the tissue, and requires neither a nucleic-acid purification step nor use of a termination buffer (&#39;stop solution&#39;). The reliability of this genotyping method is illustrated by presenting the results of a series of tests when differences are introduced with respect to the starting amount of the specimens, the age of the animals and the length of the PCR amplicons. This kit offers the advantages of automated extraction and reliability.
For the sake of being comprehensive, the use of tattooing for long-term identification of the genotyped mice is also demonstrated. Tattooing is achieved by applying tattooing ink to the dermis of the skin (under the epidermis) 7. A procedure is described for tattooing the paw pads of newborn or 1 day old mice, although tattoos can be applied to other parts of the body, such as tails and toes, and to animals of all ages. In addition, procedures will be demonstrated for plating and culturing mouse neurons at a low density, based on optimized preparation of different types of glial feeder layers 2,8.
We use a genetic mouse model of the inherited neurological disorder DYT1 dystonia &#8211; an autosomal-dominant movement disorder caused by a mutation in the gene TOR1A (c.904_906delGAG/c.907_909delGAG; p.Glu302del/p.Glu303del) 9. The encoded protein, torsinA, belongs to the &#8220;ATPases associated with diverse cellular activities&#8221; (AAA+) family of proteins, whose members generally perform chaperone-like functions, assisting in: protein unfolding, protein-complex disassembly, membrane trafficking, and vesicle fusion 10-13. The mutation results in an in-frame deletion of a codon for glutamic acid, and can lead to manifestation of &#39;early-onset generalized isolated dystonia&#39; 14,15. However, the pathophysiological mechanisms responsible for this disorder remain poorly understood. In a knock-in mouse model, the mutant allele is Tor1atm2Wtd, mentioned hereafter as Tor1a&#916;E. Heterozygous &#916;E-torsinA knock-in mice are viable and genetically mimic human patients with DYT1 dystonia, whereas homozygous knock-in mice die after birth 16,17, with the latency to postnatal death affected by genetic background 18. The early death of homozygous knock-in mice necessitates that both the genotyping of animals and the establishment of neuronal cultures are completed rapidly. As another example of genotyping, Tfap2a (transcription factor AP-2&#945;, activating enhancer binding protein 2&#945;) will be used. The protein encoded by this gene is important in regulating multiple cellular processes, such as proliferation, differentiation, survival and apoptosis 19.

<table><tbody><tr><td>REAGENTS - tattooing</td><td></td><td></td><td></td></tr><tr><td>Machine Cleanser</td><td>Animal Identification and Marking Systems, Inc.</td><td>NMCR3</td><td>This is used to clean the needles and the holder after tattooing.</td></tr><tr><td>Machine Drying Agent</td><td>Animal Identification and Marking Systems, Inc.</td><td>NDAR4</td><td>This is used to dry the needles and holder after cleaning.</td></tr><tr><td>Neonate Tattoo Black Pigment</td><td>Animal Identification and Marking Systems, Inc.</td><td>NBP01</td><td></td></tr><tr><td>Skin Prep Applicator</td><td>Animal Identification and Marking Systems, Inc.</td><td>NSPA1</td><td>Q-tip.</td></tr><tr><td>Skin Prep solution</td><td>Animal Identification and Marking Systems, Inc.</td><td>NSP01</td><td>This reagent delivers a thin layer of oil that enhances the efficiency of tattooing and prevents tattoo fading, by (information from vendor): 1) preventing non-tattooed skin from being stained temporarily, thereby allowing the quality of a paw pad tattoo to be easily evaluated before the pup is returned to its home cage &amp;ndash; the stained skin surface can be confused with the tattooed skin, 2) reducing skin damage during tattooing &amp;ndash; softening the skin and lubricating the needle will help the needle penetrate the skin without causing skin damage, and 3) preventing molecular oxygen from entering the skin, thereby reducing inflammatory responses to reactive oxygen species that can be generated.</td></tr><tr><td>REAGENTS - genotyping</td><td></td><td></td><td></td></tr><tr><td>EZ Fast Tissue/Tail PCR Genotyping Kit (Strip Tube Format)</td><td>EZ BioResearch LLC</td><td>G2001-100</td><td></td></tr><tr><td>2X PCR Ready Mix II</td><td>EZ BioResearch LLC</td><td>G2001-100</td><td>A red, loading dye for electrophoresis is included in the 2X PCR Ready Mix solution.</td></tr><tr><td>Tissue Lysis Solution A</td><td>EZ BioResearch LLC</td><td>G2001-100</td><td>Prepare DNA Extraction Solution by mixing 20 &amp;micro;l of Tissue Lysis Solution A and 180 &amp;micro;l of Tissue Lysis Solution B per specimen.</td></tr><tr><td>Tissue Lysis Solution B</td><td>EZ BioResearch LLC</td><td>G2001-100</td><td>Prepare DNA Extraction Solution by mixing 20 &amp;micro;l of Tissue Lysis Solution A and 180 &amp;micro;l of Tissue Lysis Solution B per specimen.</td></tr><tr><td>Acetic acid, glacial</td><td>VWR</td><td>BDH 3092</td><td></td></tr><tr><td>Agarose optimized grade, molecular biology grade</td><td>rpi</td><td>A20090-500&amp;nbsp;</td><td>We use 2% agarose gels in TAE buffer containing the SYBR Safe DNA gel stain (diluted 10,000-fold) or ethidium bromide (0.5 &amp;micro;g/ml gel volume).</td></tr><tr><td>Ethidium bromide</td><td>Sigma-Aldrich</td><td>E7637-1G</td><td></td></tr><tr><td>Ethylenediamine tetraacetic acid, disodium salt dihydrate (EDTA)</td><td>Fisher</td><td>BP120-500</td><td></td></tr><tr><td>Filtered Pipet Tips, Aerosol-Free, 0.1-10 &amp;micro;l</td><td>Dot Scientific Inc</td><td>UG104-96RS&amp;nbsp;</td><td>Use pipette tips that are sterile and free of DNA, RNase and DNase. For all steps involving DNA, use filtered pipette tips to avoid cross-contamination.</td></tr><tr><td>Filtered Pipet Tips, Premium Fit Filter Tips, 0.5-20 &amp;micro;l</td><td>Dot Scientific Inc</td><td>UG2020-RS</td><td>Use pipette tips that are sterile and free of DNA, RNase and DNase. For all steps involving DNA, use filtered pipette tips to avoid cross-contamination.</td></tr><tr><td>Filtered Pipet Tips, Premium Fit Filter Tips, 1-200 &amp;micro;l</td><td>Dot Scientific Inc</td><td>UG2812-RS</td><td>Use pipette tips that are sterile and free of DNA, RNase and DNase. For all steps involving DNA, use filtered pipette tips to avoid cross-contamination.</td></tr><tr><td>Molecular weight marker, EZ DNA Even Ladders 100 bp</td><td>EZ BioResearch LLC</td><td>L1001</td><td>We use either of these three molecular weight markers.</td></tr><tr><td>Molecular weight marker, EZ DNA Even Ladders 1000 bp</td><td>EZ BioResearch LLC</td><td>L1010</td><td></td></tr><tr><td>Molecular weight marker, TrackIt, 100 bp DNA Ladder</td><td>GIBCO-Invitrogen</td><td>10488-058</td><td></td></tr><tr><td>PCR tubes, 8-tube strips with individually attached dome top caps, natural, 0.2 ml&amp;nbsp;</td><td>USA Scientific</td><td>1402-2900</td><td>Use tubes that are sterile and free of DNA, RNase and DNase. An 8-tube strip is easy to handle and to group the specimens than individual tubes.</td></tr><tr><td>PCR tubes, Ultraflux Individual&amp;nbsp;</td><td>rpi</td><td>145660</td><td>Use tubes that are sterile and free of DNA, RNase and DNase.</td></tr><tr><td>Seal-Rite 0.5 ml microcentrifuge tube, natural</td><td>USA Scientific</td><td>1605-0000</td><td>Use tubes that are sterile and free of DNA, RNase and DNase.</td></tr><tr><td>SYBR Safe DNA gel stain * 10,000x concentration in DMSO</td><td>GIBCO-Invitrogen</td><td>S33102</td><td></td></tr><tr><td>Tris base</td><td>rpi</td><td>T60040-1000</td><td></td></tr><tr><td>Primers for amplifying Tor1a gene in &amp;Delta;E-torsinA knock-in mice</td><td></td><td></td><td>5&#039;-AGT CTG TGG CTG GCT CTC CC-3&#039; (forward) and 5&#039;-CCT CAG GCT GCT CAC AAC CAC-3&#039; (reverse) (reference 18). These primers were used at a final concentration of 1.0 ng/&amp;micro;l (~0.16 &amp;micro;M) (reference 2).</td></tr><tr><td>Primers for amplifying Tfap2a gene in wild-type mice</td><td></td><td></td><td>5&#039;-GAA AGG TGT AGG CAG AAG TTT GTC AGG GC-3&#039; (forward), 5&#039;-CGT GTG GCT GTT GGG GTT GTT GCT GAG GTA-3&#039; (reverse) for the 498-bp amplicon, 5&#039;-CAC CCT ATC AGG GGA GGA CAA CTT TCG-3&#039; (forward), 5&#039;-AGA CAC TCG GGC TTT GGA GAT CAT TC-3&#039; (reverse) for the 983-bp amplicon, and 5&#039;-CAC CCT ATC AGG GGA GGA CAA CTT TCG-3&#039; (forward), 5&#039;-ACA GTG TAG TAA GGC AAA GCA AGG AG-3&#039; (reverse) for the 1990-bp amplicon. These primers are used at 0.5 &amp;micro;M.</td></tr><tr><td>REAGENTS - cell culture</td><td></td><td></td><td></td></tr><tr><td>5-Fluoro-2&amp;prime;-deoxyuridine</td><td>Sigma-Aldrich</td><td>F0503-100MG</td><td>See comments section of uridine for more information.</td></tr><tr><td>B-27 supplement</td><td>GIBCO-Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>Cell Culture Dishes 35 x 10 mm Dishes, Tissue Culture-treated</td><td>BD falcon</td><td>353001</td><td></td></tr><tr><td>Cell Culture Flasks, T25, Tissue Culture-treated, Canted-neck, plug-seal cap, 25 cm&lt;sup&gt;2&lt;/sup&gt; Growth Area, 70 ml</td><td>BD falcon</td><td>353082</td><td></td></tr><tr><td>Cell Culture Flasks, T75, Tissue Culture-treated, Canted-neck, vented cap, 75 cm&lt;sup&gt;2&lt;/sup&gt; Growth Area, 250 ml</td><td>BD falcon</td><td>353136</td><td></td></tr><tr><td>Conical Tube, polypropylene, 15 ml</td><td>BD falcon</td><td>352095</td><td></td></tr><tr><td>Countess (cell number counter) chamber slides</td><td>GIBCO-Invitrogen</td><td>C10312</td><td></td></tr><tr><td>Cytosine &amp;beta;-D-Arabinofuranoside hydrochloride (Ara-C hydrochloride)</td><td>Sigma-Aldrich</td><td>C6645-100mg</td><td></td></tr><tr><td>D-(+)-Glucose (Dextrose) anhydrous, SigmaUltra, 99.5% (GC)</td><td>Sigma-Aldrich</td><td>G7528-250G</td><td></td></tr><tr><td>Dish, Petri glass 100 x 15 mm</td><td>Pyrex</td><td>3160-101</td><td></td></tr><tr><td>Distilled water</td><td>GIBCO-Invitrogen</td><td>15230-147</td><td></td></tr><tr><td>DNase Type II</td><td>Sigma-Aldrich</td><td>D4527-200KU</td><td>Stock solution is prepared at 1,500 units/20 &amp;mu;l = 75,000 units/ml in distilled water.</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM), high glucose, GlutaMAX, pyruvate</td><td>GIBCO-Invitrogen</td><td>10569-010, 500 ml</td><td></td></tr><tr><td>Fast PES Filter Unit, 250 ml, 50 mm diameter membrane, 0.2 &amp;micro;m Pore Size</td><td>Nalgene</td><td>568-0020</td><td></td></tr><tr><td>Fast PES Filter Unit, 500 ml, 90 mm diameter membrane, 0.2 &amp;micro;m Pore Size</td><td>Nalgene</td><td>569-0020</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>GIBCO-Invitrogen</td><td>26140-079</td><td></td></tr><tr><td>Glass coverslip, 12 mm Round, thickness 0.09&amp;ndash;0.12 mm, No. 0</td><td>Carolina</td><td>633017</td><td></td></tr><tr><td>GlutaMAX-I</td><td>GIBCO-Invitrogen</td><td>35050-061</td><td></td></tr><tr><td>Hanks&#039; Balanced Salts</td><td>Sigma-Aldrich</td><td>H2387-10X</td><td></td></tr><tr><td>HEPES, &amp;ge;99.5% (titration)</td><td>Sigma-Aldrich</td><td>H3375-250G</td><td></td></tr><tr><td>Hydrochloric acid, 37%, A.C.S reagent</td><td>Sigma-Aldrich</td><td>258148-100 ML</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I5500-250 mg</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate, MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;(7H&lt;sub&gt;2&lt;/sub&gt;O), BioUltra, &amp;ge;99.5% (Fluka)</td><td>Sigma-Aldrich</td><td>63138-250G</td><td></td></tr><tr><td>Matrigel Basement Membrane Matrix solution, Phenol Red-Free</td><td>BD Biosciences</td><td>356237</td><td>This is the coating material for coverslips and flasks. 1) To prepare it, thaw the Matrigel Basement Membrane Matrix solution on ice, which usually takes ~1 day. Using a pre-cooled pipette, aliquot the thawed solution into pre-cooled T25 flasks on ice, and store the flasks at -20 &amp;deg;C. To prepare the working Matrigel solution, thaw the aliquotted Matrigel in a flask on ice, dilute 50-fold by adding pre-cooled MEM solution and keep the diluted solution at 4 &amp;deg;C. It is important to pre-cool all cultureware and media that come into contact with Matrigel, except during and after the coating of coverslips, to prevent it from prematurely forming a gel. 2) To coat the glass coverslips or culture flasks with Matrigel, apply the Matrigel solution to the surface. Before plating cells, it is important to completely dry up the surface. For this purpose, it might be helpful to aspirate Matrigel during the cellular centrifugation immediately before plating the cells and to allow enough time for drying.</td></tr><tr><td>Minimum Essential Medium (MEM)</td><td>GIBCO-Invitrogen</td><td>51200-038</td><td></td></tr><tr><td>MITO+ Serum Extender, 5 ml</td><td>BD Biosciences</td><td>355006</td><td></td></tr><tr><td>Multiwell Plates, Tissue Culture-treated 24-well plate</td><td>BD falcon</td><td>353047</td><td></td></tr><tr><td>Multiwell Plates, Tissue Culture-treated 6-well plate</td><td>BD falcon</td><td>353046</td><td></td></tr><tr><td>Neurobasal-A Medium (1X), liquid</td><td>GIBCO-Invitrogen</td><td>10888-022</td><td></td></tr><tr><td>Nitric Acid</td><td>VWR</td><td>bdh 3044</td><td></td></tr><tr><td>NS (Neuronal Supplement) 21&amp;nbsp;</td><td>prepared in the lab</td><td></td><td>Source: reference 69</td></tr><tr><td>Pasteur pipets, 5 &amp;frac34;&amp;rdquo;&amp;nbsp;</td><td>Fisher</td><td>13-678-6A</td><td>Use this cotton-plugged 5 &amp;frac34;&amp;rdquo; Pasteur pipette for cellular trituration. Fire-polish the tip beforehand to smooth the cut surface and to reduce the internal diameter to 50-80% of the original. Too small a tip will disrupt the cells and reduce cell viability, but too large a tip will decrease the efficiency of trituration.</td></tr><tr><td>Pasteur pipets, 9&amp;rdquo;&amp;nbsp;</td><td>Fisher</td><td>13-678-6B</td><td></td></tr><tr><td>Potassium chloride (KCl), SigmaUltra, &amp;ge;99.0%</td><td>Sigma-Aldrich</td><td>P9333-500G</td><td></td></tr><tr><td>Serological pipet, 2 ml</td><td>BD falcon</td><td>357507</td><td></td></tr><tr><td>Serological pipet, 5 ml&amp;nbsp;</td><td>BD falcon</td><td>357543</td><td></td></tr><tr><td>Serological pipet, 10 ml</td><td>BD falcon</td><td>357551</td><td></td></tr><tr><td>Serological pipet, 25 ml</td><td>BD falcon</td><td>357525</td><td></td></tr><tr><td>Serological pipet, 50 ml&amp;nbsp;</td><td>BD falcon</td><td>357550</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO3, Sodium hydrogen carbonate), SigmaUltra, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>S6297-250G</td><td></td></tr><tr><td>Sodium chloride (NaCl), SigmaUltra, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>S7653-250G</td><td></td></tr><tr><td>Sodium hydroxide (NaOH), pellets, 99.998% trace metals basis</td><td>Sigma-Aldrich</td><td>480878-250G</td><td></td></tr><tr><td>Sodium phosphate dibasic heptahydrate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;(7H&lt;sub&gt;2&lt;/sub&gt;O)), &amp;ge;99.99%, Aldrich</td><td>Sigma-Aldrich</td><td>431478-250G</td><td></td></tr><tr><td>Sucrose, SigmaUltra, &amp;ge;99.5% (GC)</td><td>Sigma-Aldrich</td><td>S7903-250G</td><td></td></tr><tr><td>Syringe filter, sterile, 0.2 &amp;micro;m</td><td>Corning</td><td>431219</td><td></td></tr><tr><td>Syringe, 3 ml</td><td>BD falcon</td><td>309585</td><td></td></tr><tr><td>Transferrin, Holo, bovine plasma</td><td>Calbiochem</td><td>616420</td><td></td></tr><tr><td>Trypan Blue stain, 0.4%</td><td>GIBCO-Invitrogen</td><td>T10282</td><td>This is used for counting live/dead cells. Renew an old trypan blue solution if it is re-used many times (&lt;em&gt;e.g.&lt;/em&gt; several times a week for several weeks), because it will form precipitates and result in erroneous readouts of cellular density.</td></tr><tr><td>Trypsin, type XI</td><td>Sigma-Aldrich</td><td>T1005-5G</td><td></td></tr><tr><td>Trypsin-EDTA solution, 0.25%&amp;nbsp;</td><td>GIBCO-Invitrogen</td><td>25200-056</td><td></td></tr><tr><td>Uridine</td><td>Sigma-Aldrich</td><td>U3003-5G</td><td>Stock solution is prepared at 50-mg 5-fluoro-2&#039;-deoxyuridine and 125 mg uridine in 25 ml DMEM (8.12 and 20.48 mM, respectively).</td></tr><tr><td>REAGENTS - immunocytochemistry</td><td></td><td></td><td></td></tr><tr><td>Antibody, rabbit polyclonal anti-MAP2</td><td>Merck Millipore</td><td>AB5622</td><td></td></tr><tr><td>Antibody, mouse monoclonal anti-GFAP cocktail</td><td>Merck Millipore</td><td>NE1015</td><td></td></tr><tr><td>EQUIPMENT - tattooing</td><td></td><td></td><td></td></tr><tr><td>AIMS</td><td>Animal Identification and Marking Systems, Inc.</td><td>NEO&amp;#8211;9</td><td>This Neonate Rodent Tattooing System is an electric system that works by rapidly moving 1- or 3-point tattoo needles vertically into the skin. Activate the tattoo machine once for approximately 0.5 sec, while the tattoo needle tips are kept perpendicular to the skin surface. We prefer three-needle tattooing to maximize the tattooed area, but one-needle tattooing is effective on narrower areas, &lt;em&gt;e.g.&lt;/em&gt; the toes, or if fine mechanical control is necessary, &lt;em&gt;e.g.&lt;/em&gt; when numbers are tattooed. Two rounds of tattooing at the slowest speed (setting &quot;1&quot; out of 3 steps) are typically sufficient to produce a visible and long-lasting tattoo of the paw pads.</td></tr><tr><td>EQUIPMENT - genotyping</td><td></td><td></td><td></td></tr><tr><td>Electrophoresis system, horizontal, Wide Mini&amp;#8211;Sub Cell GT</td><td>BIO&amp;#8211;RAD</td><td>170&amp;#8211;4405</td><td>Typical electrophoresis parameters are electrical field strength at 6 V/cm and 25 min duration for a 10 cm gel.</td></tr><tr><td>FluorChem 8800</td><td>ProteinSimple</td><td>FluorChem 8800</td><td></td></tr><tr><td>PCR, MJ Mini Thermal Cycler</td><td>BIO-RAD</td><td>PTC-1148EDU</td><td>Our PCR reactions for the Tor1a gene in &amp;#916;E-torsinA knock-in mice are as follows: 1 cycle of denaturation at 94 &amp;#176;C for 3 min, 35 cycles of denaturation at 94 &amp;#176;C for 30 sec, annealing at 58 &amp;#176;C for 30 sec, extension at 72 &amp;#176;C for 2 min. This is followed by final extension at 72 &amp;#176;C for 10 min, and holding at 4 &amp;#176;C.</td></tr><tr><td>Power supply, PowerPac Basic</td><td>BIO-RAD</td><td>164-5050</td><td></td></tr><tr><td>EQUIPMENT - cell culture</td><td></td><td></td><td></td></tr><tr><td>Automated cell counter, Countess</td><td>GIBCO-Invitrogen</td><td>C10310</td><td>This automated cell counter separately measures the densities of live and dead cells (non stained and stained by trypan blue, respectively). It is important to know the optimal range of density measurements: the counter that we use has the highest accuracy in the range from 1 x 10&lt;sup&gt;5&lt;/sup&gt; to 4 x 10&lt;sup&gt;6&lt;/sup&gt; cells/ml. If the measured cell density values fall outside the recommended range, adjust the resuspension volume appropriately.</td></tr><tr><td>Biological Safety Cabinet, Class II, Type A2</td><td>NUAIRE</td><td>NU-425-400</td><td>This hood is used for all cell culture procedures, except for brain dissection.</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator, AutoFlow, Humidity Control Water Jacket</td><td>NUAIRE</td><td>NU-4850</td><td></td></tr><tr><td>Horizontal Clean Bench</td><td>NUAIRE</td><td>NU-201-330</td><td>This clean bench is used for brain dissection (steps 3.1.1 and 3.1.2 of &quot;Brain Dissection and Cellular Dissociation)&quot;.</td></tr><tr><td>Orbit LS Low Speed Shaker</td><td>Labnet</td><td>S2030-LS-B</td><td></td></tr><tr><td>SORVALL RC-6 Plus Superspeed Centrifuge</td><td>Fisher</td><td>46910 (centrifuge)/46922 (rotor)</td><td></td></tr></tbody></table>

rapid genotyping of animals followed by establishing primary cultures of brain neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

As an example of the application of this protocol, representative results are shown for labeling mice by tattooing, reliable genotyping under various experimental conditions, and establishing primary neuronal cultures on glial feeder layers.
Tattooing
Newborn pups were labeled on the paw pads using a tattooing system (&#39;Newborn&#39; in Figure 1). The labels remained clearly visible at 3 weeks (&#39;3-week-old&#39;) and 32 weeks o...

Watch this Scientific Journal Video about Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons at JoVE.com

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

rapid-genotyping-animals-followed-establishing-primary-cultures-brain

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

16.2K Views.  University of Iowa Carver College of Medicine. We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

Video: Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

Developmental Biology

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

Genetics

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Embryonic Mouse Brain Mixed Cell Cultures to Study Infection and Innate Immunity

Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods.

Author Spotlight: Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity  

This protocol presents a unique way of generating central nervous system cell cultures from embryonic day 17 mouse brains for neuro(immuno)logy research. This model can be analyzed using various experimental techniques, including RT-qPCR, microscopy, ELISA, and flow cytometry.

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of ...

 Primary Microglia and Neuron Co-Culture Protocol for CNS Interaction Studies

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

Author Spotlight: In Vitro Co-Culture Model for Studying Microglia-Neuronal Interactions in Disease Conditions

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS ...

Primary Neuronal Cultures

The complexity of the brain often requires neuroscientists to use a simpler system for experimental manipulations and observations. One powerful approach is to generate a primary culture by dissecting nervous system tissue, dissociating it into single cells, and growing those cells in vitro. Primary cultures make neurons and glia easily accessible to the experimental tools required for techniques like genetic manipulation and time-lapse imaging. Furthermore, these cultures represent a highly controllable environment in which to study complex phenomena such as cell-cell interactions.
This video provides an overview of the major steps in producing primary neuronal cultures, which include selecting and dissecting the tissue of interest, mechanically and chemically breaking down the tissue to produce a single cell suspension, plating the cells, and maintaining the cultures in the appropriate media. Several example experiments are also presented to show how cultured cells can be used to investigate protein trafficking, morphological changes, and electrophysiology in living neurons.

Primary Neuronal Cultures and Electrophysiology

The complexity of the brain often requires neuroscientists to use a simpler system for experimental manipulations and observations. One powerful approach ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

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Primary Neuronal Cultures