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 border="0" cellpadding="0" cellspacing="0">
	<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 &#8211; the stained skin surface can be confused with the tattooed skin, 2) reducing skin damage during tattooing &#8211; 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 &#181;l of Tissue Lysis Solution A and 180 &#181;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 &#181;l of Tissue Lysis Solution A and 180 &#181;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&#160;</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 &#181;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 &#181;l</td>
			<td>Dot Scientific Inc</td>
			<td>UG104-96RS&#160;</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 &#181;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 &#181;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&#160;</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&#160;</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 &#916;E-torsinA knock-in mice</td>
			<td></td>
			<td></td>
			<td>5&#39;-AGT CTG TGG CTG GCT CTC CC-3&#39; (forward) and 5&#39;-CCT CAG GCT GCT CAC AAC CAC-3&#39; (reverse) (reference 18). These primers were used at a final concentration of 1.0 ng/&#181;l (~0.16 &#181;M) (reference 2).</td>
		</tr>
		<tr>
			<td>Primers for amplifying Tfap2a gene in wild-type mice</td>
			<td></td>
			<td></td>
			<td>5&#39;-GAA AGG TGT AGG CAG AAG TTT GTC AGG GC-3&#39; (forward), 5&#39;-CGT GTG GCT GTT GGG GTT GTT GCT GAG GTA-3&#39; (reverse) for the 498-bp amplicon, 5&#39;-CAC CCT ATC AGG GGA GGA CAA CTT TCG-3&#39; (forward), 5&#39;-AGA CAC TCG GGC TTT GGA GAT CAT TC-3&#39; (reverse) for the 983-bp amplicon, and 5&#39;-CAC CCT ATC AGG GGA GGA CAA CTT TCG-3&#39; (forward), 5&#39;-ACA GTG TAG TAA GGC AAA GCA AGG AG-3&#39; (reverse) for the 1990-bp amplicon. These primers are used at 0.5 &#181;M.</td>
		</tr>
		<tr>
			<td>REAGENTS - cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5-Fluoro-2&#8242;-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 cm2 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 cm2 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 &#946;-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 1500 units/20 &#956;l = 75000 units/ml in distilled water.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;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 &#181;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 &#181;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&#8211;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&#39; Balanced Salts</td>
			<td>Sigma-Aldrich</td>
			<td>H2387-10X</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES, &#8805;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, MgSO4&#8226;(7H2O), BioUltra, &#8805;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&#176;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&#176;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&#160;</td>
			<td>prepared in the lab</td>
			<td></td>
			<td>Source: reference 69</td>
		</tr>
		<tr>
			<td>Pasteur pipets, 5 &#190;&#8221;&#160;</td>
			<td>Fisher</td>
			<td>13-678-6A</td>
			<td>Use this cotton-plugged 5 &#190;&#8221; 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&#8221;&#160;</td>
			<td>Fisher</td>
			<td>13-678-6B</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl), SigmaUltra, &#8805;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&#160;</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&#160;</td>
			<td>BD falcon</td>
			<td>357550</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3, Sodium hydrogen carbonate), SigmaUltra, &#8805;99.5%</td>
			<td>Sigma-Aldrich</td>
			<td>S6297-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl), SigmaUltra, &#8805;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 (Na2HPO4&#8226;(7H2O)), &#8805;99.99%, Aldrich</td>
			<td>Sigma-Aldrich</td>
			<td>431478-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose, SigmaUltra, &#8805;99.5% (GC)</td>
			<td>Sigma-Aldrich</td>
			<td>S7903-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter, sterile, 0.2 &#181;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 (e.g. 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%&#160;</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&#39;-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>
	</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

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 ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

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 ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...