Name: reliable identification of living dopaminergic neurons in midbrain cultures using rna sequencing and th-promoter-driven egfp expression
Uploaded: 2017-02-10T12:30:00
Description: Watch this Scientific Journal Video about Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression at JoVE.com

This work was supported by grants from the U.S. National Institutes of Health (DA017279, AG033954, DA037743), the Michael J. Fox Foundation, and the Caltech Innovation Initiative funding the Millard and Muriel Jacobs Genetics and Genomics Laboratory at the California Institute of Technology. We thank Brian Williams for optimising the RNA-Seq library protocol and for providing assistance. We thank Henry Amrhein for computational training. We thank Barbara J. Wold for the use of her equipment and laboratory space. We thank Igor Antoshechkin for library sequencing, and for facility management.

NOTE: All experiments were conducted in accordance with the guidelines for care and use of animals provided by the National Institutes of Health, and protocols were approved by the Institutional Animal Care and Use Committee at the California Institute of Technology.
1. Derivation of Primary Dopaminergic Cell Cultures from Embryonic Mouse Brain
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	<li>Solutions and Culture Medium
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		<li>Preparation of Ascorbic Acid Stock Solution
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			<li>Weigh out 352&#160;mg of ascorbic acid. ...

<ol>
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Here we isolate single cells from a heterogeneous population using a fluorescent tag, then study each cell with single-cell RNA-seq. We report that 100% of the live TH-eGFP cells that we harvested and sequenced were indeed dopaminergic neurons, based on the presence of the following three DA-related gene transcripts, TH, DDC and&#160;slc6a3. All of the TH-eGFP positive cells expressed these three genes as assessed by RNA-Seq. This was concordant with electrophysiological studies that showed that each TH-eGFP cell also di...

Parkinson&#39;s Disease (PD) is an incurable, age-related neurodegenerative disorder. The cause of this relatively common disorder remains poorly understood. There is widespread neuronal loss throughout the brain, with pronounced neuronal degeneration of dopaminergic neurons in the Substantia Nigra (SNc), leading to diagnostic clinical features of bradykinesia, rigidity and tremor.
Primary mixed cultures containing SNc dopaminergic neurons are especially relevant for Parkinson&#39;s disease. Ventral Tegmental Area (VTA) dopaminergic neurons have been implicated in reward and addiction. Ventral Mesencephalic (VM) primary mixed embryonic cultures contain both SNc and VTA dopaminergic (DA) neurons and GABAergic neurons. VM primary cultures can be useful for neuroprotection assays and to elucidate the selective vulnerability of dopaminergic neurons. There is no reliable way to identify dopaminergic cells in culture based on morphology. Here we develop techniques to identify and harvest single dopaminergic neurons, and construct single-cell, high-yield RNA sequencing libraries.
We report representative RNA transcriptome data for single dopaminergic and GABAergic neurons isolated from midbrain cultures. This protocol can be used for neuroprotection, neurodegeneration, and pharmacological assays to study the effects of various treatments on the DA/GABA transcriptome. Because dopaminergic neurons represent a small minority of the neurons expressed in primary VM cultures, the ability to reliably identify these neurons in living cultures will enable an enhanced range of single-cell studies. These novel techniques will facilitate advances in understanding the mechanisms taking place at the cellular level and may have applications elsewhere in the fields of neuroscience and molecular biology.

<table border="0" cellpadding="0" cellspacing="0" width="921">
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			<td height="40" style="height:40px;width:395px;">Papain</td>
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			<td height="20" style="height:20px;width:395px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:241px;">Sigma&#160;</td>
			<td style="width:108px;">A7030-50G</td>
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			<td height="20" style="height:20px;width:395px;">Medium: Neurobasal medium</td>
			<td style="width:241px;">Gibco&#160;</td>
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			<td height="40" style="height:40px;width:395px;">Culture media that contains a stabilized form of L-glutamine, L-alanyl-L-glutamine: GlutaMAX</td>
			<td style="width:241px;">Gibco&#160;</td>
			<td style="width:108px;">35050-061</td>
			<td style="width:177px;">0.5 mM final concentration.</td>
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			<td height="20" style="height:20px;width:395px;">L-Ascorbic acid</td>
			<td style="width:241px;">Sigma&#160;</td>
			<td style="width:108px;">A7506</td>
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			<td height="40" style="height:40px;width:395px;">B27</td>
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			<td style="width:241px;">Sigma&#160;</td>
			<td style="width:108px;">P4957</td>
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			<td height="40" style="height:40px;width:395px;">Laminin</td>
			<td style="width:241px;">Sigma&#160;</td>
			<td style="width:108px;">L2020</td>
			<td style="width:177px;">Stored at -20&#176;C in 20 &#181;L aliquots</td>
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			<td height="20" style="height:20px;width:395px;">Deoxyribonuclease I (DNase)</td>
			<td style="width:241px;">Sigma&#160;</td>
			<td style="width:108px;">DN25</td>
			<td style="width:177px;"></td>
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			<td height="20" style="height:20px;width:395px;">Blue pipette tips</td>
			<td style="width:241px;">Sorenson Bioscience,</td>
			<td style="width:108px;">&#160;Inc. 10130</td>
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			<td height="20" style="height:20px;width:395px;">P1000</td>
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			<td height="20" style="height:20px;width:395px;">10 mm shallow Petri dish</td>
			<td style="width:241px;">VWR&#160;</td>
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			<td height="20" style="height:20px;width:395px;">37&#176;C Water bath</td>
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			<td height="20" style="height:20px;width:395px;">37&#176;C, 5% humidity incubator</td>
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			<td height="20" style="height:20px;width:395px;">16% paraformaldehyde (PFA)</td>
			<td style="width:241px;">Electron Microscopy Sciences&#160;</td>
			<td style="width:108px;">15710</td>
			<td style="width:177px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Triton X-100</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:108px;">&#160;X100-500ML</td>
			<td style="width:177px;"></td>
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			<td height="40" style="height:40px;width:395px;">Donkey serum</td>
			<td style="width:241px;">Equitech&#160;</td>
			<td style="width:108px;">SD-0100HI</td>
			<td style="width:177px;">100% stock solution, 1% final concentration.</td>
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			<td height="21" style="height:21px;width:395px;">Standard Pattern surgical scissors</td>
			<td style="width:241px;">Fine Science Tools</td>
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			<td style="width:177px;"></td>
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			<td height="20" style="height:20px;width:395px;">10X PBS, pH 7.4</td>
			<td style="width:241px;">Gibco&#160;</td>
			<td style="width:108px;">70011-044</td>
			<td style="width:177px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Dulbecco&#39;s Phosphate Buffered solution (DPBS) 1X</td>
			<td style="width:241px;">Gibco</td>
			<td style="width:108px;">14190-144</td>
			<td style="width:177px;">500 ml&#160;</td>
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			<td height="20" style="height:20px;width:395px;">21 guage needle&#160;</td>
			<td style="width:241px;">BD PrecisionGlide Needle</td>
			<td style="width:108px;">305167</td>
			<td style="width:177px;">100 needles</td>
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			<td height="20" style="height:20px;width:395px;">Micropipette puller</td>
			<td style="width:241px;">Sutter Instrument.</td>
			<td style="width:108px;">model P-87</td>
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			<td height="20" style="height:20px;width:395px;">Micromanipulator&#160;</td>
			<td style="width:241px;">Sutter Instrument</td>
			<td style="width:108px;">&#160;MP-285</td>
			<td style="width:177px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:395px;">Sequencing Kit: SMARTer Ultra Low RNA Kit for Illumina Sequencing</td>
			<td style="width:241px;">Clontech</td>
			<td style="width:108px;">634936</td>
			<td style="width:177px;">100rnxs,incl Advantage2PCR Kit</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:395px;">oligonucleotide (12 &#181;M): SMARTer IIA oligonucleotide (12 &#181;M)</td>
			<td style="width:241px;"></td>
			<td style="width:108px;"></td>
			<td style="width:177px;">From the SMARTer Kit&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Reverse transcriptase (100 units)</td>
			<td style="width:241px;">SMARTcribe reverse transcriptase</td>
			<td style="width:108px;"></td>
			<td style="width:177px;">From the SMARTer Kit&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Primer 1</td>
			<td style="width:241px;">3&#8217; CDSIIa primer&#160;</td>
			<td style="width:108px;"></td>
			<td style="width:177px;">From the SMARTer Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Primer 2</td>
			<td style="width:241px;">IS PCR primer</td>
			<td style="width:108px;"></td>
			<td style="width:177px;">From the SMARTer Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">50X 2 polymerase mix</td>
			<td style="width:241px;">50X Advantage 2 polymerase mix</td>
			<td style="width:108px;"></td>
			<td style="width:177px;">From the SMARTer Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">10X PCR buffer</td>
			<td style="width:241px;">10X Advantage 2 PCR buffer</td>
			<td style="width:108px;"></td>
			<td style="width:177px;">From the SMARTer Kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">RNA Spikes</td>
			<td style="width:241px;">ThermoFisher</td>
			<td style="width:108px;">4456740</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">PCR cooler rack</td>
			<td style="width:241px;">IsoFreeze PCR cooler rack.</td>
			<td style="width:108px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">DNA Sample Preparation Kit&#160;</td>
			<td style="width:241px;">(Illumina/Nextera) Nexter</td>
			<td style="width:108px;">FC-121-1030</td>
			<td style="width:177px;">24 Samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">PCR master mix</td>
			<td style="width:241px;">Nextera PCR master mix (NPM)</td>
			<td style="width:108px;"></td>
			<td style="width:177px;">From the Nextera Kit&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">PCR primer cocktail (PPC)&#160;</td>
			<td style="width:241px;">Nextera PCR primer cocktail (PPC)&#160;</td>
			<td style="width:108px;"></td>
			<td style="width:177px;">From the Nextera Kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Fluorometer</td>
			<td style="width:241px;">The Qubit ThermoFisher</td>
			<td style="width:108px;">Q33216</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">HS DNA BioAnalyzer kit</td>
			<td style="width:241px;">Agilent</td>
			<td style="width:108px;">5067-4626</td>
			<td style="width:177px;">110rxns</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Electrophoresis system&#160;</td>
			<td style="width:241px;">Agilent 2100 Bioanalyzer.</td>
			<td style="width:108px;">G2938C</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Magnetic beads: Agencourt AMPure&#160;</td>
			<td style="width:241px;">Beckman Coulter</td>
			<td style="width:108px;">A63880</td>
			<td style="width:177px;">5ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">RNAse wipe: RNAse Zap wipes</td>
			<td style="width:241px;">Ambion</td>
			<td style="width:108px;">AM9786</td>
			<td style="width:177px;">Size 100 Sheets</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Qubit ds HS Assay Kit</td>
			<td style="width:241px;">Molecular probes&#160;</td>
			<td style="width:108px;">Q32854</td>
			<td style="width:177px;">500 assays</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Gel extraction kit: Qiaquick Gel Extraction Kit</td>
			<td style="width:241px;">Qiagen&#160;</td>
			<td style="width:108px;">28704</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Glass capillary tubing</td>
			<td style="width:241px;">(Kimax-51, 1.5&#8211;1.8 mm o.d.)</td>
			<td style="width:108px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Microforge</td>
			<td style="width:241px;">Narishige (or equivalent)&#160;</td>
			<td style="width:108px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Sequencer</td>
			<td style="width:241px;">Illumina HiSeq instrument</td>
			<td style="width:108px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:395px;">GENSAT tyrosine hydroxylase-eGFP mouse strain&#160;</td>
			<td style="width:241px;"></td>
			<td style="width:108px;">MMRRC stock number: 000292-UNC</td>
			<td style="width:177px;">Stock Tg(Th-EGFP)DJ76Gsat/Mutant Mouse Regional Research Center</td>
		</tr>
	</tbody>
</table>

reliable identification of living dopaminergic neurons in midbrain cultures using rna sequencing and th-promoter-driven egfp expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a tyrosine hydroxylase promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their RNA transcriptome using RNA-seq (Figure 1). The data showed that 100% of the TH-eGFP-positive cells that were harvested and sequenced were dopaminergic neurons, based on the presence of the following three DA-related gene transcripts, TH, dopa decar...

Watch this Scientific Journal Video about Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression at JoVE.com

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

The overall goal of this procedure is to reliably identify living dopaminergic neurons in midbrain cultures using tyrosine hydroxlase driven eGFP expression, single cell harvesting, and RNA sequencing library generation. This method can help answer key questions in the Parkinson's disease and drug addiction fields, because it makes it possible to perform gene expression and electrophysiological assays on identified living dopaminergic neurons in culture. The advantage of this technique is that it provides a means to reliably identify living rather than fixed dopaminergic neurons in primary ventral mesencephalic cultures.Demonstrating the first part of the procedure will be Charlene Kim, a technician from Henry Lester's laboratory. Begin by stabilizing the head of a mouse embryo with forceps firmly placed on the snout, so that the dorsal surface is accessible. Using forceps held in the other hand pinch the layer of skin and the skull just before the ridge of the mesencephalon and peel back the skin and the skull caudelly along the midline.Place the forceps around the ridge with one tip between the cortex and mesoncephalon and the other over the cerebellum. Pinch and remove the entire midbrain. Place the midbrain in a Petri dish with cold HBSS under a dissecting microscope.Under the dissecting microscope, flip the brain segment so that the ventral side is now accessible. There are now four quadrants visible. Remove all of the meninges and vasculature by gently grasping the meninges with forceps and pulling upward away from the brain.Next, place one tong of forceps into the ventricle approximately at the center of the segment. Then to separate the midbrain, make a pinch dorsally, and then pinch medially on each side. Take the lower two quadrants by making laterally cuts on both sides.The ventral tegmental area and the substantia nigra pars compacta are in the ventral inferior section, which appears dense. Place the dissected tissue containing both the VTA and SNC in fresh HBSS in a separate area of the Petri dish. After dissecting all the brain segments, use forceps and a number 11 scalpel to quarter each midbrain section into pieces of approximately equal size.Then, use a wide bore P1000 pipette tip to transfer all of the quartered midbrain segments, to a 15 milliliter conical tube. After allowing the tissue to settle and removing the HBSS add 500 microliters of papain solution. Incubate in a 37 degree Celsius water bath for 15 minutes.Following the incubation, use a wide bore P1000 tip to transfer only the midbrain segments into a one milliliter aliquot of DNase I solution and allow the segments to settle to the bottom of the tube. Then, transfer only the midbrain segments to a 15 milliliter tube containing one milliliter of cold stop solution. Replace the second rinse with one milliliter of fresh stop solution and pipette up and down seven times with a P1000 pipette tip to triturate the cells.Then underlay the cell suspension by slowly pipetting 200 microliters of 4%BSA solution into the bottom of the tube. After centrifugation at 280 x g for six minutes, aspirate the supernatant with a P1000 and resuspend the cells in one milliliter of plating medium. Perform a cell count using hemocytometer and then dilute the cell suspension to 1000 cells per microliter with plating medium.Plate 120 microliters of the cell suspension onto poly-D-lysine, poly-L-ornithine, and laminin coated culture dishes immediately after aspiration of laminin, to ensure that the coating does not dry and form an uneven surface. Then, after one hour of incubation, carefully add three milliliters of culture medium to an unseeded area of the culture dish to minimize disruption of cells. After three weeks of culture, rinse the culture dish with two milliliters RNase-free Dulbecco's PBS.Remove the DPBS. Then replace with one milliliter of fresh DPBS for harvesting. Use the GFP filter set and 40X objective on an inverted epifluoresence microscope to identify fluorescent TH positive neurons in the cultures.Use the micromanipulator to the pipette over the cell soma. Then use plastic tubing attached to the side port of the micropipette holder to apply gentle suction in order to aspirate the neuron into the glass micropipette. Immediately remove the micropipette containing the cell from the bathing solution and place the tip inside a 0.2 millilter PCR tube collection containing reaction buffer.Break the tip against the side of the tube near the bottom. Then place a sterile 21 gauge syringe needle in the back of the micorpipette and apply pressure to expel the remaining fluid from the broken tip of the micropipette. Centrifuge the collection tube for five seconds using a desktop microcentrifuge and then freeze it in dry ice.While working in the PCR cabinet with reagents on ice, or on a chiller block, prepare the first strain master mix plus 10%additional volume at room temperature. And one microliter of three prime primer one and one microliter of quantified RNA Spikes to the sample. Then incubate the sample in the thermocycler for three minutes at 72 degrees Celsius and then put the samples directly onto the PCR cooler rack.Next, add 5.5 microliters of the prepared master mix to the reaction tube in a PCR cooler rack and mix by pipetting gently. After centrifuging the tube for five seconds incubate in a thermocycler set to the parameters now shown on screen. To purify the first-strand cDNA add 25 microliters of vortexed room temperature magnetic beads to the sample.Mix by pipetting the entire volume up and down at least 10 times. Then incubate at room temperature for eight minutes to allow the cDNA to bind to the beads. Then place the samples and magnetic beads on the magnetic separation device until the liquid appears completely clear and there are no beads left in the supernatant.Aspirate and discard the supernatant. Spin the sample for five seconds to collect the liquid from the side of the tube. Place the samples on the magnetic separation device for 30 seconds.Then remove all the remaining supernatant with the P10 pipetter to leave just the beads with bound DNA. Add 50 microliters of the ds-cDNA PCR master mix and then run the second strand synthesis PCR program to obtain the double stranded cDNA. This histogram shows the number of protein coding genes detected in single cell dopaminergic RNA sequencing libraries generated from TH-eGFP positive cells in fragments per kilobase of transcript per million mapped reads.6, 000 to 8, 000 protein genes were detected in the single cell libraries. Once mastered this technique can be done in four to five weeks, including time to derive the cells, allow for the maturation of the cells in culture, the cell harvesting and the library generation steps, the sequencing of the libraries, and the preliminary analysis. While attempting this procedure, it's important to remember to keep an RNAse-free workspace for the RNA seek library generation and cell harvesting, to maintain sterile techniques through the cell culturing and to make pipettes with appropriately sized diameter for the single cell harvesting.Don't forget that working with paraformaldehyde can be extremely hazardous and precautions such as using a fume hood, avoiding skin and eye contact, keeping away from heat and open flames, and wearing the appropriate personal protective clothing should always be taken. Following this procedure, other methods like gene expression and gene network analysis can be performed in order to answer additional questions such as how drug treatments effect protein expression in dopaminergic cells.

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Research

JoVE Journal

Neuroscience

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

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 ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

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 ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...