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

Summary

In Parkinson'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.

Abstract

In Parkinson'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 "tagmentation" to produce single cell RNA-Seq libraries^1,2,3,4,5. 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.

Introduction

Parkinson'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's disease. Ventral Tegmental Area (VTA) dopaminergic neurons have been implicated in reward and addiction. Ventral Mesencephalic (VM) primary mixed embryonic cult....

Protocol

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

1. Solutions and Culture Medium
   1. Preparation of Ascorbic Acid Stock Solution
      1. Weigh out 352 mg of ascorbic acid. Add sterile H₂O to a final total volume of 20 mL. Place in 37 °C water bath to dissolve. Filter through 0.2 µm syringe tip and st....

Results

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.......

Discussion

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 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Papain	Worthington Biochemical Corporation	LS003126
Hanks-balanced Salt Solution (HBSS), 1x	Gibco	14175-095
Donor Equine Serum	Thermo Scientific	SH30074.03
Bovine Serum Albumin (BSA)	Sigma	A7030-50G
Medium: Neurobasal medium	Gibco	21103-049
Culture media that contains a stabilized form of L-glutamine, L-alanyl-L-glutamine: GlutaMAX	Gibco	35050-061	0.5 mM final concentration.
L-Ascorbic acid	Sigma	A7506
B27	Gibco	17504-044	50x stock solution, 1x final concentration.
35 mm glass bottom dishes	MatTek	P35GC-1.5-10-C
Poly-L-Ornithine	Sigma	P4957
Laminin	Sigma	L2020	Stored at -20 °C in 20 µL aliquots
Deoxyribonuclease I (DNase)	Sigma	DN25
Blue pipette tips P1000	Sorenson Bioscience	Inc. 10130
10 mm shallow Petri dish	VWR	25384-324
37 °C Water bath
37 °C, 5% humidity incubator
16% paraformaldehyde (PFA)	Electron Microscopy Sciences	15710
Triton X-100	Sigma	X100-500ML
Donkey serum	Equitech	SD-0100HI	100% stock solution, 1% final concentration.
Standard Pattern surgical scissors	Fine Science Tools	14000-14
Forceps - 2x3 teeth	Fine Science Tools	11022-14
Forceps - Dumont #55	Fine Science Tools	11295-51
Forceps - Dumont #5	Fine Science Tools	11252-23
10x PBS, pH 7.4	Gibco	70011-044
Dulbecco's Phosphate-buffered solution (DPBS) 1x	Gibco	14190-144	500 mL
21 G needle	BD PrecisionGlide Needle	305167	100 needles
Micropipette puller	Sutter Instrument	model P-87
Micromanipulator	Sutter Instrument	MP-285
Sequencing Kit: SMARTer Ultra Low RNA Kit for Illumina Sequencing	Clontech	634936	100 rnxs,incl Advantage2PCR Kit
oligonucleotide (12 µM): SMARTer IIA oligonucleotide (12 µM)			From the SMARTer Kit
Reverse transcriptase (100 units)	SMARTcribe reverse transcriptase		From the SMARTer Kit
Primer 1	3’ CDSIIa primer		From the SMARTer Kit
Primer 2	IS PCR primer		From the SMARTer Kit
50x 2 polymerase mix	50x Advantage 2 polymerase mix		From the SMARTer Kit
10x PCR buffer	10x Advantage 2 PCR buffer		From the SMARTer Kit
RNA Spikes	ThermoFisher	4456740
PCR cooler rack	IsoFreeze PCR cooler rack
DNA Sample Preparation Kit	(Illumina/Nextera) Nexter	FC-121-1030	24 Samples
PCR master mix	Nextera PCR master mix (NPM)		From the Nextera Kit
PCR primer cocktail (PPC)	Nextera PCR primer cocktail (PPC)		From the Nextera Kit
Fluorometer	The Qubit ThermoFisher	Q33216
HS DNA BioAnalyzer kit	Agilent	5067-4626	110 rxns
Electrophoresis system	Agilent 2100 Bioanalyzer.	G2938C
Magnetic beads: Agencourt AMPure	Beckman Coulter	A63880	5 mL
RNAse wipe: RNAse Zap wipes	Ambion	AM9786	Size 100 Sheets
Qubit ds HS Assay Kit	Molecular probes	Q32854	500 assays
Gel extraction kit: Qiaquick Gel Extraction Kit	Qiagen	28704
Glass capillary tubing	(Kimax-51, 1.5 - 1.8 mm o.d.)
Microforge	Narishige (or equivalent)
Sequencer	Illumina HiSeq instrument
GENSAT tyrosine hydroxylase-eGFP mouse strain		MMRRC stock number: 000292-UNC	Stock Tg(Th-EGFP)DJ76Gsat/Mutant Mouse Regional Research Center