title: Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression.
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 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.
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology (Caltech); bhenley@caltech.edu.
authors.forename: Beverley M
authors.initials: BM
authors.lastname: Henley
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology (Caltech).
authors.forename: Bruce N
authors.initials: BN
authors.lastname: Cohen
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology (Caltech).
authors.forename: Charlene H
authors.initials: CH
authors.lastname: Kim
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology (Caltech).
authors.forename: Heather D
authors.initials: HD
authors.lastname: Gold
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology (Caltech).
authors.forename: Rahul
authors.initials: R
authors.lastname: Srinivasan
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology (Caltech).
authors.forename: Sheri
authors.initials: S
authors.lastname: McKinney
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology (Caltech).
authors.forename: Purnima
authors.initials: P
authors.lastname: Deshpande
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology (Caltech).
authors.forename: Henry A
authors.initials: HA
authors.lastname: Lester
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004298:Dopamine / Q000378:metabolism; D059290:Dopaminergic Neurons / Q000166:cytology* / Q000378:metabolism; D059330:GABAergic Neurons / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics*; D008636:Mesencephalon / Q000166:cytology*; D051379:Mice; D009474:Neurons / Q000502:physiology; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine