title: Purity and enrichment of laser-microdissected midbrain dopamine neurons.
abstract: The ability to microdissect individual cells from the nervous system has enormous potential, as it can allow for the study of gene expression in phenotypically identified cells. However, if the resultant gene expression profiles are to be accurately ascribed, it is necessary to determine the extent of contamination by nontarget cells in the microdissected sample. Here, we show that midbrain dopamine neurons can be laser-microdissected to a high degree of enrichment and purity. The average enrichment for tyrosine hydroxylase (TH) gene expression in the microdissected sample relative to midbrain sections was approximately 200-fold. For the dopamine transporter (DAT) and the vesicular monoamine transporter type 2 (Vmat2), average enrichments were approximately 100- and 60-fold, respectively. Glutamic acid decarboxylase (Gad65) expression, a marker for GABAergic neurons, was several hundredfold lower than dopamine neuron-specific genes. Glial cell and glutamatergic neuron gene expression were not detected in microdissected samples. Additionally, SN and VTA dopamine neurons had significantly different expression levels of dopamine neuron-specific genes, which likely reflects functional differences between the two cell groups. This study demonstrates that it is possible to laser-microdissect dopamine neurons to a high degree of cell purity. Therefore gene expression profiles can be precisely attributed to the targeted microdissected cells.
authors.affiliation: Neurobiology of Ageing Laboratory, School of Biomedical Sciences and Pharmacy, Centre for Translational Neuroscience and Mental Health Research, University of Newcastle, Newcastle, NSW 2308, Australia.
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Brown
authors.forename: Trevor A
authors.initials: TA
authors.lastname: Day
authors.forename: Christopher V
authors.initials: CV
authors.lastname: Dayas
authors.forename: Doug W
authors.initials: DW
authors.lastname: Smith
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D059290:Dopaminergic Neurons / Q000166:cytology* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D007834:Lasers; D008636:Mesencephalon / Q000166:cytology*; D042282:Microdissection; D016133:Polymerase Chain Reaction; D013378:Substantia Nigra / Q000166:cytology; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism; D017557:Ventral Tegmental Area / Q000166:cytology; D050493:Vesicular Monoamine Transport Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; D014446:Tyrosine 3-Monooxygenase