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.
Highlights
The midbrain dopamine system, comprising the nigrostriatal, mesocortical, and mesolimbic pathways, is involved in many brain functions, such as motor control, cognition, and reward behaviors [1]
RNA was extracted from dopamine neuron-containing, whole midbrain sections located on the same microscope slide as sections used for dopamine neuron microdissection
Consistent with inter- and intranuclei differences, we found tyrosine hydroxylase (TH) and NURR1 expression to be significantly higher in ventral tegmental area (VTA) relative to substantia nigra (SN) dopamine neurons but no differences in dopamine transporter (DAT) or vesicular monoamine transporter type 2 (Vmat2) expression levels (Figure 3)
Summary
The midbrain dopamine system, comprising the nigrostriatal, mesocortical, and mesolimbic pathways, is involved in many brain functions, such as motor control, cognition, and reward behaviors [1]. Dysfunction or degeneration of midbrain dopamine neurons is associated with a number of neurological conditions including Parkinson’s disease (PD), cognitive impairment, and addiction. One way to probe the mechanisms associated with both normal and abnormal functions of the dopamine system is to characterize the molecular profile of dopamine neurons themselves. Dopamine neurons have been microdissected, and gene expression profiles have been compared between ventral tegmental area (VTA) subpopulations [2]. While various neuronal identification and laser microdissection methods have been employed to segregate dopamine neurons from surrounding brain tissue, the critical issue of sample purity has not been adequately addressed
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