Visualization, direct isolation, and transplantation of midbrain dopaminergic neurons

Abstract

To visualize and isolate live dopamine (DA)-producing neurons in the embryonic ventral mesencephalon, we generated transgenic mice expressing green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase gene promoter. In the transgenic mice, GFP expression was observed in the developing DA neurons containing tyrosine hydroxylase. The outgrowth and cue-dependent guidance of GFP-labeled axons was monitored in vitro with brain culture systems. To isolate DA neurons expressing GFP from brain tissue, cells with GFP fluorescence were sorted by fluorescence-activated cell sorting. More than 60% of the sorted GFP ؉ cells were positive for tyrosine hydroxylase, confirming that the population had been successfully enriched with DA neurons. The sorted GFP ؉ cells were transplanted into a rat model of Parkinson's disease. Some of these cells survived and innervated the host striatum, resulting in a recovery from Parkinsonian behavioral defects. This strategy for isolating an enriched population of DA neurons should be useful for cellular and molecular studies of these neurons and for clinical applications in the treatment of Parkinson's disease.

The migratory behavior of immature enteric neurons

“…The 9kb 5 0 upstream region of the rat TH gene was combined with a GFP coding variant, and the transgene injected into fertilized mouse eggs to produce a TH-GFP transgenic line. GFP expression was confirmed in dopaminergic cells of the brain (Sawamoto et al, 2001). The TH-GFP mice were on a C57BL6J background.…”

“…The generation of the TH-GFP transgenic mice has been described previously (Sawamoto et al, 2001;Matsushita et al, 2002). The 9kb 5 0 upstream region of the rat TH gene was combined with a GFP coding variant, and the transgene injected into fertilized mouse eggs to produce a TH-GFP transgenic line.…”

“…These cells represent approximately 15% of neural crest-derived cells in the gut of E10.5 mice . In the current study, transgenic mice in which GFP is expressed under the control of the TH promoter (Sawamoto et al, 2001;Matsushita et al, 2002) were used to image immature enteric neurons in live tissue. In TH-GFP mice, GFP expression coincides with TH immunoreactivity in the midbrain (Sawamoto et al, 2001); however, GFP expression in the gut of embryonic TH-GFP mice has not previously been characterized.…”

“…In the current study, transgenic mice in which GFP is expressed under the control of the TH promoter (Sawamoto et al, 2001;Matsushita et al, 2002) were used to image immature enteric neurons in live tissue. In TH-GFP mice, GFP expression coincides with TH immunoreactivity in the midbrain (Sawamoto et al, 2001); however, GFP expression in the gut of embryonic TH-GFP mice has not previously been characterized. Wholemount preparations of gut from E10.5 and E11.5 TH-GFP mice were fixed and processed for immunohistochemistry using antisera to the pan-neuronal proteins, neurofilament-M, and neuronal class III b-tubulin (Tuj1).…”

“…As immature enteric neurons transiently express TH, we used transgenic mice that express green fluorescence protein (GFP) under the control of the TH promoter (Sawamoto et al, 2001) to examine the migratory behavior of immature enteric neurons in the embryonic mouse gut. We show that some immature enteric neurons do migrate, but at a slower speed than the entire population of ENCCs.…”