GFP is expressed selectively in NPY neurons. GFP is expressed selectively in NPY neurons. A, Micrographs of coronal sections from the arcuate nucleus (Arc; A–C). GFP expression in the NPY-Renilla GFP mice is green, and immunostaining for NPY is red. C shows the merged image; green GFP- positive cells also show red NPY immunofluorescence. D–F, In contrast, immunoreactive α-MSH (red) is not colocalized in green neurons expressing GFP. Scale bars: A, 15 μm; D, 20 μm. G, Single-cell RT-PCR was used to test for NPY mRNA expression in GFP- positive cells from the arcuate nucleus and GFP-negative neurons from the ventromedial nucleus (VMH). All GFP-expressing neurons also had NPY mRNA message. In contrast, GFP-negative cells harvested from the VMH showed no NPY mRNA, but were positive for β-actin used as a control. A final lane used water as an additional control and was negative, as expected. H, In additional experiments, three GFP-positive arcuate neurons from the NPY-GFP mouse (lanes 1–3) were compared with GFP-negative neurons from the arcuate nucleus from the same mouse (lanes 4–7) and GFP-positive cells from a different transgenic mouse in which the POMC neurons expressed GFP (lanes 8–13). A final lane served as a water control. Only the three GFP-positive neurons from the NPY-GFP mouse expressed NPY mRNA, whereas the other control cells were all negative. I, Four GFP-positive cells from the arcuate nucleus were tested for NPY and AgRP. All four showed mRNA coding for NPY and AgRP, further substantiating the view that the green cells were the NPY neurons. Anthony N. van den Pol et al. J. Neurosci. 2009;29:4622-4639 ©2009 by Society for Neuroscience