(A) A confocal image of double immunostaining for proinsulin and FG in the PVN of mice i.p. injected with FG. Open arrowheads indicate single proinsulin labeling. Solid arrowheads show colocalization. (B and C) Taken 2 weeks after injection of GFP-expressing lentivirus into the PVN, a confocal image of GFP expression in the PVN (B) and of double immunostaining for C-peptide and GFP in the ME (C). Solid arrowheads show colocalization. (D–G) Confocal images of the PVN and ME from fed and 24-hour fasted mice and qRT-PCR analysis of Ins2 mRNA levels in the microdissected PVN. The PVN was immunostained for proinsulin (D) and the ME for C-peptide (E). Quantification of fluorescence intensity of proinsulin-immunoreactive cells in the PVN and C-peptide–immunoreactive nerve terminals in the ME (F). Relative levels of Ins2 mRNA in the PVN (G). (H–J) Confocal images of double immunostaining for mCRH and C-peptide 48 hours after vehicle or colchicine injection. PVN (H) and ME (I) at low magnification (H). PVN at high magnification (J). (K) Double immunogold labeling showing colocalization of mCRH and C-peptide within the same neurosecretory granules in an axon nerve terminal located in the ME. An arrowhead indicates an mCRH-immunoreactive 12 nm gold particle. An arrow denotes a C-peptide–immunoreactive 30 nm gold particle. Data are shown as mean ± SEM, 2-tailed unpaired Student’s t test. ** P < 0.01, n = 4 mice/group (F), n = 6 mice/group (G). Scale bars: 50 μm (A, B, D, H, and I), 20 μm (C, E, and high-magnification inset images in A), 10 μm (J), 100 nm (K).