Fig 1: mHb-selective lesions in mHb:DTA mice. (A) Neurons visualized by Nissl staining in the mHb and lHb of control and mHb:DTA mice. (B) Relative areas of Nissl stained neurons in the mHb and lHb of control and mHb:DTA mice (mice were 7–8 months old, n = 5/group, t-test). (C) Expression pattern of Tac1 (tachykinin 1, substance P) mRNA (red) and ChAT protein (green) in control and mHb:DTA mice at 3–4 months. ChAT localized to the cell bodies of the ventral area of the mHb and fr. Tac1 and ChAT signals were diminished in mHb:DTA mice, suggesting local interaction between subsets of mHb neurons. (D) Levels of acetylcholine in the IPN, ventral forebrain (VF), striatum, and dorsal tegmental nucleus (DTN; mice were 4–5 months old, n = 8/group, t-test). The left y-axis scale relates to the IPN and the right y-axis scale relates to other regions. (E) Quantitative analysis of the IPN area. Areas of the IPN, delineated by strong tyrosine hydroxylase (TH) immunostaining in surrounding tissue in sequential sections, were summed. The left y-axis indicates the volume of the IPN and the right y-axis represents the ratio of cell nuclear volume. The IPN volume was significantly smaller in mHb:DTA mice than in controls, while there were no differences between genotypes in the cell nuclear areas revealed by DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride) signals. (F) Confocal images of TH-positive puncta in the IPN. There were more detectable puncta in mHb:DTA mice. (G) Relative values showing the areas of TH-positive puncta in the IPN of control and mHb:DTA mice, normalized to 1.0 for control mice. TH-positive puncta were significantly larger and higher in number in mHb:DTA mice than in controls, indicating enhanced sprouting of axon terminals from the locus coeruleus (LC; mice were 7–8 months old, n = 3/group, t-test). a.u., arbitrary unit. Data are shown normalized to 1.0 for the control mice. Scale bars indicate 500 μm (A,C) and 75 μm (F). Data represent mean ± standard error of the mean (SEM).