Figure 3: Mecamylamine-precipitated withdrawal activates neurons within the IPI of the IPN through a CRF1 receptor-dependent mechanism.

(a) Photomicrographs from coronal sections immunolabeled for c-Fos (green). Sections are taken from control or chronic nicotine-treated mice given i.p. injections of saline, mecamylamine (Mec, 3 mg kg⁻¹) or antalarmin (Ant, 10 mg kg⁻¹) before mecamylamine as indicated in the left-hand column. IPN sub-regions are indicated by dotted lines. (b) Each bar graph represents the average total number of c-Fos(+) IPI neurons after a mecamylamine i.p. injection or an injection of antalarmin 15 min before mecamylamine in control-treated (n=4 mice per treatment, 16 slices per mouse) or chronic nicotine-treated mice (n=3 and 5 mice per treatment, 16 slices per mouse). (c) Photomicrograph from representative coronal sections after FISH with a control (left) or CRF1 receptor probe. Note a robust CRF1 receptor signal is predominantly localized to the IPI sub-region of the IPN. (d) Photomicrograph of coronal sections from chronic nicotine-treated mice given a CRF infusion (300 ng) into the IPN. Sections are double-labelled for CRF1 receptor mRNA (using FISH, red) and c-Fos protein (using immunohistochemistry, green). Merged sections imaged at 10 × magnification are shown (middle). Scale bar: 200 μm. Outside sections shown at 63 × magnification illustrate localization of c-Fos in CRF1 receptor-expressing IPI neurons. Scale bar: 20 μm. Data are expressed as mean±s.e.m. Two-way ANOVA, Bonferroni post hoc test. ***P<0.001 compared with saline preinjection within groups. ^^P<0.01 chronic nicotine-treated compared with control-treated mice.