FIG. 4.
Localization of leptin receptors by ICC, in situ hybridization (ISH), and in situ TSA hybridization. ICC with antibodies reactive with all leptin receptor isoforms shows the presence of Ob-R proteins in the hypothalamic nuclei (VMH, DMH, and ARN) (c and d). In situ hybridization, using a dATP 35S (a) or 11-dUTP digoxigenin (b and e to h) 3′-end-labeled oligonucleotide designed to hybridize to mRNA coding Ob-Rb (Table 1), demonstrates the presence of these transcripts in hypothalamic areas of the Swiss mouse. Leptin receptor (Ob-Rb) expression is especially evident in the VMH and ARN (a) and in the PVN (b), as indicated by the schematic drawing of a coronal section (i) of the mouse brain at the level shown in panels a, c, and d, where most leptin receptor-positive cells are located. The cells were labeled at the level of the cell bodies (b and e to h); some neuronal processes were also decorated (b; indicated by arrows). The pictures are representative of the features seen in infected and uninfected mice during the acute stage of infection (a to d). The localization of Ob-R proteins matches that of Ob-Rb gene mRNAs. Panels a and d are in opposite orientations (left versus right hemisphere). The dotted lines represent schematic limits of the hypothalamic nuclei. Ob-Rb gene mRNAs were mainly found in neurons (with labeling at the cellular level [insert in panel b]) defined according to morphological criteria, namely, the sizes (larger than 20 μm) and the anatomical locations of labeled cells in precise hypothalamic nuclei (b and e to h), as seen with in situ TSA hybridization (see Materials and Methods). At 10 months p.i., the number of Ob-Rb-expressing cells seems to be lower in the hypothalamus of an obese mouse (e and g) than in that of a nonobese infected mouse (f and h). The bars in panels e to h indicate the medial (M) and dorsal (D) positions of the photographs. Magnification = ×28 (a and d), ×70 (b and c), ×112 (e to h), and ×280 (insert). ME, median eminence; TH, thalamus; HI, hippocampus; DG, dentate gyrus; AM, amygdala; IC, interne capsula; LV, lateral ventricle; Viii, third ventricle.