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Immunocytochemistry and Laser Capture Microdissection for Real-time Quantitative PCR Identify Hindbrain Neurons Activated by Interaction Between Leptin and Cholecystokinin

Diana L. Williams, Michael W. Schwartz, L. Scot Bastian, James E. Blevins and Denis G. Baskin

Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine (DLW,MWS,JEB,DGB) and Department of Biological Structure (DGB), University of Washington, Seattle, Washington, and Medical Research Service, Department of Veterans Affairs Puget Sound Health Care System, Seattle, Washington (LSB,JEB,DGB)

Correspondence to: Diana L. Williams, University of Washington, Harborview Medical Center, 325 9th Avenue, Box 359675, Seattle, WA 98104. E-mail: dianalw{at}u.washington.edu

Current evidence suggests that leptin reduces food intake in part by enhancing the hindbrain neuronal response to meal-related gastrointestinal signals, including cholecystokinin (CCK), but the phenotypes of the relevant cells are not known. To identify neurons that participate in this interaction in the rat nucleus of the solitary tract (NTS), we induced c-Fos gene expression in NTS neurons with leptin and CCK. We focused on NTS catecholamine neurons because these cells have been implicated in the feeding response to CCK. Hindbrain sections from rats that received CCK with or without leptin pretreatment were immunostained for c-Fos and tyrosine hydroxylase (TH) by a double immunofluorescence procedure. Leptin pretreatment increased the number of NTS cells expressing c-Fos-like immunoreactivity (cFLI) 3-fold relative to CCK alone, but the number of TH-positive cells with cFLI was increased 6-fold. Next, cells detected by immunofluorescence for TH were collected by laser capture microdissection and pooled for real-time quantitative PCR of c-Fos mRNA. Here, neither le0ptin nor CCK alone affected the relative amount of mRNA in the TH cell–enriched samples, but leptin plus CCK substantially increased c-Fos mRNA content. These histochemical findings identify hindbrain catecholamine cells as potential mediators of the interaction between leptin and CCK. (J Histochem Cytochem 56:285–293, 2008)

Key Words: leptin • cholecystokinin • catecholamine neurons • food intake • satiety • c-Fos • energy homeostasis

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