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

Diana L. Williams ^1*, Michael W. Schwartz ¹, L. Scot Bastian ¹, James E. Blevins ¹ and Denis G. Baskin ¹

¹ 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, VA Puget Sound Health Care System, Seattle, Washington (LSB,JEB,DGB)

^* To whom correspondence should be addressed. E-mail: dianalw{at}u.washington.edu .

Submitted on August 1, 2007
Accepted on 13 November 2007

	Abstract

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 leptin nor CCK alone affected the relative amount of c-Fos 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.

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

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