Immuno-laser capture microdissection of rat brain neurons for real time quantitative PCR.

Laser capture microdissection (LCM) is a technical approach for obtaining microscopic samples as small as individual cells from tissues for molecular analysis. While the principles and details of the operation of LCM instruments, the technical requirements for obtaining identified cells for LCM "picking", all share the common feature of using a laser in combination with a microscope to microdissect and remove cells from tissue slices (or cultured cells) mounted on a glass slide. The use of LCM is becoming widespread in pathology laboratories and is increasingly being used for gene expression studies in cell biology. The approach is particularly powerful when used in conjunction with immunostaining techniques to obtain enriched RNA samples from cells that have been collected by picking and gathering phenotypically similar cells from anatomically complex organs such as the brain. In the present chapter, we describe an approach for combining immunocytochemistry with LCM to obtain RNA for real time quantitative PCR.

Immunocytochemistry and laser capture microdissection for real-time quantitative PCR identify hindbrain neurons activated by interaction between leptin and cholecystokinin.

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.