( (Anthony Lombardino Rockefeller U...)Laser Capture Microdissection (LCM) and Expression Profiling of Long-Range Projection Neurons

(Anthony Lombardino, Rockefeller University)

Abstract
Brain tissue is notoriously heterogeneous, and accurate analysis of neural gene expression requires procurement of specific cell types. In this application note a technique is described for using Laser Capture Microdissection (LCM) and in-vivo labeling to obtain purified populations of projection neurons from the discrete brain region HVC of the songbird Taeniopygia guttata. HVC contains two types of projection neurons connecting to two distant brain regions, the Robust nucleus of the Archistriatum (nucleus RA) and Area X of the paraolfactory lobe. The cell bodies of these HVC projection neurons sit at the apex of a neural circuit controlling the learning and production of birdsong, offering insight into how a defined neural circuit relates to a learned behavior (Nottebohm et al., 1982). Despite this complex neuronal architecture, in-vivo labeling enabled the laser capture of two distinct interspersed cell types. Differential gene expression between these cell types was then assessed by directly comparing the amplified RNA obtained from these neurons on cDNA microarrays. Genes over-expressed in each cell type were identified on the microarrays, and confirmation of the true expression patterns of these genes was shown by in situ hybridization and Taqman quantitative RT-PCR. Herein we demonstrate how the combination of LCM, RNA amplification and gene expression assays helps to define cell types within identified neural circuits as well as gives molecular information about these circuit elements, both important goals in neuroscience research.

Introduction
The brain is a highly heterogeneous organ, with a wide variety of cell types identifiable by location, morphology, physiology, and connectivity. Connectivity, or a cells place in a neural circuit, is an important defining trait for neurons, as it is central to the precise c
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