( genes were also validated by Taqman qu...)Laser Capture Microdissection (LCM) and Expression Profiling of Long-Range Projection Neurons

genes were also validated by Taqman quantitative PCR (Fig. 4), which was performed on the same amplified RNA generated from the original set of birds, as well as on new aRNA from other birds added to the experiment. The results show a significant difference in the expression of these genes by neuron subtype and validate our microarray findings.

Using cell sorting, only a single report has previously shown that projection neurons can be successfully purified for expression profiling (Arlotta et al., 2005; Dougherty and Geschwind, 2005). The results presented here demonstrate that it is possible to use LCM (Bonner et al., 1996) to microdissect closely apposed projection neurons by guiding the microdissection with fluorescent, retrogradely transported neuronal tracers. These tracers accumulate outside the nucleus of the cell body, where the pools of mRNA of interest are located. This approach eliminates the need for lengthy tissue dissociation and related steps needed for cell sorting, thus helping to preserve the original, in vivo profile of mRNA expression. The in vivo labeling approach also minimizes the time needed for tissue preparation for LCM, further preserving RNA integrity. The within-animal design of the microarray experiments makes for highly consistent results, with most genes expressed at similar levels, helping to more easily identify those genes that are most differentially expressed. The generation of a large pool of aRNA with these methods allows for many assessments of gene expression, whether by microarrays for gene discovery or Taqman for quantification of known genes. The combination of these techniques should greatly facilitate the molecular characterization of specific cellular components of neural circuits.

References
1. Nottebohm, F., Kelley, D.B. & Paton, J.A. (1982) Connections of vocal control nuclei in the
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