Expression profiling of intermingled long-range projection neurons harvested by laser capture microdissection.

Gene expression data are most useful if they can be associated with specific cell types. This is particularly so in an organ such as the brain, where many different cell types lie in close proximity to each other. We used zebra finches (Taeniopygia guttata), fluorescent tracers and laser capture microdissection (LCM) to collect projection neurons and their RNAs from two interspersed populations from the same animal. RNA amplified from each cell class was reverse transcribed, fluorescently labeled, and hybridized to cDNA microarrays of genes expressed in the zebra finch brain. We applied strict fold-expression criteria, supplemented by statistical analysis, to single out genes that showed the most extreme and consistent differential expression between the two cell classes. Confirmation of the true expression pattern of these genes was made by in situ hybridization and Taqman quantitative PCR (qPCR). High quality RNA was obtained, too, from backfilled neurons birth-dated with bromodeoxyuridine (BrdU). We also quantified changes in the levels of three genes after singing behavior using qPCR. Thus, we have brought together a combination of techniques allowing for the molecular profiling of intermingled populations of projection neurons of known connectivity, age and experience, which should constitute a powerful tool for CNS research.

Replaceable neurons and neurodegenerative disease share depressed UCHL1 levels.

Might there be systematic differences in gene expression between neurons that undergo spontaneous replacement in the adult brain and those that do not? We first explored this possibility in the high vocal center (HVC) of male zebra finches by using a combination of neuronal tracers, laser capture microdissection, and RNA profiling. HVC has two kinds of projection neurons, one of which continues to be produced and replaced in adulthood. HVC neurons of the replaceable kind showed a consistent and robust underexpression of the deubiquitination gene ubiquitin carboxyl-terminal hydrolase (UCHL1) that is involved with protein degradation. Singing behavior, known to increase the survival of adult-born HVC neurons in birds, significantly up-regulated the levels of UCHL1 in the replaceable neurons but not in their equally active nonreplaceable counterparts. We then looked in the mouse brain and found relatively low UCHL1 expression in granule neurons of the hippocampus and olfactory bulb, two well characterized types of replaceable neurons in mammals. UCHL1 dysfunction has been associated with neurodegeneration in Parkinson's, Alzheimer's, and Huntington's disease patients. In all these instances, reduced UCHL1 function may jeopardize the survival of CNS neurons.