Cell-type-specific global gene expression profiling: Applications and quantitative comparison of methods

Brain function arises from the interaction of cells with diverse morphology, connectivity, and electrical and chemical signaling properties. Given that gene expression largely determines cell phenotype, expression profiling of restricted neural populations using microarrays can facilitate the classification of neural cell types and provide insight into the transcriptional regulation of cellular properties that ultimately shape neural activity. The main thrust of the work presented here is the application of a cell-type-specific mRNA purification method developed by our lab to study the postnatal development and activity-dependent maturation of one particular cell type, cortical fast-spiking (FS) GABAergic interneurons. Using a transgenic mouse line in which a subset of cortical FS cells can be identified by green fluorescent protein (GFP) expression, we employed both microarrays and whole-cell patch clamp recordings to characterize developmental gene regulation and electrophysiological maturation in parallel. We found that thousands of transcripts are dynamically regulated between postnatal day 7 (P7) and P40, and that FS cell physiology likewise undergoes profound changes during this time. This study confirmed the developmental regulation of several genes known to mediate specific aspects of FS cell phenotype and uncovered numerous novel candidates for the regulation of others. For two particular genes encoding two-pore potassium leak channels we were able to show correlation between the regulation of the transcripts, the corresponding proteins, and the functional channels. We then tested the activity-dependence of the maturation of FS interneuron identity by inactivating a region of motor cortex for 48 hrs in vivo. As a result of this perturbation FS cells displayed immature characteristics, such as spike frequency adaptation and lower firing rates. However, activity deprivation had only a modest effect on transcript expression as measured by microarrays, suggesting that activity-dependent modifications of physiological properties may be mediated by post-transcriptional mechanisms. Finally, I present the results of a quantitative comparison study of different cell-type-specific mRNA harvesting methods. We show that existing methods differ significantly with respect to purity and stress effects, and demonstrate techniques for detecting and filtering out contaminating genes in order to focus on meaningful expression differences.