Molecules and Membrane Activity: Single-Cell RT-PCR and Patch-Clamp Recording from Central Neurons
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2006 Springer Science+Business Media, Inc. All rights reserved. This chapter summarizes methods for characterizing mRNA expression and electrophysiological properties of central neurons using patch-clamp recording and single-cell reverse-transcription/polymerase chain reaction (scRT-PCR). A simple scRT-PCR protocol can be used to identify neurons by the expression of phenotypic marker mRNAs. The combination of these methods allows for the correlation of functional properties with molecular expression. Somewhat more complex methods are available for quantitation of mRNA expression. Both traditional gel-based PCR identification and real-time fluorescent PCR identification methods can be employed. Advantages and requirements of various methods are discussed. Different types of tissue preparations are presented with emphasis on methods used in our laboratories for acutely dissociated or cultured basal forebrain and amygdala neurons. The basal forebrain contains a heterogeneous population of cholinergic and GABAergic neurons, while the amygdala displays neurons with a complex receptor subunit composition. Investigation of neurons with this type of molecular diversity benefits from techniques such as scRT-PCR for cell identification. We also illustrate how these PCR methods can be combined with more complex experimental protocols, such as calcium buffering measurements using fluorescent dyes in dissociated neurons from aged animals. The capacity to combine scRT-PCR with a variety of experimental protocols allows the identification of unique cell types and relationships between physiology and gene expression.
