| Description |
?-gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system. The GABA-A receptor is a ligand gated chloride channel and is one of the major targets for GABA released in the synapse. The receptor is thought to be formed as a pentameric combination of different GABA-A receptor subunits. Currently 15 possible subunits have been cloned and sequenced, as well as a significant number of splice variants of those subunits. The subunit, or molecular, composition of the GABA-A receptor determines its functional properties. Although some of this relationship between molecular composition and functional properties has been studied, the physiological ramifications of seemingly limitless possible combinations of different subunits has much left to be defined. In this study, the molecular and functional properties of GABA-A receptors are studied using single cell reverse transcription polymerase chain reaction (RT-PCR) to determine the subunit expression in individual cells in combination with patch clamp electrophysiological recordings to define the functional receptor characteristics. The design, optimization, and examination of a single cell RT-PCR protocol for its effectiveness in detecting the expression of GABA-A subunit mRNA, and its use in conjunction with patch clamp recording are discussed herein. Briefly, it was found that a nested PCR approach using primers capable of amplifying families of subunits in the first round of PCR, and subunit specific primers in the second round, was necessary to achieve sufficient detection sensitivity. Multiple primer pairs are used in the first round of the nested PCR as a multiplex PCR reaction to amplify all of the GABA-A receptor subunits of interest. The type I and type II error rates for this single cell RT-PCR protocol are experimentally determined and methods of improving the statistical power are discussed. Single cell RT-PCR was also performed in conjunction with ultrafast ligand exchange patch clamp recordings in an effort to correlate molecular (subunit expression) and functional GABA-A receptor properties in cultured fetal mouse cortical neurons. Neurons that had similar functional responses also had similar RT-PCR results. From the data obtained, it appears that subunit mRNA expression and receptor function are related in neurons in primary culture. |
