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Guide ch 05 Chapter ADVANCED METHODS IN ELECTROPHYSIOLOGY Recording from Xenopus Oocytes The Xenopus oocyte expression system was introduced by Gurdon in as a means to study various aspects of the control of gene expression Injection of exogenous DNA into

Chapter ADVANCED METHODS IN ELECTROPHYSIOLOGY Recording from Xenopus Oocytes The Xenopus oocyte expression system was introduced by Gurdon in as a means to study various aspects of the control of gene expression Injection of exogenous DNA into the oocyte nucleus or mRNA into the cytoplasm led to the expression of functional proteins by the oocytes Early studies dealt with the expression of proteins that are of little interest to electrophysiologists such as globin interferon and various viral proteins Consequently electrophysiologists and biophysicists paid scant attention to studies of protein expression in oocytes However beginning in Miledi and co-workers demonstrated that various types of ion channels and receptors could also be expressed in oocytes after injection of mRNAs that had been isolated from the appropriate tissue For example injection of Torpedo electric organ mRNA led to the expression of functional nicotinic acetylcholine receptors while injection of rat brain mRNA resulted in the expression of a large number of di ?erent voltage- and ligand-gated ion channels among them the voltage-gated Na channels NMDA and non-NMDA subtypes of glutamate receptors and GABAA receptors Since these initial experiments a number of workers have adopted this expression system to study various aspects of the structure and function of ion channels and receptors Some of the more common types of studies that have employed expression of exogenous ion channels and receptors in oocytes include analyzing the properties of mutated channels as a means to understand structure-function relationships in ion channels studying the post-translational processing and the assembly of multisubunit channels and receptors comparing the properties of channels from various tissues expressed in a common environment examining the modulation of channel and receptor function by various second messenger systems analyzing various aspects of receptor-e ?ector coupling and functional screening of cloned genes that encode channels and receptors expression cloning Most of these experiments involve electrophysiological recording from oocytes This chapter describes the mechanics of recording from oocytes and points out some oocyte features of which one should be aware in order to obtain high-quality recordings AXON GUIDE C Chapter ?ve What is a Xenopus Oocyte Xenopus oocytes are egg precursors that upon proper hormonal stimulation pass through the frog's oviduct and become eggs which can then be fertilized to make more frogs Oocytes are stored in the abdominal cavity and can be removed surgically for experimental purposes They go through six developmental stages termed stages I to VI Most researchers use only the large stage V and VI oocytes which can be used interchangeably in electrophysiological experiments Oocytes are found in clumps called ovarian lobes which are made up of oocytes connective tissue blood vessels and follicle cells Figure - shows an individual stage V or VI oocyte as found in an ovarian lobe Follicle Cell Layer Animal Pole Vitelline Membrane Vegetal Pole Figure - Stage V or VI Oocytes as Found in an Ovarian Lobe The oocyte is a large cell with a diameter of approximately - mm It is surrounded by the vitelline membrane which