Gramicidin perforated patch recording technique

Abstract

Cl- is one of the major ionic constituents of cells and extracellular spaces. Intracellular Cl- plays an important role in regulating the cell volume and pH, in both salt secretion and reabsorption, in membrane excitability, and G-protein-dependent intracellular signal transduction. GABA and glycine are the primary inhibitory neurotransmitters. Such agonist- stimulated responses are affected by the intracellular Cl- concentration (Cl-](i)). However, it was difficult to make an electrical recording of the physiological Cl- response from cells with native intracellular Cl- activity because of the limitations of present recording techniques using conventional glass-microelectrode, whole-cell patch and nystatin perforated patch recording modes. Recently, this difficulty was overcome by developing the gramicidin perforated patch recording mode in our laboratory. Gramicidin is a polypeptide antibiotic that forms pores in the cell membrane as well as nystatin but allows only monovalent cations to permeate the membrane, enabling both [Cl-](i) and the second messenger system to remain undisturbed. Here, I would like to primarily focus on the GABA- and glycine- induced Cl- responses to in mammalian CNS neurons maintaining native cellular [Cl-](i) under normal and pathological neuronal conditions by use of gramicidin perforated patch recording configuration. Age-related and developmental changes in neuronal [Cl-](i) are also described in detail.