A METHOD FOR DIRECT PATCH-CLAMP RECORDING FROM SMOOTH-MUSCLE CELLS EMBEDDED IN FUNCTIONAL BRAIN MICROVESSELS

The aim of this project was to develop a method to enable routine appl ication of all patch-clamp configurations to smooth muscle cells while they remain embedded in blood vessels. Small blood vessels were isola ted from rabbit brain using an enzymatic and mechanical procedure. Ves sels were identified under a microscope and the majority were small ar terioles with a mean external diameter, in Ca2+-containing (1.5 mM) so lution, of 29 mu m and variable lengths of 100 mu m or more. Arteriole s excluded trypan blue, constricted in response to 60 mM K+ and dilate d in response to levcromakalim. Patch-clamp gigaOhm seals were made re gularly on smooth muscle cells embedded in arterioles. The membrane po tential recorded using amphotericin-B-containing patch pipettes averag ed -72 mV. Short arteriolar segments could be voltage-clamped. Injecti on of depolarising current or bath application of 10 mM Ba2+ induced c onstriction of the entire arteriolar segment. Cell-attached patch, ins ide-out patch and outside-out patch recordings were made readily and K + channel unitary currents were studied. The method is readily applied and has several advantages over previous methods for the study of ion channels in smooth muscle cells. Notably, avoidance of single-cell is olation means that enzymatic treatment is minimised and cells can be s tudied within their normal environment of the blood vessel wall.