Low-noise recording of single-vesicle capacitance steps in cell-attached patches.
Capacitance recording provides a readout of membrane area that can be used to monitor exo- and endocytosis in neurons and secretory cells in real time. By interfacing a lock-in amplifier to a patch-clamp amplifier, the capacitance of cell-attached membrane patches can be measured with sufficient sensitivity to reveal the fusion and retrieval of single vesicles as unitary stepwise changes in capacitance. The small size of many secretory vesicles, especially of synaptic vesicles, places a premium on the reduction of noise in a capacitance recording. With care, the capacitance noise in cell-attached patches can be reduced to below 10 aF root-mean-square (rms), thus bringing into view steps resulting from the fusion of vesicles as small as about 18 nm in diameter. Thus, the lowest achievable noise level enables the resolution of changes in capacitance associated with the smallest secretory vesicles. This chapter presents the method of capacitance recording from cell-attached patches with an emphasis on noise reduction. It also addresses the closely related issue of extracting fusion pore properties from these recordings.
Klyachko
Vitaly
V
Howard Hughes Medical Institute and Molecular Neurobiology Laboratory, The Salk Institute, La Jolla, CA, USA.
Zhang
Zhenjie
Z
Jackson
Meyer
M
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D025802:Electric Capacitance; D005089:Exocytosis; D066298:In Vitro Techniques; D008561:Membrane Fusion; D008564:Membrane Potentials; D008954:Models, Biological; D009479:Neuropeptides / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010904:Pituitary Gland, Posterior / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D022142:Secretory Vesicles / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000378:metabolism; D013997:Time Factors
D009479:Neuropeptides