Nanoscale-Targeted Patch-Clamp Recordings of Functional Presynaptic Ion Channels

Pavel Novak,Julia Gorelik,Umesh Vivekananda,Andrew I Shevchuk,Yaroslav S Ermolyuk,Russell J Bailey,Andrew J Bushby,Guy W.J Moss,Dmitri A Rusakov,David Klenerman,Dimitri M Kullmann,Kirill E Volynski,Yuri E Korchev

doi:10.1016/j.neuron.2013.07.012

Abstract

SummaryDirect electrical access to presynaptic ion channels has hitherto been limited to large specialized terminals such as the calyx of Held or hippocampal mossy fiber bouton. The electrophysiology and ion-channel complement of far more abundant small synaptic terminals (≤1 μm) remain poorly understood. Here we report a method based on superresolution scanning ion conductance imaging of small synapses in culture at approximately 100–150 nm 3D resolution, which allows presynaptic patch-clamp recordings in all four configurations (cell-attached, inside-out, outside-out, and whole-cell). Using this technique, we report presynaptic recordings of K+, Na+, Cl−, and Ca2+ channels. This semiautomated approach allows direct investigation of the distribution and properties of presynaptic ion channels at small central synapses.Video

Highlights

Current knowledge of neurotransmitter release mechanisms relies mainly on studies of large synapses, such as the calyx of Held or hippocampal mossy fiber bouton (Bischofberger et al, 2006; Schneggenburger and Forsythe, 2006), which can be patch clamped to control the presynaptic membrane potential and to manipulate or measure Ca2+ concentrations
Recent years have witnessed substantial progress in identifying the molecules involved in activity-dependent exo- and endocytosis at such synapses (Rizo and Rosenmund, 2008; Sudhof and Rothman, 2009), a quantitative understanding of ion channel properties in small presynaptic boutons remains poorly understood (Debanne et al, 2011)
The conventional patch-clamp technique relies on diffractionlimited optical microscopy to navigate a glass pipette to the target structure

Summary

Introduction

Current knowledge of neurotransmitter release mechanisms relies mainly on studies of large synapses, such as the calyx of Held or hippocampal mossy fiber bouton (Bischofberger et al, 2006; Schneggenburger and Forsythe, 2006), which can be patch clamped to control the presynaptic membrane potential and to manipulate or measure Ca2+ concentrations. The conventional patch-clamp technique relies on diffractionlimited optical microscopy to navigate a glass pipette to the target structure. This imposes a lower limit on the size of the subcellular compartment that can be targeted for recording. Recordings from narrow axons have recently been obtained using pipettes coated with fluorescently conjugated albumin; this method only allows cell-attached recordings of action-potential (AP) waveforms (Sasaki et al, 2012)

Results

Discussion

Conclusion