Nucleated, Outside-Out, Somatic, Macropatch Recordings in Native Neurons.

Abstract

Patch-clamp recordings are a powerful tool for the live measurement of the plasma membrane biophysical properties, with the ability to discriminate fast events such as fast inactivating Na+ currents (<1ms c.a.). It can be used in virtually every cell-type, including cardiomyocytes, skeletal muscles, neurons, and even epithelial cells and fibroblasts. Voltage-clamp, patch-clamp recordings can be used to measure and characterize the pharmacological and biophysical profile of membrane conductances, including leak, voltage-gated, and ligand-gated ion channels. This technique is particularly useful in studies carried out in cell-lines transfected with the gene expressing the conductance under investigation. However, voltage-clamp measures conducted on the soma of a native, adult neuron, for example in an acute brain slice or in the brain of a live individual, are subject to three major limitations: (1) the branching structure of the neuron causes space-clamp errors, (2) ion channels are differentially expressed across different neuronal compartments (such as soma, dendrites, and axons), and (3) the complex geometry of neurons makes it challenging to calculate current densities. While not preventing the experimenter to conduct patch-clamp, voltage-clamp recordings in native neurons, these limitations make the measures poorly standardized and hence often unusable for testing specific hypotheses.To overcome the limitations outlined above, outside-out, patch-clamp recordings can be carried out instead (See Chap. 1, Sect. 3.5); however, the signal-to-noise ratio in outside-outs from native, adult neurons is usually too low for obtaining accurate measurements.Here we describe how to carry out nucleated, outside-out, somatic, macropatch recordings (from now on abbreviated into "macropatch recordings") to obtain accurate and standardized measures of the biophysical and pharmacological properties of somatic, neuronal membrane conductances.