Optical measurement of physiological sodium currents in the axon initial segment.

KEY POINTS  We optically measured the axonal Na+ fluorescence underlying an action potential in the axon initial segment at unprecedented temporal resolution.  The measurement allowed resolving the kinetics of the Na+ current at different axonal locations.  The distinct components of the Na+ current were correlated with the kinetics of the action potential.  NEURON simulations from a modified published model qualitatively predicted the experimentally measured Na+ current.  The present method permits the direct investigation of the kinetic behaviour of native Na+ channels under physiological and pathological conditions. ABSTRACT In most neurons of the mammalian central nervous system, the action potential (AP) is generated in the axon initial segment (AIS) by a fast Na+ current mediated by voltage-gated Na+ channels. While the axonal Na+ signal associated with the AP has been measured using fluorescent Na+ indicators, the insufficient resolution of these recordings did not allow resolving the Na+ current kinetics underlying this fundamental event. In this article, we report the first optical measurement of Na+ currents in the AIS of pyramidal neurons of the layer-5 of the somatosensory cortex from brain slices of the mouse. This measurement was obtained by achieving the temporal resolution of 100 μs in the Na+ imaging technique, with a pixel resolution of half a micron, and by calculating the time-derivative of the Na+ change corrected for longitudinal diffusion. We identified a subthreshold current before the AP, a fast-inactivating current peaking during the rise of the AP and a non-inactivating current during the AP repolarisation. We established a correlation between the kinetics of the non-inactivating current at different distances from the soma and the kinetics of the somatic AP. We quantitatively compared the experimentally measured Na+ current with the current obtained by computer simulation of published NEURON models demonstrating how the present approach can lead to the correct estimate of the native behaviour of Na+ channels. Finally, we discuss how the present approach can be used to investigate the physiological or pathological function of different channel types during AP initiation and propagation. This article is protected by copyright. All rights reserved.