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

Abstract

Τhe axonal Na+ fluorescence underlying an action potential in the axon initial segment was optically measured at unprecedented temporal resolution. The measurement allowed resolution of 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. 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 has not allowed tracking 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 layer 5 of the somatosensory cortex from brain slices of the mouse. This measurement was obtained by achieving a temporal resolution of 100μs in the Na+ imaging technique, with a pixel resolution of 0.5μm, 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 repolarization. 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.