Don't curse the darkness, light a candle: fluorescence studies of axon excitability.

Axons arguably remain one of the black boxes of CNS cellular neuroscience. Our view is dimmed by the small size, tortuous trajectories, and distant targets of axons, all of which limit accessibility. On the other hand, because electrical transmission along axons to axon terminals is of key importance in neuronal signalling, axons have been intensely studied since the earliest days of neurophysiology. Still, many of our tools do not directly illuminate important features of axonal signal initiation and propagation. For instance, with a few notable exceptions, most fibres remain inaccessible to patch pipettes and to other conventional electrophysiology techniques. Instead of cursing the darkness, Popovic et al. (2011) in this issue of The Journal of Physiology have lit a fluorescent candle. The authors report refinements of dye-based measurements that elucidate the behaviour of layer 5 cortical axons and their small, branched collaterals. Popovic et al. exploit a voltage-sensitive dye that is loaded into single cells and that responds with sufficiently rapid and large changes in fluorescence to resolve the time course and amplitude of action potentials. The authors used cortical slices from a mouse line in which layer 5 cortical neurons were labelled with EGFP. This allowed them to identify intact axons a priori. Parent cell bodies of these long axons were then targeted for dye loading through a whole-cell patch pipette. The pipette was removed to allow dye diffusion and cell recovery before imaging alone or before simultaneous imaging with whole-cell recording (performed by re-patching the cell). The use of an intracellular voltage-sensitive dye, rather than a dye bulk loaded from the extracellular solution, allowed the authors to unambiguously study a single cell. Although the general approach has been adopted before by this group and by others cited by Popovic et al., one of the strengths of the present paper is that the authors describe many details of their acquisition and analysis methodologies. This should greatly aid other investigators interested in lighting their own candles. The authors estimate that their methods offer a time resolution of 10 μs, a spatial resolution of 4 μm, and an amplitude resolution of 4 mV. These statistics are beginning to push the gold standards of temporal and amplitude resolution set by electrophysiological methods. For casual readers, the supplemental online movies supplied by the authors are probably the best way to get a feel for the technical achievement (http://jp.physoc.org/content/early/2011/06/13/jphysiol.2011.209015/suppl/DC1). The approach taken by Popovic et al. has several advantages and several drawbacks compared with competing electrophysiological methodologies. Several labs have achieved direct loose-seal, cell-attached, or whole-cell electrical recordings from axons. Generally speaking, these recordings are restricted to proximal parts of the axon, and to only one axonal site at a time. Popovic et al. show that the optical approach can be applied to fine collaterals of axons and to multiple regions of interest in the axon. Although the optical approach is good for recording changes in fluorescence, a drawback is that the DC potential of the cell (e.g. resting potential) cannot be directly deduced (also a drawback of loose seal and cell-attached recordings). The optical approach might be considered less invasive than whole-cell recordings, but Popovic et al. exercise due caution to avoid the notorious photodynamic effects of voltage-sensitive dyes on the function of voltage-gated and ligand-gated ion channels that could affect excitability. With some of these caveats in mind, it is reassuring to find evidence generally confirming electrophysiological approaches, which have demonstrated that the axon initial segment is the normal site of action potential initiation (reviewed in Debanne et al. 2011). By exploiting the ability to image more than one site within the initial segment simultaneously, Popovic et al. demonstrate that the action potential emerges first over a zone that encompasses much of the initial segment. The authors leverage the advantages of their approach to beautifully demonstrate saltatory conduction in myelinated axons and monotonic conduction in immature, unmyelinated fibres. Popovic et al.'s imaging approach also has the power to address questions of action potential fidelity that have typically been addressed only indirectly by electrophysiological approaches. For instance, fidelity is often inferred by monitoring synaptic failures in recordings from postsynaptic target cells. Popovic et al.'s imaging approach, in contrast, directly assessed fidelity by imaging multiple collaterals of the same axon. The data demonstrate strikingly high fidelity of propagation through the axonal tree, even during repetitive stimulation. The featured approach of Popovic et al. opens up the possibility of addressing spike fidelity and waveform changes in the axonal trees of multiple neuronal types, under varied physiological and pathophysiological conditions. Particularly interesting might be to explore disease models, including axonopathies, to identify the earliest changes in axon function. Is fidelity altered? Alternatively, are more subtle alterations evident in action potential waveform that might contribute to abnormal synaptic signalling? This approach would also allow one to assess the efficacy of potential therapeutic interventions in disease processes. In summary the approach of Popovic et al. and the work of others employing similar fast imaging methods (reviewed by Scanziani & Hausser, 2009) are beginning to minimize the limitations that have plagued past approaches to the study of excitability. Hopefully, the field can look forward to continued multidisciplinary input from chemists, biophysicists and physiologists that will further illuminate the smallest and least accessible elements of CNS electrical signalling.