Fast optical measurement of membrane potential changes at multiple sites on an individual nerve cell

In the past 15 years, there has been renewed interest in the detailed spatial analyses of signalling in individual neurons. The behaviour of many nerve cells is difficult to understand on the basis of microelectrode measurements from the soma. Regional electrical properties of neurons have been studied using sharp microelectrode and patch-electrode recordings from neuronal processes, high-resolution multisite optical recordings of Ca2+ concentration changes and by using models to predict the distribution of membrane potential in the entire neuronal arborization. Additional, direct evidence about electrical signalling in neuronal processes of individual cells in situ can now be obtained by recording of membrane potential changes using voltage-sensitive dyes. A number of recent studies have shown that active regional electrical properties of individual neurons are extraordinarily complex, dynamic and, in the general case, impossible to predict by present models. This places a great significance on measuring capabilities in experiments studying the detailed functional organization of individual neurons. The main difficulty in obtaining a more accurate description was that experimental techniques for studying regional electrical properties of neurons were not available. With this motivation, we worked on the development of multisite voltage-sensitive dye recording as a potentially powerful approach. The results described here demonstrate that the sensitivity of voltage-sensitive dye recording from branches of individual neurons was brought to a level at which it can be used routinely in physiologically relevant experiments. The crucial figure-of-merit in this approach, the signal-to-noise ratio from neuronal processes in intact ganglia, has been improved by a factor of roughly 150 over previously available signals. The improvement in the sensitivity allowed, for the first time, direct investigation of several important aspects of the functional organization of an individual neuron: (1) the direction and the velocity of action potential propagation in different neuronal processes in the neuropile was determined; and (2) the interaction of two independent action potentials (spike collision) was monitored directly in a neurite in the neuropile; (3) it was demonstrated that several action potentials are initiated in the same neuron at different sites (multiple spike trigger zones) by a single stimulus; (4) the exact location and the size of one of the remote spike trigger zones was determined; (5) the spread of passive subthreshold signals was followed in the neurites in the neuropile. This kind of information was not previously available. Preliminary experiments on vertebrate neurons indicate partial success in the effort to use intracellularly applied voltage-sensitive dyes to record from neurons in a mammalian brain slice preparation. The results suggest that, with further improvements, it may be possible to follow optically synaptic integration and spike conduction in the dendrites of vertebrate nerve cells. The main impact of these results is a demonstration of a new way of analysing how individual neurons are functionally organized. Limitations and prospects for the further refinement of the technique are discussed mostly in terms of the signal-to-noise ratio; both improvements in the apparatus and design of more sensitive dyes are addressed.