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 microelec trode measurements from the soma. Regional electrical properties of ne urons 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 predi ct the distribution of membrane potential in the entire neuronal arbor ization. Additional, direct evidence about electrical signalling in ne uronal processes of individual cells in situ can now be obtained by re cording of membrane potential changes using voltage-sensitive dyes. A number of recent studies have shown that active regional electrical pr operties of individual neurons are extraordinarily complex, dynamic an d, in the general case, impossible to predict by present models. This places a great significance on measuring capabilities in experiments s tudying the detailed functional organization of individual neurons. Th e main difficulty in obtaining a more accurate description was that ex perimental techniques for studying regional electrical properties of n eurons were not available. With this motivation, we worked on the deve lopment of multisite voltage-sensitive dye recording as a potentially powerful approach. The results described here demonstrate that the sen sitivity of voltage-sensitive dye recording from branches of individua l neurons was brought to a level at which it can be used routinely in physiologically relevant experiments. The crucial figure-of-merit in t his approach, the signal-to-noise ratio from neuronal processes in int act ganglia, has been improved by a factor of roughly 150 over previou sly 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 neurona l processes in the neuropile was determined; and (2) the interaction o f two independent action potentials (spike collision) was monitored di rectly in a neurite in the neuropile; (3) it was demonstrated that sev eral action potentials are initiated in the same neuron at different s ites (multiple spike trigger zones) by a single stimulus; (4) the exac t location and the size of one of the remote spike trigger zones was d etermined; (5) the spread of passive subthreshold signals was followed in the neurites in the neuropile. This kind of information was not pr eviously available. Preliminary experiments on vertebrate neurons indi cate partial success in the effort to use intracellularly applied volt age-sensitive dyes to record from neurons in a mammalian brain slice p reparation. The results suggest that, with further improvements, it ma y be possible to follow optically synaptic integration and spike condu ction in the dendrites of vertebrate nerve cells. The main impact of t hese results is a demonstration of a new way of analysing how individu al neurons are functionally organized. Limitations and prospects for t he further refinement of the technique are discussed mostly in terms o f the signal-to-noise ratio; both improvements in the apparatus and de sign of more sensitive dyes are addressed. (C) 1998 Chapman & Hall.