Simultaneous optical measurements of electrical activity from multiple sites on processes of cultured neurons.

Abstract

Optical methods using changes in fluorescence and absorption ofvoltage-sensitive dyes were developed to record electrical activity from processes of nerve cells grown in monolayer culture. For transmission measurements, a merocyanine dye was discovered that was more sensitive than others previously tested on cultured neurons. Action potentials from the somata of these cells were detected without averaging, with a signal-to-noise ratio of 20:1. With this dye, electrical responses were simultaneously recorded from many points along the arborization of neuroblastoma cells by using a 10 x 10 array ofphotodiodes positioned in the microscope image plane. Frequently different processes had different shapes of electrical responses, suggesting regional specializations. Fluorescence measurements with an oxonol dye proved to be more sensitive than transmission measurements, particularly when recording from small processes. By changing the position of the cell relative to a laser microbeam while recording electrically from the cell body, it was possible to monitor the membrane potential in the cell body and in the process simultaneously. From the delay in reponse in the process, a lower limit for the mean conduction velocity of 0.2-0.6 m/sec was found for 2-to 7-,um processes. The mean space constants of processes were estimated by comparing the amplitudes of passive voltage responses in the cell body and growth cone. A lower limit of400-950 ,um was obtained for 4-to 7-,um processes. Many questions in neurobiology require an understanding of the regional properties ofneurons. For example, the integration of multiple synaptic inputs by single nerve cells requires a knowledge ofthe passive and active electrical membrane properties ofthe dendritic branches, soma, and axon (1-4); the space constants of each of the processes and the location and nature of specialized segments with localized excitability must be determined. Also, the role that given ions play in the regulation of neuronal development requires knowledge of the electrical properties of the developing neurites (5). Yet, the electrical events in the growth cone are not known.,Nerve cells maintained in tissue culture offer a simplified system for studying these electrical parameters, yet these questions are difficult to approach by current techniques because it is hard to record intracellularly from small processes. Extracellular recording from small processes ofcultured neurons has been reported (6-8). However, this technique is inadequate when studying graded potentials or regional variations in the form of the action potential (9, 10). Furthermore, simultaneous measurements of events in different regions within one neuron are very hard with classical recording methods. Some of the limitations inherent in these methods were recently discussed (10).Optical methods for measuring membrane potential by using voltage-sensitive dyes have been developed by Cohen and coworkers (11)(12)(13)(14) and by Waggoner (15) and colleagues. These dyes act as molecular transdu...

Detection and modulation of acetylcholine release from neurites of rat basal forebrain cells in culture.

“…Such brief latencies indicate that the release sites must be very close to the patch and that the detected release was not the result of diffusion of transmitter from more distant sites. Assuming a conduction distance from the soma to a release site of between 20 and 100 ,tm and a conduction velocity of approximately 0 2 m s-' (Grinvald, Ross & Farber, 1981), spike propagation time would account for about 0 1-0 5 ms of the observed latency. In addition, there would be a delay due to the time it takes for the released transmitter to diffuse across to the detector patch.…”

Cable properties of a straight neurite of a leech neuron probed by a voltage-sensitive dye.

“…The method of choice seems to be optical recording with voltagesensitive dyes (6)(7)(8)(9). Transient electrical activity-with a time constant of -0.5 ms-was observed at spatial resolutions of 40 um (10)(11)(12)(13)(14), of 15 gm (15), and of 8 Am (16,17). Images at a resolution of 1 Am were measured only at a time scale of ""1 s (18).…”

Regional distribution of calcium influx into bursting neurons detected with arsenazo III.

“…11). Photodiode arrays allow simultaneous optical recordings from many different tissue sites (12) or from many regions of a single neuron (13)(14)(15)(16). These techniques have been combined to examine sites of voltage-dependent calcium influx in neurons of intact ganglia (17,18).…”