Optical recording of calcium and voltage transients following impulses in cell bodies and processes of identified leech neurons in culture

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Abstract
Optical methods were used to examine the spread of electrical potentials and the distribution and time course of calcium transients in individual identified nerve cells isolated from the leech. A photodiode array detected voltage transients by measuring absorbance changes of cells stained with the voltage-sensitive dye RH155 added to the bath. Calcium transients were recorded by measuring absorbance changes of the dye arsenazo III, which had been injected into the cells. In addition, Lucifer yellow was injected to outline the some and processes. Calcium changes resulting from individual action potentials were recorded from N, P, and Retzius cells without averaging. Signals from T cells and anterior pagoda (AP) cells were weaker but could be detected with averaging. These results are in accord with previous studies on calcium contributions to action potentials in these cells. For all cells, larger or wider action potentials gave bigger signals. Calcium changes from each of a train of action potentials were of equal amplitude, showing no sign of facilitation. Calcium transients from Retzius cells that had formed chemical synapses with P cells had properties similar to those of isolated cells. We were also able to detect responses from prolonged subthreshold depolarizations to -40 mV from a hyperpolarized membrane potential (-65 mV). These signals rose throughout the duration of the pulse (1–2 sec). With the photodiode array we mapped the distribution of the calcium signals. The amplitudes from each pixel are proportional to the amount of calcium entering that element in response to the stimulating pulse, if the simplifying assumption is made that the calcium buffering of the cytoplasm is uniform throughout the cell. The largest signals were detected over the axon stump left from the cell isolation procedure. Large signals were also detected from the soma. Weak signals were detected from the processes of some cells. From many Retzius cells, no signals at all were detected from the newly formed processes. Using the photodiode array, we also recorded voltage transients from the cells. Signals were recorded from all over the arborization of the neuron, with no obvious variation in time course, showing that the entire cell, including fine slender processes and broad growth cones, was essentially isopotential. Combining these observations with the measured distribution of calcium transients in the same cell suggests that the density of calcium channels in most cells is less in the outgrowing processes than in the soma or axon stump.