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Fluorescent dyes derived from anilino-pyridinium are studied with respect to their application as probes of voltage-transients in neuron membranes. Their photophysical properties are characterized in bulk solvents. The spectral features of their voltage-sensitivity in membranes are determined. A mechanistic scheme of voltage-sensitivity is proposed. The dyes are applied for optical recording in neurites of cultivated neurons of the leech. The relative sensitivity is evaluated from the fluorescence transients induced by hyperpolarization on the basis of the cable equation. The pervasion of a neuritic tree by action potentials is observed.

We measured a time-resolved map of electrical activity in a thin straight neurite (1. (24). The 100 outputs were fed into current-voltage converters (OPA 121; Burr-Brown, Filderstadt, F.R.G., with 100-Ma feedback resistors, time constant 0.4 ms) and eventually into differential sample-and-hold amplifiers. Ten diodes were selected, and their signals were stored on a tape recorder (Racal, Bergisch Gladbach, F.R.G.).The protocol of a measurement was as follows: (i) Adjustment of a segment of the neurite along a row of 10 diodes, (ii) record baselines in the dark, (iii) record fluorescence with open shutter (after a 5-ms interval) and then switch to the sample-and-hold mode with the total fluorescence as a reference. (iv) Record and store fluorescence with electrical stimulation for 70 ms: A positive current (2-4 nA) was injected for 25 ms to elicit an action potential (amplitude 60-75 mV). After an interval of 10 ms, a negative Gaussian voltage (amplitude around -100 to -160 mV) was induced by a negative and positive current pulse. (v) After an interval of 5 s the fluorescence without stimulation was recorded for 70 ms and stored (baseline of bleaching). To overcome the limited range of the diode array (-85 pum) we displaced the neuron along a row of diodes several times-starting with the terminal segment-and repeated cycles (i-v). A complete set of data was sampled from the tape at an interval of0.1 ms and a resolution of 12 bit and read into a microcomputer. The fluorescence changes without stimulation were fitted by an exponential function and subtracted from the changes with stimulation; the results were divided by the total fluorescences. The transients of depolarization were corrected for the decreased amplitude of the action potential (from 75 to 60 mV) during a set of measurements with displaced neurites.Then all transients of depolarization were normalized to constant amplitude. The transients of hyperpolarization were scaled by the same factors. Finally the signals were smoothed Abbreviation: N cell, nociceptive neuron. 4604The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

No abstract

Dendrites of pyramidal neurons from embryonic rat hippocampus are investigated in culture using a voltage-sensitive fluorescent dye. The electrical response to somatic stimulation is observed as a time-resolved map with a resolution of 0.9 microm at a time constant of 0.4 ms without signal averaging. The data are interpreted in terms of a tapering cable with Hodgkin-Huxley parametrization. The spread of short hyperpolarizing transients is damped by capacitive shunting. The invasion of an action potential is boosted by voltage-gated conductances of a low density. No irregularity is observed at a bifurcation. The passive cable parameters of internal resistance and membrane resistance at resting voltage are Ri = 300 omega cm and Rm = 40 (k)omega cm2 respectively, at a maximum sodium conductance of approximately 4.4 mS/cm2. The electrotonic length constant and the dynamic length constant at 1 kHz are 580 and 90 microm respectively. These results are compatible with electrophysiological data of dendrites in slices of adult hippocampus and with optical data of narrow processes of leech neurons in culture. The functional implications of boosting an action potential by voltage-gated channels of low density are considered.