Voltage-Sensitive Dye Recording from Networks of Cultured Neurons

Citation

Chien, Chi-Bin (1990) Voltage-Sensitive Dye Recording from Networks of Cultured Neurons. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/jwsd-2f67. https://resolver.caltech.edu/CaltechETD:etd-06082007-080125

Abstract

This thesis describes the development and testing of a sensitive apparatus for recording electrical activity from microcultures of rat superior cervical ganglion (SCG) neurons by using voltage-sensitive fluorescent dyes.

The apparatus comprises a feedback-regulated mercury arc light source, an inverted epifluorescence microscope, a novel fiber-optic camera with discrete photodiode detectors, and low-noise preamplifiers. Using an NA 0.75 objective and illuminating at 10 W/cm² with the 546 nm mercury line, a typical SCG neuron stained with the styryl dye RH423 gives a detected photocurrent of 1 nA; the light source and optical detectors are quiet enough that the shot noise in this photocurrent--about.03% rms--dominates. The design, theory, and performance of this dye-recording apparatus are discussed in detail.

Styryl dyes such as RH423 typically give signals of 1%/100 mV on these cells; the signals are linear in membrane potential, but do not appear to arise from a purely electrochromic mechanism. Given this voltage sensitivity and the noise level of the apparatus, it should be possible to detect both action potentials and subthreshold synaptic potentials from SCG cell bodies. In practice, dye recording can easily detect action potentials from every neuron in an SCG microculture, but small synaptic potentials are obscured by dye signals from the dense network of axons.

In another microculture system that does not have such long and complex axons, this dye-recording apparatus should be able to detect synaptic potentials, making it possible to noninvasively map the synaptic connections in a microculture, and thus to study long-term synaptic plasticity.

Thesis Files