Acquisition of voltage probe signals from dissociated zebrafish retinal neurons

Figure 1 (perfusion chamber)

Oxonol is a negatively charged lipophilic molecule that equilibrates across membranes according to membrane potential. When cells become depolarized, more of the negatively charged oxonol is drawn into the cytoplasm and fluorescence increases. When cells become hyperpolarized, the negative intracellular charge repels dye out of the cytoplasm and fluorescence decreases. In fact ideally the dye distributes according to the Nernst equation. Voltage calibrations suggest that a sensitivity of ~100 mV per decade change of fluorescence. This probe produces large signals, however it takes several minutes to equilibrate.

Figure 2 (voltage probe)

Voltage probe recordings from a pair of zebrafish retinal neurons. The cell on top (areas 2 and 3) is a bipolar cell, the one on the bottom (area 1)is a horizontal cell. To the right are the probe fluorescence responses to applications of glutamate, the metabotropic glutamate agonist APB, and kainate, an AMPA kainate ionotropic glutamate agonist. The bipolar cell is an ON type. It is hyperpolarized by glutamate. There are superimposed fluorescence decreases in both cell body (2) and dendrites (1). The horizontal cell is depolarized both by glutamate and by kainate. Following excitation there is a delayed hyperpolarizing action in horizontal cells (AHP, arrows). AHP is the dominant feature of the horizontal-cell voltage-probe responses to glutamate. It is blocked by oubain, and requires extracellular sodium. AHP is the hyperpolarizing action of the sodium-potassium ATPase in response to glutamate stimulation. At the end of each recording session gramicidin is applied. This permeabilizes cells, bringing membrane potentials to zero millivolts, and establishes the fluorescence level corresponding to 'zero' transmembrane potential (dotted lines). By clicking on the oxonol fluorescence image one can start a probe fluorescence movie. This illustrates the profound reduction in fluorescence following glutamate or kainate treatment in the horizontal cell (AHP), and the decrease in fluorescence during glutamate treatment in the (ON-type) bipolar cell. The frames are acquired at 30 s intervals.

Movie file

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• Figure 1
• Figure 2
• AVI movie file