Ionic Dependence of Membrane Potential and Glutamate Receptor‐Linked Responses in Synaptoneurosomes as Measured with a Cyanine Dye, DiS‐C₂‐(5)

Abstract

Membrane potentials of particles present in a subcellular brain preparation, called synaptoneurosomes, have been monitored by measurement of changes in the absorbance of a cyanine dye, DiS-C2-5. The membrane potential of the particles seems to be dependent on both Cl- and K+ diffusion potentials, as judged from dependence of the absorbance changes on the K+ equilibrium potential across the membrane in the presence of Ba2+ or when Cl- was replaced with gluconate. The apparent high Cl- permeability of the membrane preparation was reduced in the presence of picrotoxin, a finding suggesting endogenous activation of receptor-linked Cl- channels. Glutamate and kainate caused depolarization of the membranes present in the preparation. This effect was only seen if K+ channels had been blocked in the presence of Ba2+ or 4-aminopyridine. No responses were observed with other glutamate receptor agonists (quisqualate or N-methyl-D-aspartate). The membrane potential of particles present in conventional synaptosomal preparations neither had a high Cl- permeability nor reacted to glutamate or kainate in the present conditions. The results suggest that synaptoneurosome preparations may be used for functional studies on postsynaptic neurotransmitter receptor-linked membrane potential changes with optical probes of membrane potential.

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“…Upon depolarization, the dye moves out and, hence, the fluorescence increases. 24 Synaptosomes were preincubated and resuspended as described for the glutamate release experiments. After 3-min incubation, 5 lM DiSC 3 (5) was added and allowed to equilibrate before the addition of CaCl 2 (1 mM) after 4-min incubation.…”

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“…At high concentrations, the dye molecules accumulate and the fluorescence is quenched. Upon depolarization, the dye moves out, and the fluorescence increases (Akerman et al, 1987). Synaptosomes were preincubated and resuspended as described for the glutamate release experiments.…”

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“…At high concentrations, the dye molecules accumulate and the fluorescence is quenched. On depolarization, the dye moves out, and hence, the fluorescence increases (Akerman et al, 1987). Synaptosomes were resuspended in HBM and incubated in a stirred and thermostated cuvette at 37°C in a PerkinElmer LS-55 spectrofluorimeter.…”