Abstract
The phototransduction enzymatic cascade in cones is less understood than in rods, and the zebrafish is an ideal model with which to investigate vertebrate and human vision. Therefore, here, for the first time, the zebrafish green cone photoresponse is characterized also to obtain a firm basis for evaluating how it is modulated by exogenous molecules. To this aim, a powerful method was developed to obtain long-lasting recordings with low access resistance, employing pressure-polished patch pipettes. This method also enabled fast, efficient delivery of molecules via a perfusion system coupled with pulled quartz or plastic perfusion tubes, inserted very close to the enlarged pipette tip. Sub-saturating flashes elicited responses in different cells with similar rising phase kinetics but with very different recovery kinetics, suggesting the existence of physiologically distinct cones having different Ca2+ dynamics. Theoretical considerations demonstrate that the different recovery kinetics can be modelled by simulating changes in the Ca2+-buffering capacity of the outer segment. Importantly, the Ca2+-buffer action preserves the fast response rising phase, when the Ca2+-dependent negative feedback is activated by the light-induced decline in intracellular Ca2+.
Highlights
G-protein-coupled enzyme cascades are ubiquitous signalling systems: they transduce physical or chemical stimuli, triggered by photon absorption or ligand binding, into intracellular messages that elicit adaptive responses [1, 2]
The small drift in the baseline was due to small changes in the current flowing through the inner segment channels rather than in changes in the light sensitive current flowing through the cGMP channels, because the amount of current suppressed by saturating flashes was the same from the beginning to the end of these recordings
The whole-cell patch-clamp recordings from zebrafish green cones performed with pressurepolished pipettes had reproducible sensitivity, waveform photoresponse kinetics and light adaptation
Summary
G-protein-coupled enzyme cascades are ubiquitous signalling systems: they transduce physical or chemical stimuli, triggered by photon absorption or ligand binding (e.g. ions, hormones, neurotransmitters, or odorants), into intracellular messages that elicit adaptive responses [1, 2]. The most direct, most sensitive method for studying these cascades is to precisely control the stimulus with light (for instance, by using optogenetics or caged agonists [3,4,5]), and recording the stimulus response with electrophysiological methods which are suitable for those.
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