Abstract
Phototransduction is a process in which light is converted into electrical signals used by the central nervous system. Invertebrate phototransduction is a process mediated by the phosphoinositide signaling cascade, characterized by Phospholipase C (PLC) as the effector enzyme and the Transient Receptor Potential (TRP) channels as its target. The great advantage of using invertebrate photoreceptors is the simplicity of the preparation, the ease of light stimulation, the robust expression of key molecular components, and most importantly, the ability to apply the power of molecular genetics. This last feature is mainly attributed to Drosophila melanogaster as a preferred animal model.The Electroretinogram (ERG) is an extracellular voltage recording from the entire eye, which reflects the total electrical activity arising from the retina in response to a light stimulation. The Drosophila ERG light response is robust and easily obtained, thus making it a convenient method to identify defects in the light response as a result of mutations. The Prolonged Depolarizing Afterpotential (PDA) is a useful ERG phenomenon that can be recorded from white-eyed flies following intense blue light. It is induced by a massive photo-conversion of the photopigment rhodopsin to its dark stable state called metarhodopsin, due to failure of light response termination. Unlike the light coincident ERG recording, which declines quickly to the dark baseline after the cessation of the light stimulus, the PDA response continues long (hours) after light offset. However, this response can be suppressed to the dark baseline at any time by photo-conversion of metarhodopsin back to rhodopsin, by application of an intense orange light stimulus (see Figure 7; Minke, 2012). The PDA has been used as an important tool to screen for visual defective mutant (Minke, 2012).
Keywords: Drosophila photoreceptors, Electroretinogram (ERG), Photoresponse, Drosophila mutants, in vivo electrical recordings
Materials and Reagents
Equipment
Procedure
The main components of the light response recorded by the ERG are (1) the extracellularly recorded photoreceptor potential, (2) the “on” and “off” transients, at the beginning and the end of the light pulse, arising from the second order lamina neurons, and (3) the slow response of the pigment (glia) cells. The photoreceptor potential is the physiological response to light arising from the light-induced openings of the Transient Receptor Potential (TRP) and TRP-Like (TRPL) channels. In response to intense step of light, the photoreceptor’s component of the ERG is composed of an initial corneal negative fast transient, which declines to a lower steady state phase due to light adaptation. The depolarization of the photoreceptor cell triggers the induction of the corneal positive “on” transient, via sign inverting synapse between the photoreceptor axon and the large monopolar neurons of the lamina. The response of the pigment cells appears as a corneal negative slow rise and slow decay of the ERG after light on and off, respectively. These slow components arise from an increase in extracellular K+, resulting from K+ efflux from the photoreceptor cells via the TRP and TRPL channels, which depolarize the pigment cells surrounding the photoreceptor cells (see Figure 6; Minke, 1982).
Notes
Recipes
Acknowledgments
We thank Anatoly Shapochnikov for constructing the Wax Filament Heater. The construction of the ERG protocol was supported by the Israel Science Foundation (ISF).
References
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