Methods

Electrophysiology. Electrographic (ERG) methodology analysis has been standard in this laboratory for decades (Stark, 1973) (McKay et al, 1995). For this work, the stimulus was from a 150 W Xenon Arc in an Opti-Quip housing and 1600 power supply and the readout was via a PowerLab 410 feeding into a Macintosh computer running operating system 9.2. To photograph the deep pseudopupil (DPP) before running the ERG, the cover slip the flies were fixed to was optically fused to a microscope slide using a drop of water, and the DPP was viewed with a 10x air objective. For the ERG, a long wavelength, 585 nm, was chosen for two reasons: (1) potential differences between the effects of eye color pigments of experimental and control flies would be minimized (Stark, 1973); and (2) visualization of on- and off transients would be optimized (Stark & Wasserman, 1972). Data acquisition was expedited by quickly obtaining responses to a defined sequence of seven stimuli of 1.0 s 0.3 to 0.6 log units apart (an intensity response sequence). After obtaining ERGs, the cover slip was again placed on a slide, and rhabdomeres were photographed using oil immersion and a 40x objective.

Results

Figure

ERGs of GGA knock-downs were surprisingly consistent with the expectation for "normal" flies. Near threshold, i.e. when the ERG component attributed to receptor depolarization was 0-3 mV, there were on- and off-transients that were fairly large. For higher intensities, the receptor wave was larger, and the transients were diminished. At the highest intensities, the receptor component sometimes settled back rather than just swing up to a steady state. Newly emerged GGA knockdown flies were run as well as flies of various ages out to 22 days, and they all had similar ERG responsivities and sensitivities. ERG on- and off-transients not only imply a high level of R1-6 function but also that the synaptic connections of R1-6 in the first order optic neuropile, the lamina ganglionaris, are functional. GGA knock downs, (top) and driver-only controls (second row) are shown. Newly emerged (left), 6-8 days (second column), 15 days (3rd column) and 22 days (right). For comparison, wild-type newly emerged is at the bottom left. The vertical scales have been adjusted to be equivalent

Newly eclosed, knockdown and driver are about the same sensitivity and about 0.6 log units than wild type. With age, the knockdown stays about the same while the driver only stock increases to wild type, consistent with the increase in rhabdomere size in only the driver-only stock. Assuming the lower sensitivity of the GGA knockdown were based on strictly on a lower amount of rhodopsin, then the inference is that the GGA knockdown has about 0.25 x the normal rhodopsin amount since sensitivity and visual pigment content were shown to be linearly related in the invertebrate (Hamdorf & Schwemer, 1975). Contrast the approximate 0.6 log unit diminution og the GGA knockdown's sensitivity with the over 2 log unit decrease of sensitivity of vitamin A deprived flies (Stark & Zitzmann, 1976).


References

Hamdorf K, Schwemer J (1975) Photoregeneration and the adaptation process in insect photoreceptors of invertebrates. In Photoreceptor optics, Snyder AW, Menzel R (eds), pp 263-289. Berlin: Springer

McKay RR, Chen DM, Miller K, Kim S, Stark WS, Shortridge RD (1995) Phospholipase C rescues visual defect in norpA mutant of Drosophila melanogaster. J Biol Chem 270: 13271-13276

Stark WS (1973) The effect of eye colour pigments on the action spectrum of Drosophila. J Insect Physiol 19: 999-1006

Stark WS, Wasserman GS (1972) Transient and receptor potentials in the electroretinogram of Drosophila. Vision Res 12: 1771-1775

Stark WS, Zitzmann WG (1976) Isolation of adaptation mechanisms and photopigment spectra by vitamin A deprivation in Drosophila. J Comp Physiol 105: 15-27

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