Name: electrophysiological methods for measuring photopigment levels in drosophila photoreceptors
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This research was supported by grants from the Israel Science Foundation (ISF), and the United States-Israel Binational Science Foundation (BSF). We thank Mr. Anatoly Shapochnikov for the construction of the wax filament heater.

1. Measuring the intensity response relationship, prolonged depolarizing afterpotential (PDA), and the early receptor potential (ERP) using the electroretinogram
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	<li>Suitable rearing conditions for D. melanogaster preparation
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		<li>Raise D. melanogaster flies in bottles containing standard yellow corn containing food in an incubator maintained at a temperature of 24 &#176;C and in a 12 h dark/light cycle</li>
		<li>Keep the fly bottles in the dark at least 24 h prior to th...

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Comparative Physiology and Evolution of Vision in Invertebrates. 7, 145-224 (1979).</li><li>Gagn&#233;, S., Roebroek, J. G., Stavenga, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enigma+of+early+receptor+potential+in+fly+eyes.">Enigma of early receptor potential in fly eyes.</a> Vision Research. 29 (12), 1663-1670 (1989).</li><li>Pak, W. L., Shino, S., Leung, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDA+(prolonged+depolarizing+afterpotential)-defective+mutants:+the+story+of+nina's+and+ina's--pinta+and+santa+maria,+too.">PDA (prolonged depolarizing afterpotential)-defective mutants: the story of nina's and ina's--pinta and santa maria, too.</a> Journal of Neurogenetics. 26 (2), 216-237 (2012).</li><li>Wang, X., Wang, T., Jiao, Y., von, L. 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L., Breakfield, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neurogenetics, Genetic Approaches to the Nervous System. , 67-99 (1979).</li><li>Minke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+reduction+in+excitation+efficiency+in+the+trp+mutant+of+Drosophila.">Light-induced reduction in excitation efficiency in the trp mutant of Drosophila.</a> The Journal of General Physiology. 79, 361-385 (1982).</li><li>Minke, B., Kirschfeld, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+electrical+potentials+arising+from+activation+of+metarhodopsin+in+the+fly.">Fast electrical potentials arising from activation of metarhodopsin in the fly.</a> The Journal of General Physiology. 75 (4), 381-402 (1980).</li><li>Stephenson, R. 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C., Minke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+trp+gene+is+essential+for+a+light-activated+Ca2++channel+in+Drosophila+photoreceptors.">The trp gene is essential for a light-activated Ca2+ channel in Drosophila photoreceptors.</a> Neuron. 8, 643-651 (1992).</li><li>Niemeyer, B. A., Suzuki, E., Scott, K., Jalink, K., Zuker, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+light-activated+conductance+is+composed+of+the+two+channels+TRP+and+TRPL.">The Drosophila light-activated conductance is composed of the two channels TRP and TRPL.</a> Cell. 85 (5), 651-659 (1996).</li><li>Phillips, A. M., Bull, A., Kelly, L. 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N., Minke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+genetics+of+photoreceptors+desensitization+in+Drosophila+flies.">Mechanisms and genetics of photoreceptors desensitization in Drosophila flies.</a> Biochimica et Biophysica Acta. 1179, 283-299 (1993).</li><li>Minke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+of+the+prolonged+depolarizing+afterpotential+(PDA)+and+its+role+in+genetic+dissection+of+Drosophila+phototransduction.">The history of the prolonged depolarizing afterpotential (PDA) and its role in genetic dissection of Drosophila phototransduction.</a> Journal of Neurogenetics. 26 (2), 106-117 (2012).</li><li>Satoh, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arrestin+translocation+is+stoichiometric+to+rhodopsin+isomerization+and+accelerated+by+phototransduction+in+Drosophila+photoreceptors.">Arrestin translocation is stoichiometric to rhodopsin isomerization and accelerated by phototransduction in Drosophila photoreceptors.</a> Neuron. 67 (6), 997-1008 (2010).</li><li>Byk, T., Bar Yaacov, M., Doza, Y. N., Minke, B., Selinger, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+arrestin+cycle+secures+the+fidelity+and+maintenance+of+the+fly+photoreceptor+cell.">Regulatory arrestin cycle secures the fidelity and maintenance of the fly photoreceptor cell.</a> Proceedings of the National Academy of Sciences of the United States of America. 90, 1907-1911 (1993).</li><li>Dolph, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arrestin+function+in+inactivation+of+G+protein-coupled+receptor+rhodopsin+in+vivo.">Arrestin function in inactivation of G protein-coupled receptor rhodopsin in vivo.</a> Science. 260, 1910-1916 (1993).</li><li>Belusic, G., Pirih, P., Stavenga, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+responses+of+fruitflies+with+normal+and+reduced+arrestin+content+studied+by+simultaneous+measurements+of+visual+pigment+fluorescence+and+ERG.">Photoreceptor responses of fruitflies with normal and reduced arrestin content studied by simultaneous measurements of visual pigment fluorescence and ERG.</a> Journal of Comparative Physiology. A, Neuroethology, Sensory, Neural, and Behavioral Physiology. 196 (1), 23-35 (2010).</li><li>Stavenga, D. G., Hardie, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metarhodopsin+control+by+arrestin,+light-filtering+screening+pigments,+and+visual+pigment+turnover+in+invertebrate+microvillar+photoreceptors.">Metarhodopsin control by arrestin, light-filtering screening pigments, and visual pigment turnover in invertebrate microvillar photoreceptors.</a> Journal of Comparative Physiology. A, Neuroethology, Sensory, Neural, and Behavioral Physiology. 197 (3), 227-241 (2011).</li><li>Stark, W. S., Zitzmann, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+adaptation+mechanisms+and+photopigment+spectra+by+vitamin+A+deprivation.">Isolation of adaptation mechanisms and photopigment spectra by vitamin A deprivation.</a> Journal of Comparative Physiology. 105, 15-27 (1976).</li><li>Minke, B., Kirschfeld, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+a+sensitizing+pigment+to+the+photosensitivity+spectra+of+fly+rhodopsin+and+metarhodopsin.">The contribution of a sensitizing pigment to the photosensitivity spectra of fly rhodopsin and metarhodopsin.</a> The Journal of General Physiology. 73, 517-540 (1979).</li><li>Cone, R. A., Cobbs, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodopsin+cycle+in+the+living+eye+of+the+rat.">Rhodopsin cycle in the living eye of the rat.</a> Nature. 221 (5183), 820-822 (1969).</li><li>Murakami, M., Pak, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellularly+recorded+early+receptor+potential+of+the+vertebrate+photoreceptors.">Intracellularly recorded early receptor potential of the vertebrate photoreceptors.</a> Vision Research. 10 (10), 965-975 (1970).</li><li>Hodgkin, A. L., Obryan, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+recording+of+the+early+receptor+potential+in+turtle+cones.">Internal recording of the early receptor potential in turtle cones.</a> The Journal of Physiology. 267 (3), 737-766 (1977).</li><li>Yasin, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+expression+of+mouse+melanopsin+in+Drosophila+photoreceptors+reveals+fast+response+kinetics+and+persistent+dark+excitation.">Ectopic expression of mouse melanopsin in Drosophila photoreceptors reveals fast response kinetics and persistent dark excitation.</a> Journal of Biological Chemistry. 292 (9), 3624-3636 (2017).</li><li>Hardie, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photolysis+of+caged+Ca+2++facilitates+and+inactivates+but+does+not+directly+excite+light-sensitive+channels+in+Drosophila+photoreceptors.">Photolysis of caged Ca 2+ facilitates and inactivates but does not directly excite light-sensitive channels in Drosophila photoreceptors.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 15, 889-902 (1995).</li><li>Hardie, R. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+mutant+defective+in+extracellular+calcium-dependent+photoreceptor+deactivation+and+rapid+desensitization.">A Drosophila mutant defective in extracellular calcium-dependent photoreceptor deactivation and rapid desensitization.</a> Nature. 354, 230-232 (1991).</li><li>Pak, W. L., Lidington, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+electrical+potential+from+a+long-lived,+long-wavelength+photoproduct+of+fly+visual+pigment.">Fast electrical potential from a long-lived, long-wavelength photoproduct of fly visual pigment.</a> The Journal of General Physiology. 63 (6), 740-756 (1974).</li><li>Katz, B., Gutorov, R., Rhodes-Mordov, E., Hardie, R. 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A., Tsacopoulos, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+recovery+from+a+transient+inhibition+of+the+oxidative+metabolism+of+the+photoreceptors+of+the+drone+(Apis+mellifera).">The recovery from a transient inhibition of the oxidative metabolism of the photoreceptors of the drone (Apis mellifera).</a> Journal of Experimental Biology. 119, 165-181 (1985).</li><li>Agam, K., Frechter, S., Minke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+Drosophila+TRP+and+TRPL+channels+requires+both+Ca+2++and+protein+dephosphorylation.">Activation of the Drosophila TRP and TRPL channels requires both Ca 2+ and protein dephosphorylation.</a> Cell Calcium. 35 (2), 87-105 (2004).</li></ol>

The major advantage of using the Drosophila photoreceptor preparation is its accessibility, the ease and accuracy of light stimulation, and, most importantly, the ability to apply the power of molecular genetics7. Extensive genetic studies have established Drosophila as an extremely useful model system for the genetic dissection of complex biological processes7. The relatively simple structure of the Drosophila genome (consisting of only four chro...

The light-activated rhodopsin (R), which is a G-protein-coupled receptor (GPCR), is composed of a 7 transmembrane protein (opsin) and a chromophore. In Drosophila melanogaster (fruit fly), photon absorption induces isomerization of the 11-cis-3-OH-retinal chromophore to all-trans-3-OH-retinal1, promoting the conformational change of the rhodopsin to metarhodopsin (M, Figure 1A). Unlike vertebrate rhodopsin, the predominant fraction of invertebrate&#160;chromophore does not dissociate from the opsin, resulting in the physiologically active dark-stable pigment state M. In turn, additional photon absorption by the all-trans-3-OH-retinal chromophore induces isomerization of the chromophore2,3, generating the R pigment state with the 11-cis-3-OH-retinal chromophore. The R state is a dark, stable, and physiologically non-active photopigment. In addition to the extremely fast photon regeneration route of the chromophore4, much like vertebrate&#160;photopigments, an alternative enzymatic slow route for chromophore regeneration exists in invertebrates, in which some of the stages are performed in retinal cells surrounding the photoreceptors cells5,6.
Drosophila&#160;entails great advantages as a model organism for studying invertebrate photoreceptors. In particular, the accessibility of the preparation and the ability to apply molecular genetics have made Drosophila a powerful model system7. Hence, several in vivo and ex vivo experimental methods for studying phototransduction in general and photopigment levels, in particular, have been established. The simplest in-vivo method exploits the relatively large extracellularly recorded voltage response to light of the Drosophila eye. Accordingly, light stimulation evokes an electrical voltage response in the entire eye that can be measured using extracellular electroretinogram (ERG) recording, which is ~3 orders of magnitude larger than the ERG response to light of vertebrate eyes8,9. The Drosophila ERG response is robust and easily obtained, which makes it a convenient method for identifying abnormalities in light response due to mutations. The ERG response to light arises mainly from the photoreceptors, pigment (glia) cells, and secondary neurons of the lamina (see Figure 1B). The main components of the ERG are (i) the extracellular voltage response of the photoreceptors, (ii) the &#34;on&#34; and &#34;off&#34; transients at the beginning and end of the light stimulus that arise from the lamina neurons (Figure 2A,&#160;inset, ON, OFF), (iii) the slow response of the glia cells (Figure 2A, inset, arrows), and (iv) the brief and transient response, resulting from charge displacement during photopigment activation that precedes the ON transient10 (Figure 2C [inset],&#160;D,&#160;E). This brief response is composed of two phases (M1 and M2, Figure 2C [inset]) and can be induced only by extremely strong light stimulation, which activates simultaneously millions of photopigment molecules. It is neither observed under blue stimulation (Figure 2D, blue trace) nor in mutants with highly reduced photopigment levels (Figure 2E, red trace), but its amplitude is mildly enhanced in a mutant that abolishes PLC activity (Figure 2E, orange trace). The M1 phase is a typical ERP of the fly arising from the activation of M in the photoreceptors. The M1 phase, which has a positive polarity (intracellularly), releases a neurotransmitter in the normal way in a sign-inverting synapse and activates the lamina neurons, which respond to the photoreceptor depolarization by generating the synaptically amplified M2 phase. Thus, both M1 and M2 phases reflect M activation10,11.
The depolarization of the photoreceptor generates the corneal-positive &#34;on&#34; transient, arising from the sign-inverting synapse between the photoreceptor axon and the monopolar neurons of the lamina10,11 (Figure 1B). The slow rise and decay of the ERG arise from the depolarization of the pigment cells (Figure 2A, inset, arrows) mainly due to K+ efflux from the photoreceptor cells12 via the transient receptor potential (TRP) and TRP-like (TRPL) channels13,14,15. These slow kinetic components largely mask and distort the waveform of the photoreceptor response when compared to intracellular or whole-cell recordings of the photoreceptor response to light9,10. In addition, at very strong illuminations, an additional transient response, which precedes and partially fuses with the &#34;on&#34; transient, may be observed (Figure 2C [inset],D,E). This signal originates directly from the massive activation of the photopigment10.
Several light regime protocols using neutral density (ND) and color filters, as well as strong illuminating flashes, have been developed to investigate the eye in general and the phototransduction cascade in particular. These protocols have also been used to investigate the properties of the photopigment.
The intensity-response protocol measures the peak amplitude of the ERG voltage response of the entire eye to increasing light intensities (Figure 2A,B). This protocol assists in detecting changes in the sensitivity of the photoreceptor cells to light9.
The prolonged depolarizing afterpotential (PDA) protocol&#160;exploits the differences in the absorption spectra of rhodopsin and metarhodopsin that allows, in Drosophila, a massive photopigment conversion of R to its physiologically active and dark-stable intermediate M state2. In the ERG voltage response, a relatively short pulse of saturating light is given, and the resulting voltage response is recorded. Under this condition, a ceiling (reversal potential) is reached by the depolarization signal because activation of a fraction of a percent of the huge amount of rhodopsin molecules (~1 x 108) is sufficient to reach the ceiling. The presence of the phototransduction components in great abundance ensures that this ceiling will be reached even in mutants with a significant reduction in concentration or subtle malfunction of the phototransduction components. This situation precludes the isolation of these mutants. Pak et al. introduced the PDA screening7 seeking a reliable and revealing test to isolate visual mutants. In Drosophila, the PDA response is brought about by genetically removing the red screening pigment, which allows photopigment conversion, and the application of blue light, which is preferentially absorbed by rhodopsin (Figure 3A) and, thus, results in a large net conversion of the R to the M photopigment state. Phototransduction termination is disrupted at the level of the photopigment by a large net conversion of R to M, which, in turn, results in sustained excitation long after the light is turned off (Figure 2C, Figure 4A [top]). During the PDA period, the photoreceptors are less sensitive to subsequent test lights and are partially desensitized (inactivated). The PDA detects even minor defects in rhodopsin biogenesis and tests the maximal capacity of the photoreceptor cell to maintain excitation for an extended period. Since it strictly depends on the presence of high concentrations of rhodopsin, it easily scores for deficient replenishment of the phototransduction components. Remarkably, the PDA screen has yielded many new and very important visual mutants (reviewed in Pak et al.7). Thus, the PDA mutants isolated by Pak et al.7 are still extremely useful for analyzing the Drosophila visual system.
The PDA is induced in Drosophila by saturating blue light, resulting in continuous depolarization long after light offset (Figure 4A [top]). After saturating PDA-inducing blue light, the peripheral photoreceptors (R1-6) remain continuously active in the dark at their maximal capacity, reaching saturation. Additional saturating blue lights during the PDA do not produce any additional response in R1-6 cells for many seconds but induce a response in R7-8 cells that is superimposed on the PDA. The superimposed responses are explained by the different absorption spectra of the photopigments expressed in these cells (R7-8)16. The PDA can be suppressed by the photoconversion of M back to R with saturating orange light (Figure 4A [top]). The ability of the PDA to bring the photoreceptor cells to their maximal active capacity, a situation that cannot be achieved by intense white light, explains why it has been a major tool to screen for visual mutants of Drosophila. This is because it allows the detection of even minor defects in proteins involved in the biogenesis of normal photopigment levels17,18. Two groups of PDA defective mutants have been isolated: neither inactivation nor afterpotential (nina) mutants and inactivation but not afterpotential (ina) mutants. The phenotype of the former is a lack of a PDA and the associated inactivation arising from a large reduction in the photopigment levels (Figure 4A [middle]). The phenotype of the latter shows inactivation but no dark depolarization after blue light due to a still-unknown mechanism in the mutant with normal rhodopsin levels but lacking proteins interacting with the TRP channels (Figure 4A [bottom]).
The PDA arises from the difference in the amount of photopigment relative to arrestin (ARR2), which binds and terminates M activity19,20,21 (Figure 1A). In Drosophila photoreceptors, the amount of the photopigment is about fivefold larger than the amount of ARR219. Thus, ARR2 levels are insufficient to inactivate all the M molecules generated by a large net photoconversion of R to M, leaving an excess of M constantly active in the dark17,19,20,22,23. This mechanism explains the elimination of the PDA response by mutations or by carotenoid deprivation24,25, causing a reduction in photopigment level, but does not affect arrestin levels. Moreover, this explanation also accounts for the phenotypes of null ARR2 (arr23)&#160;mutant allele21, in which PDA could be achieved at ~10 fold dimmer blue light intensities19,20,21 (Figure 4B,C). The PDA is not a unique feature of fly photoreceptors, and it appears in every tested species that has dark stable M with an absorption spectrum different from that of the R state, allowing sufficient photoconversion of the photopigment from the R to the M state. A thoroughly investigated species in which the PDA phenomenology was discovered is the barnacle (Balanus) photoreceptor, in which the absorption spectrum of the R state is in a longer wavelength than the M state2 (Figure 3B). Accordingly, unlike the situation in the fly, in the barnacle, orange-red light induces a PDA, while blue light suppresses the PDA2.
The early receptor potential (ERP) protocol exploits the charge displacement occurring during R or M activation. The visual pigment is an integral part of the surface membrane of the signaling compartment of both vertebrate&#160;and invertebrate membranes3. Accordingly, the activation process in which the photopigment molecules change from one intermediate state to the next is accompanied by a charge displacement4,26. As the photopigment molecules are electrically aligned in parallel with the membrane capacitance4, a rapid synchronized conformational change generates a fast polarization change of the surface membrane, which, in flies, occurs in the signaling compartment composed of a stack of ~30,000-50,000 microvilli called rhabdomere. This polarization then discharges passively through the membrane capacitance of the cell body until the cell membrane is equally polarized. The ERP is the extracellular recording of the charge displacement. The intracellularly recorded ERP manifests the extracellular ERP integrated by the time constant of the cell membrane4,27,28. The current activated by the visual pigment charge displacement could also be measured in whole-cell voltage-clamp recordings29,30 (Figure 5A-D), with the major advantage (in early receptor current (ERC) recordings) of minimizing the effect of membrane capacitance on the kinetics of the signal.
The protocol section describes how to perform ERG measurements from Drosophila eye9 and ERC measurements by whole-cell recordings from Drosophila isolated ommatidia31,32. We also describe specific protocols that are used to investigate phototransduction in general and photopigments in particular.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe with elongated tip</td>
			<td></td>
			<td></td>
			<td>Figure 6M</td>
		</tr>
		<tr>
			<td>1 rough tweezers</td>
			<td></td>
			<td>Dumont #5, Standard</td>
			<td>0.1 mm x 0.06 mm, length 110 mm, Inox (Figure 6H)</td>
		</tr>
		<tr>
			<td>2 condenser lenses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A/D converter</td>
			<td>Molecular Device</td>
			<td>Digidata 1200</td>
			<td>Possible replacement: any digidata from molecular devices (e.g 1440A) -Figure 7C</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Almost perfect electronics</td>
			<td></td>
			<td>Possible replacement: Warner instruments- IE251A or IE-210 (comes with headstage)- Figure 7D</td>
		</tr>
		<tr>
			<td>Anti-vibration Table</td>
			<td>Newport</td>
			<td>VW-3036-OPT-01</td>
			<td>Figure 7H</td>
		</tr>
		<tr>
			<td>Capillaries</td>
			<td>Harvard Apparatus</td>
			<td>Borosilicate glass capillaries</td>
			<td>1 mm x 0.58 mm (Figure 6O)</td>
		</tr>
		<tr>
			<td>Clampex</td>
			<td>Molecular Device</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>CO2 tank</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cold light source</td>
			<td>Schott</td>
			<td>KL1500 LCD</td>
			<td>Figure 6C</td>
		</tr>
		<tr>
			<td>Delicate wipers</td>
			<td>Kimtech</td>
			<td></td>
			<td>Kimwipes (Figure 6K)</td>
		</tr>
		<tr>
			<td>Electrode holder</td>
			<td></td>
			<td></td>
			<td>Suitable for capillary O.D. 1 mm (Figure 6N, Figure 7N, and Figure 7P)</td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>Home made</td>
			<td></td>
			<td>Electromagnetic noise shielding and black front curtain (Figure 7K)</td>
		</tr>
		<tr>
			<td>Filter (Color)</td>
			<td>Schott</td>
			<td>OG590, Edge filter</td>
			<td>Figure 7S</td>
		</tr>
		<tr>
			<td>Filter (Color)</td>
			<td>Schott</td>
			<td>BP450/40 nm</td>
			<td>Figure 7S</td>
		</tr>
		<tr>
			<td>Filter (Color)</td>
			<td>Blazers</td>
			<td>550 nm</td>
			<td>Figure 7S</td>
		</tr>
		<tr>
			<td>Filter (Color) for cold light source</td>
			<td>Schott</td>
			<td>RG630</td>
			<td>Figure 6C</td>
		</tr>
		<tr>
			<td>Filter (Heat)</td>
			<td>Schott</td>
			<td>KG3</td>
			<td>Figure 7S</td>
		</tr>
		<tr>
			<td>Filters (Neutral density filter)</td>
			<td>Chroma</td>
			<td>6,5,4,3,2,1,0.5,0.3</td>
			<td>Figure 7S</td>
		</tr>
		<tr>
			<td>Flash Lamp system</td>
			<td>Honeywell</td>
			<td></td>
			<td>Figure 7U</td>
		</tr>
		<tr>
			<td>Fly sleeper system with injector</td>
			<td>Inject + matic</td>
			<td></td>
			<td>Figure 6A-B</td>
		</tr>
		<tr>
			<td>Lamp power supply</td>
			<td>PTI</td>
			<td>LPS-220</td>
			<td>Figure 7W</td>
		</tr>
		<tr>
			<td>Light detector</td>
			<td>Home made</td>
			<td></td>
			<td>Phototransistor (Figure 7O)</td>
		</tr>
		<tr>
			<td>Light guide</td>
			<td></td>
			<td></td>
			<td>3 mm diameter, 1.3 m long (Figure 7L,M)</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td></td>
			<td></td>
			<td>High-pressure ozone-free 75 W Xenon lamp (operating on 50 W), possible replacement: Cairn research- OptoLED (Figure 7R)</td>
		</tr>
		<tr>
			<td>Low temperature melting wax</td>
			<td>Home made</td>
			<td></td>
			<td>Composed of mixture of beeswax (Tm&#8776;62 &#176;C) and paraffin at ~3:1 to reach a melting temperature of ~55&#8211;56 &#176;C (Figure 6J)</td>
		</tr>
		<tr>
			<td>Magnetic stand for flies</td>
			<td>Home made</td>
			<td></td>
			<td>Figure 6I and Figure 7Q</td>
		</tr>
		<tr>
			<td>Microelectrode preamplifier system with head-stage</td>
			<td>Almost perfect electronics</td>
			<td></td>
			<td>Impedance tester (Figure 7G)</td>
		</tr>
		<tr>
			<td>Micromanipulator (mechanical coarse)</td>
			<td>Tritech Research, Narishige</td>
			<td>M-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator (mechanical fine)</td>
			<td>Leitz Microsystems</td>
			<td>Leitz Mechanical Micromanipulator</td>
			<td>Figure 7F</td>
		</tr>
		<tr>
			<td>pCLAMP</td>
			<td>Molecular Device</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td></td>
			<td></td>
			<td>60 mm</td>
		</tr>
		<tr>
			<td>Pulse generator</td>
			<td>AMPI</td>
			<td>Master 8</td>
			<td>Figure 7A</td>
		</tr>
		<tr>
			<td>Redux cream for electrocardiography</td>
			<td>Parker Laboratories</td>
			<td>Redux Electrolyte Cr&#232;me</td>
			<td></td>
		</tr>
		<tr>
			<td>Shutter driver</td>
			<td>Uniblitz, Vincent Associates</td>
			<td>VCM-D1 Single Channel Uni-stable</td>
			<td>Figure 7V</td>
		</tr>
		<tr>
			<td>Shutter system</td>
			<td>Uniblitz, Vincent Associates</td>
			<td>LS2 2 mm Uni-stable Shutters</td>
			<td>Figure 7V</td>
		</tr>
		<tr>
			<td>Silver Wire</td>
			<td>Warner Instruments</td>
			<td></td>
			<td>0.25&#8211;1 mm diameter, needs to be chloridized</td>
		</tr>
		<tr>
			<td>Soldering iron composed of a platinum-iridium filament</td>
			<td></td>
			<td></td>
			<td>0.25 mm diameter (Figure 6F)</td>
		</tr>
		<tr>
			<td>Stereoscopic zoom Microscope</td>
			<td>Nikon</td>
			<td>SMZ-2B</td>
			<td>Figure 6D</td>
		</tr>
		<tr>
			<td>Stereoscopic zoom Microscope</td>
			<td>Wild</td>
			<td>Wild M5</td>
			<td>With 6, 12, 25 and 50 magnification settings (Figure 7E)</td>
		</tr>
		<tr>
			<td>Syringe filters</td>
			<td>Millex</td>
			<td></td>
			<td>22 &#181;m PVDF filter</td>
		</tr>
		<tr>
			<td>Vertical pipette puller</td>
			<td>Sutter/ Narishige</td>
			<td>Model P-97/PP-830</td>
			<td>Use either vertical or horizontal puller, as preferred (Figure 6L)</td>
		</tr>
		<tr>
			<td>Wax filament heater</td>
			<td>Home made</td>
			<td></td>
			<td>See figure S1 (Figure 6E-G)</td>
		</tr>
		<tr>
			<td>Xenon Flash Lamp system</td>
			<td>Dr. Rapp OptoElectronic</td>
			<td>JML-C2</td>
			<td>Figure 7X</td>
		</tr>
	</tbody>
</table>

electrophysiological methods for measuring photopigment levels in drosophila photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Figure 2&#160;exemplifies the robustness and ease of using the ERG technique. It is robust because it is recorded in the virtually intact fly by a simple technique of extracellular voltage recordings that require a simple electrophysiological setup. The robustness is manifested by obtaining recordings of light responses with relatively large amplitudes (in the millivolt range) even when mutations strongly reduce or distort the light response. Therefore, even an inexperienced experimenter can...

Watch this Scientific Journal Video about Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors at JoVE.com

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems.&#160;

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...

This protocol is particularly suitable for genetic screen of visually defective Drosophila mutants. This is because they allow the detection for even small abnormalities in Drosophila phototransduction, particularly in photopigment levels and tier peer-related functions. This technique is profound in vivo, it's easy to execute and highly robust, enabling the detection of small changes in the activity of phototransduction proteins.In my opinion, fixing the flies while maintaining the viability is probably the critical part of this method. Turn on the computer and open the Clampex software. Turn on the amplifier and the Master-8 pulse generator, then turn on the xenon lamp and shutter controller.Switch on the puller and place a glass capillary in the puller and pull it. Remove the glass capillary from the puller. Fill the glass capillary with filtered ringer solution using an elongated tip syringe.Insert the wire electrode into the glass capillary. Ensure that the solution within the capillary is in contact with the silver wire. Insert the electrode holders into the two electrode micromanipulators.Turn on the power supply of the soldering iron. Set the current to approximately 2.25 ampere. This current should heat the 0.25-millimeter platinum-iridium filament to approximately 55 to 56 degrees Celsius.Place a drop of wax with a low melting temperature on the soldering iron. Anesthetize the flies within the bottle. Pour the anesthetized flies into the sleeper container, choose one fly and cover the rest of the flies with a Petri dish.Carefully hold the fly by its wings using a sharp tweezer and place it on the fly holder. On the fly holder, place the fly lying on its side with its back toward the hand. Using tweezers, lift the fly from its wings and fix its wings to the fly holder using the soldering iron.Connect the fly's back to the stand surface with wax using the soldering iron. Lower the soldering iron's tip to the joining point of the legs, then melt the wax to cover all legs together. Place a small drop of wax between the head and the neck in the neck area.Place the fly holder in a dark faraday cage on a magnet block and ensure that the fly is approximately five millimeters from the end of the light guide. Place the recording electrode above the fly's eye and the ground electrode over the fly's upper back using the micromanipulators. Then insert the ground electrode into the back of the fly using the micromanipulators.Insert the recording electrode into the outer periphery of the fly's eye, preferably using the micro manipulators. Close the faraday cage and turn off the lights in the room to allow adaptation to dark for five minutes. Input the parameters starting from a pulse duration of 500 meters per second, a pulse interval of 60 seconds, and the number of pulses set to six.For measuring the PDA, set the Master-8 pulse generator in TRAIN mode, then give a five-second light pulse of maximal intensity using an orange filter. Check the voltage response. Replace the orange filter with a broadband blue filter and give three 5-second light pulses at maximum intensity.Check the voltage response. Wait at least 60 seconds in the dark. Replace the blue filter with the previous orange filter.Then give two 5-second light pulses with 60-second intervals. Check the voltage response. For measuring the ERP M-potential, give a continuous blue light pulse until a steady state voltage response is reached.Give a brief intense light flash of a wavelength between 350 to 700 nanometers using a 20-nanometer band-pass filter. Then measure the peak amplitude of the M1 phase of the M-potential response, which reflects the metarhodopsin absorption at this specific wavelength at photoequilibrium. The PDA responses were evaluated for different mutant flies.Wild-type flies showed a saturated depolarization in the dark that appeared as prolonged corneal negative ERG, following intense blue light stimulation. The following blue lights produced small responses superimposed on the PDA, lacking the on and off transients. In mutant flies with abnormal photopigment biogenesis such as ninaE, the voltage response returned to baseline following intense blue light stimulation, and the response to an additional blue light was not suppressed and showed normal on and off transients.The wild-type ERP of the fly is obtained by the same protocol, but an intense green flash is applied during the PDA. This green flash elicits the ERP and suppressed the PDA. The M-potential was obtained by the green flash following blue illumination, but not by a blue flash after blue illumination in the wild-type fly.This protocol was repeated in wild type, in photopigment hypomorph mutant ninaE, and in phototransduction-deficient mutant with normal photopigment levels norpA. Relative absorption spectra of fly rhodopsin and metarhodopsin were calculated from photometric measurements of the different spectrum and the photoequilibrium spectrum. It is important for the survival of the flies to pay attention to the wax viscosity by refraining from overheating.This procedure is suitable for genetic and via screening of defective visual mutants. Methods that randomly isolate and identify visual mutations may facilitate the discovery of novel proteins and mechanisms involved in Drosophila phototransduction. The PDA protocol enabled the isolation of important and novel visual mechanisms.The participation would probably have not been discovered or even anticipated otherwise.

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