Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

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 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 chromophore^2,3, 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 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 cells^5,6.

Drosophila 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 eyes^8,9. 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 "on" and "off" transients at the beginning and end of the light stimulus that arise from the lamina neurons (Figure 2A, 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], D, 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 activation^10,11.

The depolarization of the photoreceptor generates the corneal-positive "on" transient, arising from the sign-inverting synapse between the photoreceptor axon and the monopolar neurons of the lamina^10,11 (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) channels^13,14,15. 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 light^9,10. In addition, at very strong illuminations, an additional transient response, which precedes and partially fuses with the "on" 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 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 levels^17,18. 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 activity^19,20,21 (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 dark^{17,19,20,22,23}. This mechanism explains the elimination of the PDA response by mutations or by carotenoid deprivation^24,25, causing a reduction in photopigment level, but does not affect arrestin levels. Moreover, this explanation also accounts for the phenotypes of null ARR2 (arr2³) mutant allele²¹, in which PDA could be achieved at ~10 fold dimmer blue light intensities^19,20,21 (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 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 displacement^4,26. 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 membrane^4,27,28. The current activated by the visual pigment charge displacement could also be measured in whole-cell voltage-clamp recordings^29,30 (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 ommatidia^31,32. We also describe specific protocols that are used to investigate phototransduction in general and photopigments in particular.