Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

Podsumowanie

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. 

Streszczenie

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.

Wprowadzenie

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.

Protokół

1. Measuring the intensity response relationship, prolonged depolarizing afterpotential (PDA), and the early receptor potential (ERP) using the electroretinogram

1. Suitable rearing conditions for D. melanogaster preparation
   1. Raise D. melanogaster flies in bottles containing standard yellow corn containing food in an incubator maintained at a temperature of 24 °C and in a 12 h dark/light cycle
   2. Keep the fly bottles in the dark at least 24 h prior to the experiment.
2. General setup
   1. Prepare recording pipettes by pulling 1 mm x 0.58 mm (O.D x I.D) fiber-filled borosilicate glass capillaries (Figure 6L, O). The resistance of the pipettes should be 5-10 MΩ; any suitable puller can be used.
   2. Coat two silver wires with AgCl₂, inserting 0.25 mm silver wire into 3 M KCl solution connected to a custom-made 5 V power supply.
   3. Insert each coated silver wire into the electrode holders (Figure 6N).
   4. Fill the glass capillary with filtered Ringer's solution (see Table 1) using an elongated tip syringe (Figure 6M).
   5. Insert the wire electrode into the glass capillary. Ensure that the solution within the capillary is in contact with the silver wire.
   6. Insert the electrode holders (Figure 7P, N) into the two electrode micromanipulators (Figure 7G).
3. Procedure of preparing the fly for electrical recordings
   NOTE: To keep the fly under dark-adapted conditions, use only dim red light illumination during the following steps.
   1. Anesthetize the flies in the bottle with CO₂ gas using the fly sleeper system (Figure 6A, B) and pour them into the sleeper container.
   2. Choose one fly and carefully hold it by its wing using a sharp tweezer. Cover the rest of the flies with a Petri dish.
   3. Place the fly on the fly holder in the proper orientation-lying on its side, with its back toward the hand (Figure 6P).
   4. Turn ON the power supply of the soldering iron. Set the current to ~2.25 A. This current should heat the 0.25 mm platinum-iridium filament to ~55-56 °C (see Supplemental File).
   5. Place a drop of wax with a low melting temperature (~55-56 °C) on the soldering iron (Figure 6F).
   6. Using tweezers, lift the fly from its wings and fix its wings to the fly holder (Figure 6I) using the soldering iron.
   7. Using the soldering iron, connect the fly's back to the stand surface with wax (Figure 6P).
   8. Lower the tip of the soldering iron onto the joining point of the legs and melt the wax to cover all the legs together (Figure 6P).
   9. Place a small drop of wax between the head and the back in the neck area (Figure 6P).
      NOTE: Take special care to avoid overheating the fly head. Ensure that the fly is properly fixed and is unable to move during the experiment; minor movements may create artifacts in the recordings. Ensure that the trachea openings (breathing inlets) in the thorax and abdomen are not covered with wax.
   10. Place the fly holder (Figure 7Q) in a dark Faraday cage on a magnet block (Figure 7I) and ensure that the fly is ~5 mm from the end of the light guide (Figure 7L).
   11. Place the recording electrode (Figure 7P) above the fly's eye and the ground electrode (Figure 7N) over the fly's upper back using the micromanipulators.
   12. Insert the ground electrode into the back of the fly using the micromanipulators.
   13. Insert the recording electrode into the outer periphery of the fly's eye, preferably, using the micromanipulators.
       NOTE: After inserting the electrode into the eye, a small dimple will be observed; pull the electrode upwards without removing it from the eye until the dimple disappears. The electrodes can also be immersed in small droplets of electrode jelly applied at the torso and eye.
4. Intensity-response protocol
   1. Place an orange filter (590 edge filter) in front of the high-pressure Xenon lamp. Use a large (six orders of magnitude) attenuating neutral density (ND) filter.
   2. Wait 60 s in the dark and give a 5 s light pulse.
   3. Replace the ND filter with a less attenuating ND filter in one order of magnitude increments.
   4. Wait 60 s in the dark and give a second 5 s light pulse.
   5. Repeat Steps 1.4.1.-1.4.4, gradually increasing the light intensity using less attenuating ND filters (the last pulse should be generated with no ND filter at all). Ensure to use the series of ND filters in the proper direction, starting from large attenuation and reaching low attenuation.
5. PDA protocol (this protocol can only be performed on white-eyed flies)
   1. Give a 5 s light pulse of maximal intensity using an orange filter (590 edge filter, to convert maximal photopigment to the R state).
   2. Replace the orange filter with a broad-band blue (BP450/40 nm) filter and give three 5 s light pulses at maximum intensity.
      NOTE: It is also possible to give a long continuous maximum intensity blue light pulse until a steady-state voltage response is reached.
   3. Wait 60 s in the dark, replace the blue filter with the previous orange filter, and give two 5 s light pulses with 60 s intervals.
6. ERP/M-potential protocol for measuring the photo-equilibrium spectrum of M (this protocol can only be performed on white-eyed flies^10,25)
   1. Give a continuous blue (bandpass (BP) 450/40 nm) light pulse until reaching a steady-state voltage response, which converts the maximal amount of photopigment from the R state to the M state of R1-6 cells.
   2. Give a brief (<3 ms) intense light flash of a wavelength between 350-700 nm (the known absorption spectrum of R1-6 cells photopigment) using narrow (~20 nm) bandpass filters and measure the peak amplitude of the M1 phase of the M potential response (which reflects the metarhodopsin absorption at this specific wavelength at photo-equilibrium^10,25).
      NOTE: For synchronous activation of a large pool of photopigment molecules, a brief, intense flash of light is required so that the photon content will be packed in a short duration. The M potential is composed of two components: M1 (corneal negative phase), which reflects the charge displacement of the metarhodopsin in the photoreceptor, and M2 (corneal positive phase^11,33), which reflects the amplified M1 response of the photoreceptors in the lamina^9,10,11. Each of these components can be identified and measured. However, it is preferable to measure the M1 potential since it is a direct linear manifestation of the M levels. If M2 is used, make sure that its amplitude is in the linear range by using mild carotenoid deprivation^24,25.
   3. Give a continuous blue (BP450/40 nm) light pulse again, followed by a brief (<1 ms) intense light flash of a different wavelength.
   4. Repeat this protocol until the entire absorption spectrum of M at photo equilibrium is covered.

2. ERC protocol for measuring the action spectrum of R and the M states of R1-6 cells using whole-cell voltage-clamp recordings

NOTE: For a detailed protocol for using whole-cell voltage-clamp recordings, see Katz et al.³⁴. The M-potential uses the ERG to measure the activation of the M state only because the contribution of the R state is suppressed by the membrane capacitance. In contrast, the ERC measures the activation of both R (positive ERC) and M (negative ERC) states because voltage-clamp recordings remove the effect of membrane capacitance (see introduction).

1. Convert the photopigment to the desired state (R or M). For R to M conversion, first, adapt the flies by a brief (<1 ms) adaptive blue (BP450/40 nm) flash. For M to R conversion, give a short adaptive orange (OG590 edge filter) flash.
2. Give a brief light flash (<1 ms) of a wavelength between 350-700 nm and measure the maximal negative or positive amplitude of the ERC response, which reflects the M/R absorption at this specific wavelength.
   NOTE: For synchronous activation of a large pool of photopigment molecules, a brief, intense flash of light is required to pack the photon content in a short duration. The maximal photopigment conversion from R to M by blue flash can reach ~80% of the total photopigment molecules because of the overlap in the absorption spectrum of R and M (Figure 3A). Therefore, at wavelengths below ~550 nm, the ERC has two components: a negative phase that reflects the response of the metarhodopsin, and a positive phase that reflects the response of the remaining rhodopsin. The ERC depends linearly on light intensity (Figure 5D). Accordingly, to derive the spectral sensitivity of R and M states, each phase of the ERC needs to be normalized at the various wavelengths for equal energy²⁹.
3. Repeat Steps 2.1.-2.2. using flashes of different wavelengths.
4. Plot the normalized positive and negative ERC as a function of wavelength.
   NOTE: The strong flash of light induces a massive current through the light-sensitive channels leading to metabolic stress on the photoreceptor, which, in turn, causes light-independent channel opening. The ERC is superimposed on this constitutive current.

Wyniki

Figure 2 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...

Dyskusje

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...

Materiały

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