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ERG in Drosophila

Written By

Gregor Belusic

Submitted: November 15th, 2010 Published: August 9th, 2011

DOI: 10.5772/21747

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1. Introduction

The fruitfly Drosophila melanogaster has been a workhorse of genetics for over a hundred years, following the first report of a white-eyed mutant by T.H. Morgan (Morgan, 1910). Since then, systematic mutagenesis and subsequently, more sophisticated methods of genetic modifications have been implemented to isolate strains with specific defects in various proteins (Ashburner et al., 2005; Greenspan et al., 2004). Finally, the newly developed techniques enabled the researchers to mark, activate or silence specific cells or cell lines (Hampel et al., 2011; Gohl et al., 2011). Genetic modification and elimination of eye-specific proteins have led to fundamental discoveries in the visual pathway and the deciphering of the mechanisms of phototransduction in great detail (Pak, 2010). The prominent compound eyes of Drosophila have allowed for an easy selection of the visual mutants on the basis of the eye color, external eye morphology, presence of the deep pseudopupil, phototaxis, and the time course and the amplitude of ERG (Pak, 1995). Drosophila ERG is an excellent method for physiological evaluation of genetically induced protein modifications. It is a particularly robust and technically feasible technique which can yield high throughput and reproducible results. The limitations of ERG have become very obvious in the last 20 years due to the development of the whole-cell patch-clamp in isolated ommatidia (Ranganathan et al., 1991; Hardie, 1991) and the advanced application of sharp intracellular electrodes (Juusola and Hardie, 2001). However, ERG in Drosophila is an informative and well understood signal. It remains the method of choice in many studies, especially when the molecular genetic tools are extensively applied to studying cellular processes in the photoreceptor model, and when the phenotypes of numerous transgenic fruitfly strains need to be physiologically characterised in a reasonable time.


2. The visual system of Drosophila

The visual system of Drosophila consists of the three ocelli, the compound eyes and the visual ganglia – the lamina, the medulla, the lobula and the lobula plate. The retina of a compound eye contains ca. 800 ommatidia. Each ommatidium is a cluster of eight photoreceptor cells, supporting cells and pigment cells. The pigment cells contain the red screening pigment which optically isolates the ommatidia. The photoreceptor cells contain an additional, yellow screening pigment which translocates transversely in the photoreceptor soma thus conveying a pupillary response. The yellow and the red screening pigments are absent in commonly used white-eyed mutants.

Figure 1.

Left, red and white eyed Drosophila. Right, relative absorbance of the five rhodopsins expressed in peripheral photoreceptors (R1-6: Rh1) and in the central photoreceptors (R7y: Rh4; R7p: Rh3; R8y: Rh6; R8p: Rh5). Shown are only the α-bands; Rh1, Rh5 and Rh6 have an additional β-band in the UV (peak height ca. 20% of the α-band peak).

Photoreceptor cells have three morphologically distinct regions: the light sensing rhabdomere, the soma with the nucleus, endoplasmic reticulum (ER), the mitochondria and other organelles, and the axon. The soma and the axon are sometimes referred to as the light-insensitive basal membrane, i.e. the membrane portion which is equipped with voltage gated (mostly K+) channels, which dynamically shape the light response. This is not to be confused with the basement membrane (sometimes also the basal lamina), an extracellular structure separating the photoreceptor bodies from the higher order neurons in the lamina, an optical neuropil.

The light-sensitive part of the photoreceptor cell - the rhabdomere - is composed of ~30.000 microvilli, each 1-2 µm long and 60 nm wide. The microvillus harbours all the molecules that are necessary for the transduction of a successfully captured photon of light to a quantum bump – the unitary depolarization of the photoreceptor cell membrane. The microvillar membrane is packed with about 1000 visual pigment (rhodopsin) molecules, making the rhabdomere optically dense and behave as a light guide. The rhabdomeres of an ommatidium together form an open rhabdom. The rhabdomeres of the six outer photoreceptor cells R1-6 function as separate optical waveguides, which surround a central waveguide formed by the superimposed rhabdomeres of the central photoreceptors R7 (positioned distally in the ommatidium) and R8 (positioned proximally). The receptors R1-6 form an achromatic channel for detection of light contrasts and motion, while R7 and R8 receptors mediate colour vision. The R1-6 contain the Rh1 rhodopsin, which absorbs maximally in the blue, at 486 nm, and the R7&8 cells express the rhodopsins Rh3-6, maximally absorbing at 331, 355, 442 and 515 nm; the UV-sensitive rhodopsin Rh2 is expressed in the ocelli (Salcedo et al., 1999). The rhodopsin content in the eyes depends on the amount of carotenoid precursors in the food. Elimination of carotenoids from the feeding media can result in dramatic reduction of rhodopsin in the eyes (Goldsmith et al., 1964; Harris et al., 1977). On the other hand, a carotenoid enriched diet results in high sensitivity of R1-6 cells in the UV, due to the incorporation of an additional chromophore in the opsin molecule which acts as an antenna pigment molecule (Kirschfeld et al., 1983). Maximised rhodopsin content, as well as structural integrity of photoreceptors, can be easily checked by observing the clarity of the deep pseudopupil (optical superposition of rhabdomere images or shadows in the geometrical centre of the eye). Optically, the compound eyes of Drosophila are of the apposition type. However, different R1-6 cells in six adjacent ommatidia are arranged so that their visual axes are pointing to the same direction, and their axons project to a common neuron in the lamina. Such information integration, which improves the image quality in dim light, is typical of the higher dipteran insects and is named neural superposition (Kirschfeld and Franceschini, 1968). The central R7 and R8 receptor axons bypass the lamina and form chemical synapses in the medulla (Cajal and Sanchez, 1915; Fischbach and Dittrich, 1989).

Figure 2.

Left, Spectral properties of the main visual pigment, Rh1, of Drosophila. The photosensitivities (β) of the two thermostable states, rhodopsin (R) and metarhodopsin (M), normalized to the rhodopsin peak, and the metarhodopsin fraction f M in the photoequilibrium created by monochromatic stimuli with wavelenght λ. The spectra were calculated with the template functions of Govardovskii et al. (2000) using peak wavelength values 486 and 566 nm for R and M, respectively. Right, Spectral sensitivities measured via ERG of white-eyed Drosophila bred on a carotenoid-rich (P+) and carotenoid-deprived (P-) medium. The spectral sensitivity in P- exhibits the β-peak due to absorption by Rh1 in the UV, while the P+ has an much higher sensitivity in the UV with a fine vibronic structure (triple peaks) due to the incorporation of an additional chromophore into the Rh1 molecule.

Fruitflies are able to detect light photons at wavelengths between 300 nm and 650 nm. Upon photon absorption, their rhodopsins (R) are converted to a thermostabile metarhodopsin (M) isoform. In the case of Rh1, the absorbance peak of M is shifted to longer wavelengths (bathochromic shift) and the absorbance amplitude is higher by a factor of 1.6 (Ostroy et al., 1974; Salcedo et al. 1999). A M molecule can be converted back to R by another photon. The conversion of M to R is facilitated by the leaking of red stray light through the long-pass filtering red screening pigments (Stavenga et al., 1973; Stavenga, 2002). After a sufficient period of time, a photoequilibrium between M and R is created which depends on the spectral composition of the illumination. Short-wavelength monochromatic light can create large amounts of M. M, which triggers phototransduction (Hardie and Raghu, 2001), is quenched by the binding of arrestin. Rhodopsin to arrestin ratio has been estimated as 2.7:1 (Satoh et al., 2010). Thus, high fraction of M can out-titrate the available arrestin (Minke et al., 1975; Hamdorf, 1979; Dolph et al., 1993; Belušič et al., 2010; Satoh et al., 2010). In the white-eyed mutants illuminated with conventional light sources, the photoequilibrium with high metarhodopsin fraction (f M) can be established within seconds. Thus, a state of maximal receptor depolarization and absolute desensitisation is created, which can persist for many hours in the dark. This phenomenon is called the PDA, prolonged depolarizing afterpotential. A PDA can be abolished with long wavelength light. Usually, the PDA is created with blue light (460-490 nm) and interrupted with orange to red light (580-640 nm). In R7 photoreceptors, PDA can be created with UV and abolished with blue light (Stark, 1977). PDA has never been observed in R8 receptors.


3. The ERG signal

3.1. Recording preparation

The ERG is measured in immobilised living fruitflies. Fruitflies (and other flies) can tolerate extended periods of anoxia without any subsequent changes in the ERG (Agam et al., 2000). Therefore, living animals can be anaestethised by exposure to gaseous CO2 or N2 at room temperature or by chilling on ice. Subsequently, they are mounted to a holder, (e.g. a microscope slide or pipette tip) with wax, agarose or putty. Frequently, Krönig's mixture of bees wax and resin (colophony) 3:1 is used due to its excellent adherence to the chitin and its low melting temperature. The waxing is best performed with miniature soldering devices, such as a 12 V soldering needle (Stannol, Germany), driven at 3-4 V. It is important that the animal's abdomen with the stigmata is left untreated during the immobilization procedure in order to avoid suffocation. Suffocation is first indicated by the absence of the neural transient components of the ERG, but a somewhat slowed receptor component can persist for many minutes. The ERG can be recorded in virtually intact animals, and a stable measurement is possible for many hours or even days, if sufficiently high humidity is provided in the air surrounding the animal. The signal is usually recorded with glass micropipettes filled with physiological saline – 0.9% NaCl or insect salines of different compositions, yielding a resistance of a few kΩ to a few MΩ. An ERG can be recorded also with a chlorided silver wire or salinated cotton wicks. Direct illumination of the silver wire must be avoided since a photoelectric artifact of a few mV is easily introduced. The signal is amplified unipolarly in DC mode, with the recording electrode contacting the compound eye dipped into a droplet of EKG gel, or inserted just below the cornea. The grounded reference electrode is dipped into a gel droplet or inserted in a non-illuminated part of the body. Similarly, an ERG can be also obtained from the ocelli.

3.2. Stimulus

The stimulus for obtaining an ERG is typically a simple light pulse, lasting between 10 ms and 10 s. The light source can be a DC operated halogen lamp, a xenon arc lamp, or LED. AC driven lamps and conventional fluorescent bulbs are inadequate since they contain flickering components at 50 Hz or 100 Hz which a fly eye actually perceives as flicker, resulting in oscillations of the signal. The light from the lamps is usually filtered with long-pass (OG515, OG580, Schott, Germany) or short-pass light filters (BG family of filters, Schott, Germany). Monochromators inserted between the light and the preparation can be operated between 300 and 650 nm to produce stimuli for the measurements of spectral sensitivity or for selective light adaptation of different photoreceptor classes. Superluminescent LED sources can deliver light from 300 nm on, capable of saturating any fly photoreceptor. The intensity of the stimuli is adjusted with neutral density filters or metal wedges. In order to cover the entire dynamical working range of the Drosophila ERG, the intensity of the light stimuli needs to be adjusted over a range of 6 orders of magnitude, in steps no larger than 0.5 log unit. LED sources can be driven with voltage operated current sources, which allow for fast changes of intensity modulation and thus provide e.g. sinusoidally modulated stimulus or white noise, spectrally flat up to 500 Hz (fast enough even for the fastest insect photoreceptors).

Figure 3.

A simple setup for Drosophila ERG, constructed by the author and dr. Olaf Voolstra. Left, Stimulator made of two superluminescent LED sources (480 nm and 580 nm, Roithner Lasertechnik, Austria), three sets of latchable neutral density filters and a 50% neutral density filter (45° inclined) as a beam mixer. Optics and mechanics by Qioptiq, Germany. Right, Faraday cage with an optical breadboard (Thorlabs, Germany), binocular (Zeiss, Germany), a micromanipulator (Märzhauser, Germany) with the recording headstage (NPI, Germany), an improvised Drosophila yoke made from a micropipette holder (WPI, USA), with a 50 µm diameter Ag/AgCl wire (Goodfellow, UK) attached as a reference electrode. The objective lens of the stimulus setup protrudes from the left wall of the cage. Photo: O. Voolstra, University of Hohenheim, Stuttgart, Germany.

Figure 4.

The photoelectric effect. Signal in a dead animal, recorded with the Ag/AgCl wire directly illuminated with the stimulating light (stimuli corresponding to the upper trace: o - 580 nm; b - 480 nm; stimuli corresponding to the lower trace: uv – 360 nm; v – 420 nm).


3.3 Red-eyed and white-eyed fruitflies

The ERG is usually recorded in a few varieties of white-eyed Drosophila. White-eyed fruitflies are preferably used in biochemical experiments since the red pigment from the wild-type eyes colours the homogenates and the gels. In physiological experiments, white-eyed mutants are often preferred because they lack the pupillary response, in wild type mediated by the movement of yellow pigment granules within the photoreceptors. Moreover, uniform »ganzfeld« illumination of the entire compound eye is easily achieved in the translucent retina of white-eyed mutants. The angular sensitivity of the photoreceptors in the white-eyed mutants is extended more than tenfold (Streck, 1972), so that image formation is heavily impaired. The photoreceptor cells of the white-eyed mutants are otherwise physiologically unaffected. However, the red screening pigment in the wild-type flies has an important role as a long-pass, red filter so that illuminations with broadband light allow long wavelength photons reaching the photoreceptors. These photons convert metarhodopsin to rhodopsin so that the metarhodopsin levels in the photoreceptors are kept sufficiently low to be manageable by the limited amount of arrestin molecules. In the white-eyed fruitflies, this is not the case. Under white light, the metarhodopsin levels in white eyes can become so high that the photoreceptors remain depolarized for long periods of time. Furthermore, in physiological experiments, short wavelength light is frequently used as a stimulus. White eyed flies then must be regularly exposed to orange or red light in order to interrupt any possible prolonged depolarizations.

3.4. ERG waveform

The ERG recorded from the retina in the described configuration (DC, unipolar, measuring electrode in the retina, grounded reference in the body) is a rather complex signal, composed of several fast transient components, and several rising and decaying sustained components (Stark and Wasserman, 1972). The photoreceptors contribute a corneal-negative, slow, sustained plateau component at medium light intensities. The second-order neurons L1 and L2 in the lamina contribute fast transient »on« and »off« components, at the beginning and at the end of the stimulus (Coombe, 1986). At light intensities which elicit maximal responses, the ERG is more complex due to fast adaptation processes of the photoreceptor cells. The receptor response is further transformed at the synapse and in the lamina, resulting in additional transients in the ERG (Järvilehto and Zettler, 1973; Laughlin et al., 1987).

In order to understand the nature and origin of the ERG time course in detail, it is necessary to delineate the underlying responses of the photoreceptor cells and second order neurons. All photoreceptor cells (R1-8) respond to a simple light pulse with a sustained depolarization, occurring with a delay as short as 3 ms. This so-called receptor potential lasts as long as the stimulus. Its amplitude is proportional to the logarithm of the stimulus intensity. At high light intensities, the large depolarization of the photoreceptor at the onset of the pulse is reduced after 50-100 ms by fast adaptation mechanisms to a lower level. This results in a sharp spike at the beginning of the receptor potential. In light adapted flies, the fast adaptation is frequently followed by a damped oscillation of the receptor potential plateau. The receptor potential is detected by the ERG electrode in the extracellular space as a corneal-negative signal, with a somewhat smaller amplitude (e.g. -20 mV between the retina and the body, instead of the 40 mV of the transmembrane depolarization), and its time course resembles an inverted receptor potential. The photoreceptors R1-6 convey the receptor potential to second order neurons in the lamina, the L1 and L2. These neurons transform the receptor potential to a sequence of decaying transient components and thus seem to work analogously to the on- and off-bipolar cells in the vertebrate retina (Joesch et al., 2010). The laminar neurons act as high-pass filters with strong amplification at high frequencies, thus greatly amplifying small changes in the receptor potential (Autrum et al., 1970; Uusitalo and Weckström, 2000). The combined signal from L1 and L2 is very well detected by the ERG electrode and contributes a corneal-positive »on« transient at the beginning of the light pulse, and a corneal-negative »off« transient at the end of the pulse.

Figure 5.

Origin of the ERG waveform in the retina (Re), recorded in DC mode, unipolarly across the basement membrane (Bm). Left, the ERG from the photoreceptors R1-6, which is an extracellular summation of the inverted receptor potential (RP) from R1-6, and the inverted signal from the large monopolar cells (LMC) L1 and L2 in the lamina (La). Right, the ERG from the photoreceptors R7 and R8, which is a scaled-down, inverted receptor potential from R7 and R8. The large LMC transients are absent because the R7 and R8 form synapses in the medulla (Me). Sometimes, the ERG from R7 and R8 is accompanied with an on-transient (ERG R7,8, grey trace), since R7 and R8 form gap junctions (Gj) with the R1-6 axons in the lamina.

The photoreceptors R7 and R8 form chemical synapses in the medulla and the synapsing higher order interneurons do not contribute any signal to the ERG. Yet, light responses of the R7 and R8 cells are sometimes accompanied by transients, which are presumably due to electrical synapses between the R7 and R8 and the axons of R1-6 and accompanying R1-6 signals to L1 and L2 (Shaw, 1984; Juusola, personal communication).

The Drosophila ERG is probably shaped also by the pigment cells which respond to the changes in the ionic composition of the extracellular medium next to the photoreceptors by actively pumping ions (Hamdorf et al., 1978). The receptor potential plateau during long stimuli thus slowly decays, which is reflected in a not fully sustained ERG. The ERG at high light intensities is often accompanied by corneal-positive afterpotentials, in the dark after the stimulus, which is probably due to the hyperpolarizing action of the activated Na+/K+ pump in the receptor membrane (Jansonius, 1990).


4. Quantitative analysis of ERG

4.1. ERG amplitude and stimulus-response relationship

The ERG recorded with an electrode in the distal retina and the reference elsewhere in the body is actually measured as a change in the voltage drop across the basement membrane. The lateral resistance between the ommatidia is low, so that the ERG is not a local phenomenon. The basement membrane separating the retina and the lamina functions as a blood-brain barrier and has a much larger resistance; even in the dark, a voltage drop across the eye of 30-50 mV exists (Heisenberg, 1971). Illumination of only a few ommatidia of a red-eyed fruitfly creates an ERG amplitude of only a few mV, even at high light intensities. In a white-eyed fly, the ERG in a brightly illuminated eye can measure more than 40 mV peak-to-peak, with a sustained (DC) component of as much as 30 mV. The extraordinary large amplitude of the ERG in the uniformly illuminated eye is a result of the concerted activity of a large number of photoreceptors, which produce large currents running across the high resistance of the basement membrane.

Other measuring configurations allow for isolation of specific ERG components. First, if the measuring electrode is advanced into the retina, the basement membrane is penetrated at ca. 120-150 μm from the cornea. This is marked by a sudden voltage drop of ca. 15 mV and a change in the ERG shape. At a depth of between 120-200 µm below the cornea, the receptor component becomes negligible, so that the neuronal transients dominate the ERG response. Further advancement of the electrode leads to a complete loss of the ERG signal. If the reference electrode is placed deep into the receptor layer, just above the basement membrane, the ERG attains the shape of inversed receptor potentials and no neural transients are detectable in the signal. The effects of different recording configurations are described in great detail by Heisenberg (1971).

Both the photoreceptor and the neuronal ERG components are graded with respect to the stimulus intensity. The ERG amplitude is graded over a very wide range of stimulus intensities, spanning more than 6 log units. The relationship between the logarithm of light intensity and the sustained ERG component is very well described by the Hill equation

V / V m a x = I n / ( I n + I 5 0 n ) E1

where V/V max is the normalized ERG amplitude, I is the logarithm of the relative light intensity (log(I/I max ); at logI=0, V=V max,, n is the Hill slope (slope of the sigmoid curve at the half maximum) and I 50 is the light intensity producing 50% of the maximal ERG amplitude. The Hill coefficient or Hill slope n is used to describe the dynamic working range of a photoreceptor cell. If measured intracellularly, n in Drosophila photoreceptors is typically around 0.6 (Wu and Pak, 1978). The Hill slope as estimated from the ERG in white eyed fruitflies in the dark adapted state is as low as 0.49 (Belušič et al., 2010). This means that the estimated photoreceptor dynamic working range via ERG is 5-6 log units or orders of magnitude, and 3-4 log units via intracellular measurements. The dynamic working range via ERG is obviously exaggerated by 2 log units or 100-fold. The difference between the two methods is probably due to the different illumination conditions.

In intracellular measurements, a single cell is illuminated on-axis, and the whole dynamic range of a photoreceptor, from single quantum bumps to saturation is easily studied. In the ERG, oblique illumination with a beam of a limited numerical aperture is most often used, so that a population of the photoreceptors is not illuminated directly, but rather with the scattered light penetrating the retina. The photoreceptors in the white eyed mutant still retain a fraction of directional sensitivity (Streck, 1972) and are progressively recruited as the oblique beam gets stronger. Thus, the saturation state can be reached only at very high light intensities, often higher than available. The problem is even more pronounced in the red eyed flies, but can be overcome by using a hemispherical diffuser around the compound eye, providing real »ganzfeld« illumination. Anyhow, the measurement of the entire dynamic working range is feasible with proper illumination, allowing for very precise estimates of light sensitivity in different populations of the fruitfly.

Figure 6.

The stimulus-response relationship. Left, Sequence of ERG recordings from a wild-type, white-eyed fruitfly; stimulus intensity attenuated from -4.5 log (10-4.5) to 0 log (full light) in 0.5 log steps. The response is not saturated. Right, The »V-log I curve« as obtained with ERG and with intracellular recordings. Curves are plotted with the Hill equation (1) with the Hill slope n = 0.49 (ERG 1,2) and 0.66 (IC (1)), respectively, and I 50 = -3.4 (ERG 1), -2.9 (ERG 2) and -4.3 (IC). The intracellular curve (IC (1)) corresponds to a hypothetical photoreceptor in the most illuminated part of the eye in ERG 1. The ERG curves are shallower than the IC curve due to non-ideal illumination of the retina and the progressive recruitment of the shaded photoreceptors at high light intensities. The working dynamic range of the photoreceptors (3-4 orders of magnitude, IC) as measured intracellularly is exagerrated in the ERG (5-6 orders of magnitude, ERG). The light sensitivity in the flies of ERG 1 is higher than in the flies of ERG 2 by 0.5 log units, i.e. 3.16 fold. Such differences between fly strains can be reliably measured with appropriate instrumentation and skills.

The ERG in Drosophila is a very sensitive method. In the white eyed, wild-type flies, the half maximal intensity (I 50) corresponds to the photon catch rate of 2.9 × 104 photons s-1 per receptor cell (Belušič et al., 2010). Since the signal is detectable already at the light intensities that are three log units lower, i.e. 10-3 × I 50, it follows that at the threshold of the ERG, the rate of photon absorption per receptor is as low as 10 s-1, when the individual quantum bumps are still discernible in the intracellular recordings. The weak supratreshold ERG signal is however characterized only by the neural transients. Namely, the Drosophila eye belongs to the neural superposition type, typical of the higher dipteran insects. This means that the axons of the six R1-6 photoreceptors in the adjacent ommatidia which are directed into the same point in the space project to the common neuron in the lamina. Thus, a laminar neuron receives the input from a larger area, covered by six photoreceptors, without much loss in the angular resolving power, and the limited sensitivity of the eye due to the apposition optical design is greatly enhanced. In addition, laminar neurons strongly amplify the small changes in the receptor potential: even the single quantum bumps are faithfully transmitted to the brain (Dubs et al., 1981). Due to the nature of the neural summation of photoreceptor responses, the amplification and the filtering properties of the laminar neurons, i.e. the neuronal component of the ERG signal is detectable even at very dim light, where the photoreceptor response is dominated by single photon absorptions.

4.2. The dynamic properties of ERG

The dynamic properties of the ERG should be treated with great caution. Firstly, Drosophila is a poikilothermic animal and the rates of its physiological processes depend strongly on the temperature. The eyes of the flies (Calliphora vicina, Drosophila melanogaster) can operate at temperatures of up to 45°C, and the ERG can be detected even at -10°C. The ERG parameters exhibit temperature quotients Q10 between 2 and 4 (Hamdorf and Keller, 1962; Stušek et al., 2000). Therefore, it is very important to control the temperature of the preparation and avoid possible heating of the eyes with broadband light sources, which contain infrared light. Secondly, the latencies and rates of depolarization and repolarization depend strongly on the state of the light adaptation and on the effective illumination intensity and tend to become shorter and quicker with the increasing light adaptation. Neural transients are strongly high-pass filtered and amplify the fast components of the receptor response. The amount of the neural ERG transients depends on the quality of the preparation, the ability of the animal to ventilate and on the depth of the electrode within the retina. The visual system of a fruitfly can follow the stimulus flicker of up to 80 Hz at the room temperature, but the signal at the high frequencies originates exclusively in the lamina neurons. If the contribution of the neurons is diminished for any of the aforementioned reasons, the ERG waveform will become slower and the flicker response will cease (flatten) at lower stimulus frequencies.

The speed of repolarization as measured in the ERG seems to reflect the amount of arrestin available in the microvilli (Lee et al., 2003; Satoh et al., 2010) or the ability of arrestin to bind to metarhodopsin (Elsaesser et al., 2010). In the dark adapted state, microvilli contain only 25% of the total arrestin available. With light adaptation, the remaining arrestin is progressively translocated from the soma to the rhabdomere, and the repolarization is accelerated (Satoh et al., 2010).

4.3. The PDA paradigm

Extended or very bright illumination of a fruitfly eye can create any desired fraction of metarhodopsin (f M) between 0 and 0.7. The easiest way to establish a defined photoequilibrium is to use bright monochromatic light, since the absorbance characteristics of R and M are well known and allow for the precise, wavelength dependent setting of f M. f M>0.37 will create a PDA, i.e. maximally depolarize the photoreceptor cell for many hours, even in the dark. PDA can be created with light at wavelengths below 520 nm, and is quickly abolished with orange or red light above 580 nm, which brings the f M nearly to 0 and leads to repolarization of the photoreceptors. During the blue-light induced PDA, receptors R1-6 are inactivated, but the responses of R7 and R8 can still be elicited with blue, violet or UV pulses (Minke et al., 1975). In the UV-sensitive R7y and R7p receptors, PDA can be created with UV light and abolished with blue light (Stark, 1977). PDA in R8 receptors has never been observed. During the PDA in R1-6, it is not only possible to isolate the ERG of R7 and R8, but also of R8y exclusively in the mutant sevenless (sev, lacking R7y, R7p and R8p). PDA represents the maximal metabolic load to photoreceptors and reflects their ability to sustain maximal depolarization for an extended period of time.

The most frequently applied sequence of light pulses presented during a PDA assay is orange – blue – blue – orange – orange (OBBOO), each pulse 5-10 s long, with 10-20 s dark intervals. The first O pulse converts all M to R; the first B creates high f M and PDA; the second B is to make sure the f M is maximal or to further increase f M if the first B was too weak or short; the second O reconverts M back to R and and starts the repolarization; the last O pulse is applied to check if the preceding O was sufficient to bring f M next to 0. PDA has been traditionally used to characterise the phenotypes of visual mutants. This protocol also allows for separation of the photoreceptor activation from inactivation and deactivation.

Figure 7.

The PDA paradigm. Left, PDA in the wild-type. Right, PDA in the sevenless mutant. Upper row, PDA elicited with blue light (473 nm, 5 s) and abolished with orange light (580 nm, 5 s). Lower row, PDA elicited with blue light (473 nm), additional PDA in R7 cells elicited with UV light (360 nm, 5 s; grey rectangles). In the wild type, PDA isolates the response of R7 and R8; additional UV isolates the ERG of R8y and R8p (lower row, left, second UV pulse). In sevenless, R7 and R8p cells are absent; »blue PDA« isolates the response of R8y (upper row, second blue pulse; lower row, UV pulses), which do not enter PDA under any condition. Calibration bars in the lower left corner, 5 s and 5 mV.

Sometimes the blue light pulse cannot provoke a PDA, photoreceptors repolarize vigorously, and the ERG returns back to the baseline. The inability to provoke a PDA can indicate low rhodopsin content due to the lack of carotenoids in the food. Rhodopsin levels are low also in the mutants of the Rh1 gene ninaE. The ninaE mutant has been named after its inability to enter a PDA, the property named »neither inactivation, nor activation«, abbreviated into nina. PDA is absent also in certain retinal degeneration mutants during early phases of degeneration of photoreceptors R1-6 (such as in the mutant rdgC, retinal degeneration C).

Figure 8.

Left column, absence of PDA in the white-eyed, wild-type fruitfly, reared without the carotenoids in the food. Note the transients indicating that the R1-6 receptors are functional. Right column, same paradigm applied in a ninaE fly. The absence of large transients indicates that only receptors R7 and R8 are functional. R7 enter PDA in the UV. Rectangles, 5 s stimuli (orange, 580 nm; blue, 480 nm; black, 360 nm). Calibration bars on the lower left indicate 5 s and 5 mV.

Fruitfly strains can be compared with respect to the amount of blue light, required to enter PDA. The amount can be set by varying pulse intensity or duration. A more precise method is to set a defined f M in order to provoke PDA with a series of long pulses at different wavelengths. PDA creation depends on the ratio between rhodopsin and arrestin or the ability of arrestin to inactivate metarhodopsin. Thus, the quantity of arrestin can be calculated from the f M required to provoke a half-maximal PDA (Belušič et al., 2010). PDA is created at low f M in the mutants of the visual system specific arrestin, arr2.

In certain fruitfly mutants, the receptor potential cannot be sustained during the light pulse. The ERG returns to baseline, but a subsequent light pulse does not elicit a response: the photoreceptors are inactivated. Obviously, PDA cannot be created. Such ERG waveform has been named the transient receptor potential and was described in the first discovered visual mutant of the fruitfly, trp (Cosens and Manning, 1969). The mutant lacks the canonical TRP channel which mediates the major portion of the light-induced current in the photoreceptors. Similar phenotype can be observed in the mutants of the inaD class which have mutated or lack the scaffolding protein INAD. This protein anchors TRP and, as a consequence of the mutation, TRP is lost from the microvillar membrane. The abbreviation ina denotes the phenotype »inactivation – no afterpotential«, describing the absence of PDA and photoreceptor inactivation.

Figure 9.

Transient ERG caused by the transient receptor potential in a inaD 1 mutant (upper trace) and in a trp P343 mutant (lower trace). Calibration bars, 5s, 3 mV.

All the key players in Drosophila phototransduction have been so far studied via mutations which rendered them non-functional. Elimination of certain proteins resulted in very pronounced, easily discernible ERG phenotypes. These were the rhodopsin Rh1 (gene and mutant ninaE), the light operated channel TRP (trp), the scaffolding protein INAD (InaD), and the arrestin 2 (arr2). Mutation in the gene norpA which eliminated the enzyme phospholypase PLCβ resulted in the absence of receptor potential (no receptor potential; norp). However, a number of different mutations have yielded weak ERG phenotypes, converging phenotypes or phenotypes hard to explain. Such were the mutations which eliminated the TRPL channel (resulting e.g. in oscillations of the ERG), yielding an ERG phenotype similar to the one in the flies with arrestin 1 eliminated (Belušič, 2003). Elimination of the protein kinase C (eye specific PKC) gene inaC resulted in an inactivation phenotype, similar as in inaD.

A number of mutations that include protein eliminations, point mutations or ectopic protein expression result in a general degeneration phenotype. This phenotype is manifested by delayed repolarizations, responses that are diminished over time, and the gradual disappearance of the R1-6 response.

Figure 10.

ERG phenotype in a retinal degeneration mutant rdgC. The fly has been dark-raised for a week. Exposure to bright light in the ERG setup elicited rapid decay of the photoreceptors within minutes (numbers at the left of the traces), characterized by progressively slowed repolarization and decreasing amplitude.


5. Future applications of ERG

ERG in combination with other techniques

ERG in Drosophila is a feasible and robust technique that allows for simultaneous measurement of other physiological signals from the eyes. These are the measurement of ionic concentrations with the ion-selective electrodes (Agam et al., 2000), monitoring of pO2 with carbon fibre electrodes (Meglič et al., 2009), spectrophotometrical measurement of redox states of mitochondria in the photoreceptors (Zupančič, 2003; Meglič and Zupančič, 2011), photometrical estimation of metarhodopsin to rhodopsin ratio (Belušič et al. 2010), or monitoring of GFP marked proteins in the eyes (Meyer et al,. 2006). Exclusive ectopic expression of UV rhodopsin in all functional photoreceptor classes allows for separate photoreceptor stimulation at short wavelengths and optical measurements at long wavelenghts (Liu et al., 2008).

Drosophila as a model for human disease

Drosophila is an animal model for numerous human diseases (Pandey and Nichols, 2011). Nearly 75% of human disease-causing genes are believed to have a functional homolog in the fruitfly. The human diseases available for research in Drosophila include Parkinson's disease, Alzheimer's disease, several cardiac diseases, amyotrophic lateral sclerosis, fragile X syndrome, hereditary spastic paraplegia, Huntington disease, hypoparathyroidism-retardation-dysmorphism syndrome, Machado-Joseph Disease, metabolic diseases, diabetes, obesity, microcephaly, neuronal ceroid lipofuscinosis, Niemann-Pick disease, polyglutamine diseases, seizure disorders, epilepsy, spinal muscular atrophy, spinocerebellar ataxia 2, Werner syndrome, and X-linked mental retardation. Several mutations which are lethal if expressed in all somatic cells can be conveniently studied if expressed only in the visual system. This is easily achieved by the driver-reporter genetical systems (Greenspan et al., 2004), and the effects of expression can be elegantly followed by ERG.


  1. 1. Agam K. von Campenhausen. M. Levy S. Ben-Ami H. C. Cook B. Kirschfeld K. Minke B. 2000 Metabolic Stress Reversibly Activates the Drosophila Light-sensitive Channels TRP and TRPL in vivo. Journal of Neuroscience, 20 15 (August 2000), 5748 5755 . 0270-6474
  2. 2. Ashburner M. Golic G. K. Scott H. R. 2005 Drosophila: A Laboratory Handbook. Cold Spring Harbor Laboratory Press, 978-0-87969-706-8 Woodbury, USA.
  3. 3. Autrum H. Zettler F. Jarvilehto M. 1970 Postsynaptic Potentials From a Single Monopolar Neurone of the Ganglion Opticum of the Blowfly Calliphora. Zeitschrift fuer Vergleichende Physiologie, 70 4 (December 1970), 414 424 . 0004-4362X
  4. 4. Belušič G. 2003 A double role for arrestin 1? In: Proceedings of the 29th Göttingen Neurobiology Conference and the 5th Meeting of the German Neuroscience Society 2003, Elsner, N., Zimmerman, H. (eds.), Georg Thieme Verlag: Stuttgart, Germany; Abstract 459. 3-13137-351-2
  5. 5. Belušič G. Pirih P. Stavenga D. G. 2010 Photoreceptor Responses of Fruitflies With Normal and Reduced Arrestin Content Studied by Simultaneous Measurements of Visual Pigment Fluorescence and ERG. Journal of Comparative Physiology A, 196 1 (January 2010), 23 35 . 0340-7594
  6. 6. Cajal S. R. Sanchez D. 1915 Contribucion al Conocimiento de los Centros Nerviosos de los Insectos (Madrid: Imprenta de Hijos de Nicholas Moja).
  7. 7. Coombe P. E. 1986 The Large Monopolar Cells L1 and L2 are Responsible for ERG Transients in Drosophila. Journal of Comparative Physiology A, 159 5 (September 1986), 655 665 . 0340-7594
  8. 8. Dolph P. J. Ranganathan R. Colley N. J. Hardy R. W. Socolich M. Zuker C. S. 1993 Arrestin Function in Inactivation of G Protein-Coupled Receptor Rhodopsin In Vivo. Science, 260 5116 (1993), 1910 1916 . 0146-0404
  9. 9. Dubs A. Laughlin S. B. Srinivasan M. V. Single Photon Signals in Fly Photoreceptors and First Order Interneurones at Behavioral Threshold. Journal of Physiology, 317 317 No., (August 1981), 317 334 . 0022-3751
  10. 10. Elsaesser R. Kalra D. Li R. Montell C. 2010 Light-induced Translocation of Drosophila Visual Arrestin2 Depends on Rac2. Proceedings of the National Academy of Sciences of the U.S.A, 107 10 (March 2010), 4740 4745 . 0027-8424
  11. 11. Fischbach K. F. Dittrich A. P. M. 1989 The Optic Lobe of Drosophila melanogaster. I. A Golgi Analysis of Wild Type Structure. Cell and Tissue Research, 258 , 1 (January 1989), 441 475 . 0030-2766X
  12. 12. Goldsmith T. H. Barker R. J. Cohen C. F. 1964 Sensitivity of Visual Receptors of Carotenoid-Depleted Flies: A Vitamin A Deficiency in an Invertebrate. Science, 146 3640 (October 1964), 65 67 . 0146-0404
  13. 13. Govardovskii V. I. Fyhrquist N. Reuter T. Kuzmin D. G. Donner K. 2000 In Search of the Visual Pigment Template. Visual Neuroscience, 17 4 (September 2000), 509 528 . 0952-5238
  14. 14. Greenspan R. J. 2004 Fly Pushing: The Theory and Practice of Drosophila Genetics. Cold Spring Harbor Laboratory Press, 978-0-87969-711-2 Woodbury, USA.
  15. 15. Gohl D. M. Silies M. A. Gao X. J. Bhalerao S. Luongo F. J. Lin C. C. Potter C. J. Clandinin T. R. 2011 A Versatile In Vivo System for Directed Dissection of Gene Expression Patterns. Nature Methods, 8 3 (March 2011), 231 237 . 1548-7091
  16. 16. Hamdorf K. Höglund G. Schlecht P. 1978 Ion Gradient and Photoreceptor Sensitivity.
  17. 17. Journal of. Comparative Physiology. A. Vol 125 3 September 1978), 237 252 . 0340-7594
  18. 18. Hamdorf K. 1979 The Physiology of Invertebrate Visual Pigments. In: Autrum H (ed) Handbook of sensory physiology, vol VII/6A. Springer, Berlin, 145 224
  19. 19. Hamdorf K. Keller L. R. 1962 Das Verhalten des Elektroretinogramms von Calliphora im Temperaturbereich von-10°C bis +35°C. Zeitschrift fuer Vergleichende Physiologie, 45 6 (November 1962), 711 724 . 0004-4362X
  20. 20. Hamdorf K. Razmjoo S. 1979 Photoconvertible Pigment States and Excitation in Calliphora; the Induction and Properties of the Prolonged Depolarizing Afterpotential. Biophysics of Structure and Mechanism 5 137 161 . 0340-1057
  21. 21. Hampel S. Chung P. Mc Kellar C. E. Hall D. Looger L. L. Simpson J. H. 2011 Drosophila Brainbow: a Recombinase-Based Fluorescence Labeling Technique to Subdivide Neural Expression Patterns. Nature Methods, 8 3 march 2011), 253 259 . 1548-7091
  22. 22. Hardie R. C. 1991 Whole-Cell Recordings of the Light-Induced Current in Drosophila Photoreceptors: Evidence for Feedback by Calcium Permeating the Light Sensitive Channels. Proceedings of the Royal Society of London B, 245 1314 (September 1991), 203 210 . 0080-4649
  23. 23. Hardie R. C. Raghu P. 2001 Visual Transduction in Drosophila. Nature 413 6852 (September 2001), 186 193 . 0028-0836
  24. 24. Harris W. A. Ready D. F. Lipson E. D. Hudspeth A. J. Stark W. S. 1977 Vitamin A Deprivation and Drosophila Photopigments. Nature. 266 5603 (April 1977), 648 650 . 0028-0836
  25. 25. Heisenberg M. 1971 Separation of Receptor and Lamina Potentials in the Electroretinogram of Normal and Mutant Drosophila. Journal of Experimental Biology, 55 1 (August 1971), 85 100 , 0022-0949
  26. 26. Jansonius N. M. 1990 Properties of the Sodium Pump in the Blowfly Photoreceptor Cell. Journal of Comparative Physiology A, 167 4 (September 1990), 461 467 . 0340-7594
  27. 27. Järviilehto M. Zettler F. 1973 Electrophysiological-Histological Studies on Some Functional Properties of Visual Cells and Second Order Neurons of an Insect Retina. Cell and Tissue Research, 136 2 (June 1973), 291 306 . 0340-0336
  28. 28. Joesch M. Schnell B. Raghu S. V. Reiff D. F. Borst A. 2010 ON and OFF pathways in Drosophila motion vision. Nature 468 7321 (November 2010), 300 304 . 0028-0836
  29. 29. Juusola M. Hardie R. C. 2001 Light Adaptation in Drosophila Photoreceptors: I. Response Dynamics and Signaling Efficiency at 25 Degrees C. Journal of General Physiology, 117 1 (January 2001), 3 25 . 0022-1295
  30. 30. Kirschfeld K. Franceschini N. Optische Eigenschaften der Ommatidien im Komplexauge von Musca. Kybernetik, 5 5 2 (August 1968), 47 52 . 0023-5946
  31. 31. Kirschfeld K. Feiler R. Hardie R. Vogt K. Franceschini N. 1983 The Sensitizing Pigment of Fly Photoreceptors. Biophysics of Structure and Mechanism l0 1-2 , (April 1983), 81 92 . 0340-1057
  32. 32. Laughlin S. B. 1987 Form and Function in Retinal Processing. Trends in Neurosciences 10 11 (November 1987), 478 483 . 0166-2236
  33. 33. Lee S. J. Xu H. Kang L. W. Amzel L. M. Montell C. 2003 Light Adaptation Through Phosphoinositide-Regulated Translocation of Drosophila Visual Arrestin. Neuron, 39 1 (July 2003), 121 132 . 0896-6273
  34. 34. Liu C. H. Satoh A. K. Postma M. Huang J. Ready D. F. Hardie R. C. 2008 Ca2+-dependent Metarhodopsin Inactivation Mediated by Calmodulin and NINAC Myosin III. Neuron, 59 5 (September 2008), 778 789 . 0896-6273
  35. 35. Meglič A. Belušič G. Zupančič G. 2009 Using Carbon Fibre Microelectrodes to Monitor the Oxidative Metabolism of Blowfly Eyes. Acta biologica slovenica, 52 1 19 28 . 1408-3671
  36. 36. Meglič A. Zupančič G. 2011 Changes in Redox States of Respiratory Pigments Recorded From the Eyes of Live Blowflies Exposed to Light Stimuli and Hypoxia. Journal of Comparative Physiology A, 197 3 (March 2011), 301 310 . 0340-7594
  37. 37. Meyer N. E. Joel-Almagor T. Frechter S. Minke B. Huber A. 2006 Subcellular Translocation of the eGFP-tagged TRPL Channel in Drosophila Photoreceptors Requires Activation of the Phototransduction Cascade. Journal of Cell Science, 15 119 (June 2006), 2592 2603 . 0021-9533
  38. 38. Minke B. Wu C. F. Pak W. L. 1975 Isolation of Light-Induced Response of the Central Retinula Cells From the Electroretinogram of Drosophila. Journal of Comparative Physiology A, 98 4 (December 1975), 345 355 . 0340-7594
  39. 39. Morgan T. H. 1910 Sex Limited Inheritance In Drosophila. Science, 32 812 July1910). 0146-0404
  40. 40. Ostroy S. E. Wilson M. Pak W. L. 1974 Drosophila Rhodopsin: Photochemistry, Extraction and Differences in the NorpAP12 Phototransduction Mutant. Biochemichal and Biophysical Research Communications, 59 3 (August 1974), 960 966 . 0000-6291X
  41. 41. Pak W. L. 1995 Drosophila in Vision Research. The Friedenwald Lecture. Investigative Ophthalmolgy and Visual Science, 36 12 November 1995), 2340 2357 . 0146-0404
  42. 42. Pak W. L. 2010 Why Drosophila to Study Phototransduction? Journal of Neurogenetics, 24 2 July 2010), 55 66 . 0167-7063
  43. 43. Pandey U. B. Nichols C. D. 2011 Human Disease Models in Drosophila melanogaster and the Role of the Fly in Therapeutic Drug Discovery. Pharmacological Reviews, (March 2011). Online 1521-0081 1521 0081
  44. 44. Ranganathan R. Harris G. L. Stevens C. F. Zuker C. S. 1991 A Drosophila Mutant Defective in Extracellular Calcium-Dependent Photoreceptor Deactivation and Rapid Desensitization. Nature, 354 6350 November 1991), 230 232 . 0028-0836
  45. 45. Salcedo E. Huber A. Henrich S. Chadwell L. V. Chou W. H. Paulsen R. Britt S. G. 1999 Blue- and Green-Absorbing Visual Pigments of Drosophila: Ectopic Expression and Physiological Characterization of the R8 Photoreceptor Cell-Specific Rh5 and Rh6 Rhodopsins. Journal of Neuroscience, 19 No. (December 1999), 10716 10726 . 0270-6474
  46. 46. Satoh A. K. Xia H. Yan L. Liu C. H. Hardie R. C. Ready D. F. 2010 Arrestin Translocation is Stoichiometric to Rhodopsin Isomerization and Accelerated by Phototransduction in Drosophila Photoreceptors. Neuron, 67 6 (September 2010), 997 1008 . 0896-6273
  47. 47. Shaw S. R. 1984 Early Visual Processing in Insects. Journal of Experimental Biology, 112 1 (September 1984), 225 251 . 0022-0949
  48. 48. Stark W. S. 1977 Sensitivity and Adaptation in R7, an Ultraviolet Photoreceptor, in the Drosophila Retina. Journal of Comparative Physiology A, 115 1 (January 1977), 47 59 . 0340-7594
  49. 49. Stark W. S. Wasserman G. S. 1972 Transient and Receptor Potentials in the Electroretinogram of Drosophila. Vision Research, 12 10 (October 1972), 1771 1775 . 0042-6989
  50. 50. Stavenga D. G. 2002 Colour in the Eyes of Insects. Journal of Comparative Physiology A, 188 5 (June 2002), 337 348 . 0340-7594
  51. 51. Stavenga D. G. Zantema A. Kuiper J. W. 1973 Rhodopsin processes and the function of the pupil mechanism in flies. In: Langer H (ed) Biochemistry and physiology of visual pigments. Springer, Berlin, 175 180
  52. 52. Stavenga D. G. Schwemer J. 1984 Visual pigments of invertebrates. In: Ali MA (ed) Photoreception and vision of invertebrates. Plenum, New York, 11 61
  53. 53. Streck P. 1972 Der Einfluss des Schirmpigmentes auf das Sehfeld einzelner Sehzellen der Fliege Calliphora erythrocephala Meig. Zeitschrift fuer Vergleichende Physiologie, 76 4 (December 1972), 372 402 . 0004-4362X
  54. 54. Stušek P. Belušič G. Drašlar K. 2000 Temperature dependence of photoreceptor signal termination in fruitfly (Drosophila melanogaster white eyed mutant). Pflügers Archiv, 439 3 [suppl.] (December 2000), R200 0031-6768
  55. 55. Uusitalo R. O. Weckström M. 2000 Potentiation in the First Visual Synapse of the Fly Compound Eye. Journal of Neurophysiology, 83 4 (April 2000), 2103 2112 . 0022-3077
  56. 56. Zupančič G. 2003 A Method For Dynamic Spectrophotometric Measurements In Vivo Using Principal Component Analysis-Based Spectral Deconvolution. Pflügers Archiv, 447 1 (October 2003), 109 119 . 0031-6768

Written By

Gregor Belusic

Submitted: November 15th, 2010 Published: August 9th, 2011