1. Introduction
The fruitfly
2. The visual system of Drosophila
The visual system of
Photoreceptor cells have three morphologically distinct regions: the light sensing
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
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 (
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
3.3 Red-eyed and white-eyed fruitflies
The ERG is usually recorded in a few varieties of white-eyed
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.
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
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
where
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.
The ERG in
4.2. The dynamic properties of ERG
The dynamic properties of the ERG should be treated with great caution. Firstly,
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 (
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
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
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
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
All the key players in
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.
5. Future applications of ERG
ERG in
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