Documenta Ophthalmologica 104: 57–68, 2002.
© 2002 Kluwer Academic Publishers. Printed in the Netherlands.
Ganzfeld ERG in zebrafish larvae ∗
MATHIAS W. SEELIGER1, ALBRECHT RILK1 and STEPHAN C.F.
NEUHAUSS2
1 University Eye Hospital, Dept. II, Schleichstr. 12-16, D-72076 Tübingen, Germany;
2 Max-Planck-Institut für Entwicklungsbiologie, Abt. I, Spemannstr. 35/I, Tübingen,
Germany; Brain Research Institute, ETH Zurich, Winterthurerstr. 190, CH-8057 Zurich,
Switzerland
Abstract. In developmental biology, zebrafish are widely used to study the impact of mutations. The fast pace of development allows for a definitive morphological evaluation of the
phenotype usually 5 days post fertilization (dpf). At that age, a functional analysis is already
feasible using electroretinographic (ERG) methods. Corneal Ganzfeld ERGs were recorded
with a glass microelectrode in anaesthetized, dark-adapted larvae aged 5 dpf, using a platinum
wire beneath a moist paper towel as reference. ERG protocols included flash, flicker, and
ON/OFF stimuli, both under scotopic and photopic conditions. Repetitive, isoluminant stimuli
were used to assess the dynamic effect of pharmacological agents on the ERG. Single flash,
flicker, and ON/OFF responses had adequately matured at this point to be informative. Typical
signs of the cone dominance were the small scotopic a-wave and the large OFF responses.
The analysis of consecutive single traces was possible because of the lack of EKG, breathing,
and blink artefacts. After application of APB, which selectively blocks the ON channel via
the mGluR6 receptor, the successive loss of the b-wave could be observed, which was quite
different from the deterioration of the ERG after a circulatory arrest. The above techniques
allowed to reliably obtain Ganzfeld ERGs in larvae aged 5 dpf. This underlines the important
role of the zebrafish as a model for the functional analysis of mutations disrupting the visual
system.
Key words: animal model, electrophysiology, ERG, zebrafish
Introduction
Zebrafish larvae have become a standard model for the study of the genetic
control of vertebrate development. A large number of mutations affecting
specific developmental processes have been isolated in large and small-scale
mutagenesis screens [1,2], including mutations in the retina [3, 4].
Most work on the zebrafish retina so far has focused on the morphology
[5–11], but comparatively little on functional aspects. Visual mediated behavior was detected as early as 3 days post fertilization (dpf) [12–14], so that
∗ This study was supported by DFG grant SFB 430 C2, fortuene grant # 517, and the Swiss
National Science Foundation.
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visual function may be studied from that time on. Behavioral tests of visual
function include assays for an optokinetic response, optomotor response, phototaxis, dorsal light reaction and background adaptation [12–16].
These tests assay for complex neural networks including a varying degree
of central processing and efferent pathways. However, a failure in one of these
tests does not allow a conclusion about the function of the retina. In contrast,
electroretinography (ERG) is a retina specific functional test.
For most in vivo applications, the ERG, which is an electric sum potential
generated by retinal cells following exposure to light, is non-invasively measured at the corneal surface. The light stimulus can be applied by different
means, e.g. as a pulse from a constant source or a short flash from a Xenon
tube. Typically, the evoked response consists of an initial negative deflection
(a-wave), followed by a large, positive component (b-wave). Superimposed
on the ascending portion of the b-wave are the oscillatory potentials (OPs), a
set of wavelets oscillating with approximately 4–5 times the frequency of
the a- and b-wave. Finally, a prolonged positive component (c-wave) follows, which takes several seconds to develop (reviewed in [17]). If a flash
of long duration is applied, the b-wave separates into a ‘true’ b-wave at the
onset of light due to depolarization of ON-bipolar cells, and the d-wave as
an off-response mainly due to hyperpolarization of OFF-bipolar cells and
photoreceptors [18].
There are not many reports describing the use of the ERG to examine
zebrafish visual system development [6, 19] and mutant analysis [14, 20–22].
All authors so far have recorded ERGs elicited by light flashes of several
milliseconds duration obtained from a constant light source, and delivered
to the eye via a fiber optic. In such a configuration, the intensity of light on
the retina and the illuminated area cannot be easily controlled. Consequently,
the representation of receptor types in the ERG may vary with the area stimulated, which is also likely to cause additional variability in the results between
specimens.
In a Ganzfeld setup, the stimulus (usually a flash of less than 1 ms duration
to counteract a substantial separation of ON-and OFF components) passes
through a diffusor that yields a relatively homogeneous distribution of light
intensity on the inside of a bowl. The term ‘Ganzfeld’ meaning ‘full field’
denotes that if a subject looks through an opening inside the bowl, the stimulus reaches practically all parts of the retina and its intensity is approximately
equal across that area. As opposed to focal stimulation, the Ganzfeld-ERG
measures the contributions of both rods and cones regardless of regional
variations in receptor distribution.
The purpose of this work is to describe a setting for recording Ganzfeld
ERGs in zebrafish larvae and to point to different fields of application. A
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developmental study and the analysis of specific mutants using this system
are in progress and will be presented separately.
Materials and methods
Animals and anesthesia
Fish were maintained and bred as previously described [23]. Larvae from
natural spawning were staged by days after fertilization (dpf) at 28.5 ◦ C.
Wild-type larvae of the Tübingen strain [21] were tested at an age of 5 dpf.
Specimen were dark-adapted for a minimum of 4 h prior to the measurements and subsequently handled under dim red illumination. They were anesthetized with 0.02% buffered 3-aminobenzoic acid methyl ester (MESAB;
Sigma, St. Louis, USA) and paralyzed with 0.8 mg/ml Esmeronl’ (Organon
Teknika, Eppelheim, Germany). After the experiments, larvae were sacrificed
using a lethal dose of MESAB.
Experimental setup
Anesthetised larvae were placed in a lateral position on a wet paper towel
sitting on a platinum wire utilized as reference electrode. Under visual control via a standard microscope equipped with red illumination (Stemi 2000
C, Zeiss, Oberkochen, Germany), a glass microelectrode with an opening
of approximately 20 µm at the tip was placed on the center of the cornea
(Figure 1A). This electrode was filled with E3 medium (5 mM NaCl, 0.17
mM KCl, 0.33 mM CaCl, and 0.33 mM MgSO4), the same in which the
embryos were raised and held [2]. The electrode was moved with a micromanipulator (M330R, World Precision Instruments Inc., Sarasota, USA)
mounted in a wooden box (Figure 1C). The microelectrode holder was directly connected to a voltage follower (VF2, World Precision Instruments Inc.,
Sarasota, USA), which in turn was connected to an input channel of the ERG
amplifier. The wooden box including the micromanipulator was subsequently
transferred into the Ganzfeld bowl for recording (Figure 1D).
Ganzfeld-electroretinography
Stimulation and data acquisition were performed with a commercially available ERG setup (Toennies Multiliner Vision, Jaeger/Toennies, Höchberg, Germany) featuring a Ganzfeld bowl, a DC amplifier, and a personal computer
for stimulus generation and data management.
The ERG protocol included a scotopic (dark adapted) and a photopic (light
adapted) part as described below. Bandpass filter cutoff frequencies were
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Figure 1. Zebrafish larva and Ganzfeld ERG recording setup. (A) Zebrafish larva at 5 dpf.
The arrowhead indicates the glass microelectrode (diameter 20 µm) positioned on the center
of the cornea. (B) Histological section of the zebrafish eye at 5 dpf. The different layers are
already well organized, which is in accordance with the functional performance. (C) Close-up
view of the micromanipulator mounted in the recording box. (D) Ganzfeld bowl carrying the
recording box. The box was tilted for better visualization.
1 and 300 Hz for all measurements. The scotopic session included single
flash stimuli increasing from 0.1 mcds/m2 to 25 cds/m2 ). Ten responses per
intensity level were averaged, with an inter-stimulus interval (ISI) of 5 (0.1,
1, 3, 10, 30, 100 mcds/m2 ) or 17 s (1, 3, 10, 25 cds/m2 ).
Upon completion, a custom-made stimulator (as suggested by Sieving
[24]) was invoked to provide light pulses of 1 s duration for the separation
of ON and OFF responses. It uses a 50 W halogen bulb (Philips, Eindhoven,
Netherlands) and a fast shutter (Uni-Blitz Model D122, Vincent Associates,
Rochester, NY, USA) driven by a delay unit interfaced to the main ERG
recording setup. A separate opening in the Ganzfeld bowl was used to accomplish a light distribution comparable to that of the Xenon flashes and
the background, with a maximum intensity of 240 cd/m2 on the inner bowl
surface. Neutral density filters were inserted in four steps of one logarithmic
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unit to achieve an intensity series from 0.24 to 240 cd/m2 . Three responses per
intensity level were averaged, with an ISI of 15 (0.24 cd/m2 and 2.4 cd/m2 ),
30 (24 cd/m2 ), or 60 s (240 cd/m2 ).
After the end of the scotopic session, a homogeneous background illumination of 30 cd/m2 was turned on. Following the light adaptation of 10 min,
the photopic session began with single flash recordings as above but with
a reduced number of light intensities (0.03, 0.1, 1, 3, 10 and 25 cds/m2 ).
Subsequently, flicker ERGs were obtained with flashes of 3 cds/m2 using
frequencies of 1, 2, 5, 10, 15, 20, 25 and 30 Hz. The photopic session was
concluded with a repetition of the ON/OFF protocol as described earlier.
ERG single trace series
In some animals, a series of isoluminant flashes with an ISI of 10 sec was
used to assess the dynamic effect of pharmacological agents on the ERG.
These drugs were applied externally directly on the eye using an Eppendorf
pipette. The compounds used in this study were 2-amino-4-phosphonobutyric
acid (APB, 10 mM), a lethal dose of MESAB (0.5%), and plain medium as
control.
Results
Single flash and flicker ERG
A typical scotopic intensity series is shown in Figure 2A. At around 1 –
3 mcds/m2 , a negative deflection similar to the scotopic threshold response
(STR) becomes visible. From around 3 to 10 mcds/m2 on, the positive components (b-wave) begin to increase. The initial, negative a-wave is very small
or absent, and can be masked by a flash artefact observable at intensities
above 10 cds/m2 . Oscillatory potentials become apparent especially at higher
intensities.
In comparison, the photopic intensity series featured an abrupt rise in
b-wave amplitude between 0.1 and 1 cds/m2 (Figure 2B). The flicker ERG
(Figure 2C) revealed a transition from a monophasic waveform (1 and 2 Hz)
to complicated waveforms (5 – 15 Hz), and back to a monophasic one (20 Hz
and above). Most larvae at 5 dpf could not follow flicker frequencies above
20 Hz, although a few specimens reached substantial amplitudes even at 30
Hz.
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Figure 2. Typical ERG record. Intensity series obtained under (A) dark-adapted and (B)
light-adapted conditions. (C) Flicker frequency series obtained at 3 cds/m2 .
Figure 3. Amplitude vs. log intensity (VlogI) and implicit time plot. Statistical evaluation of
scotopic intensity series from 20 fish (A) amplitude and (B) implicit time). Boxes indicate the
25–75% quantile range, the whiskers the 5% and 95% quantiles, and the asterisks connected
by the black line the medians of the data. See text for details.
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Figure 4. Typical ON/OFF-ERG record. Intensity series obtained under (A) dark-adapted and
(B) light adapted conditions. Stimulus intensities (from top to bottom): 0.24, 2.4, 24 and 240
cd/m2 .
Amplitude vs. log. Intensity (VlogI) function
A VlogI function based on scotopic single flash recordings in 20 fish is shown
in Figure 3. The curve is biphasic, i.e. there is a ‘notch’ at about 3 cds/m2 ,
indicating a superposition of two different components.
ON/OFF-ERG
Responses associated with the on- and offset of light were recorded both
under scotopic and photopic conditions, and for four increasing stimulus
intensities (Figure 4). The scotopic section (Figure 4A) showed a gradual
increase of the positive going ON response, whereas the OFF response diminished and flattened after an initial increase, and often turned completely
negative. In contrast, the photopic ON response was less sensitive, but again
featured a sudden rise in amplitude between 2.4 and 24 cd/m2 (Figure 4B).
In addition, the OFF response did not diminish at higher stimulus intensities,
and stayed usually positive.
ERG single trace series
To evaluate the sequence of changes following drug application, repeated
measurements with a standard flash and an ISI of 10 sec were performed
(Figure 5). If a control solution was used, no changes were detectable (Figure
5, left). APB in the given dose caused a steady decline of the b-wave, which
in turn led to an increase in both a-wave amplitude and implicit time (Figure
5, center). One minute after the APB treatment, the b-wave peak did not
exceed the zero level anymore. After approximately 3 min, a stable period
followed, featuring a so-called negative ERG. Using lower doses, this process was found to be reversible. In contrast, application of a high dose of
the anaesthetic MESAB, the usual way to sacrifice the larvae, caused a more
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Figure 5. Evaluation of changed following drug application, using a flash intensity of 3
cds/m2 . Recordings start with the top trace at t=0 after drug application. The time interval
between traces was 10 s. See text for details.
gradual, unspecific loss of electrical activity (Figure 5, right) likely due to a
circulatory arrest.
Discussion
In this work, we have introduced Ganzfeld electroretinography as a tool for
the analysis of the retinal function in zebrafish larvae. Especially in the early
developmental stages with strong regional variations in receptor distribution,
the Ganzfeld method has the advantage not to be susceptible to the alignment
of light sources and measures the contributions of the whole rod and cone
population. The age of 5 dpf was chosen both for physiological and technical
reasons. Below that age, the insufficient functional maturation of the retina
[25] and the smaller sized eye make reliable recordings more difficult. Above
that age, oxygen supply by diffusion may not be sufficient, and nutrition becomes a problem as the yolk sac has been used up. For these reasons, reports
about ERG recordings in zebrafish older than 7 dpf are rare [26].
It appears that at 5 dpf both rods and cones contribute to the scotopic
ERG. The small scotopic a-wave [27] (Figure 2A) and the large OFF responses (Figure 4) indicate that the cones dominate. However, the responses
obtained at light intensities below cone threshold and the ‘notch’ in the VlogI
curve (Figure 3), like the one in the human luminance-response function [28],
point to some rod involvement. In favour of this hypothesis – despite the
morphologically still immature rods – is the beginning staining of rhodopsin
in immunohistochemistry [29, 30]. Alternatively, differently sensitive cone
subpopulations could also account for the notch.
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Following 10 min of adaptation with a rod-desensitizing background of 30
cd/m2 , the photopic ERG (Figure 2B) was recorded. As expected from other
species, it appeared less sensitive to light stimuli.
The flicker ERG frequency series (Figure 2C) appears to be useful in
mutant detection. In particular, mutants with a negative, PIII-like ERG often
have a reduced flicker fusion frequency (unpublished data). This is also true
for wild-type larvae at younger ages, as the developing retina responds much
slower than its mature analogue [31, 32].
The ON/OFF-ERG is another very useful test in mutant analysis, as it
reveals whether a response is associated with the onset or offset of the stimulus. For example, some of the mutants with a negative, PIII-like ERG have
no OFF response (unpublished data).
An important feature of the zebrafish larva model is that many watersoluble substances permeate well into tissues after external application. Thus,
the dynamic processes induced by retina-specific drugs can be examined in
vivo by repetitive ERG measurements using flashes of equal intensity. The
analysis of consecutive single traces is possible because of the lack of an
EKG, breathing and blink artefacts. The temporal resolution achievable is dependent on stimulus intensity, because the ISI must be chosen large enough to
avoid adaptation. If the substances tested are not eye-specific, then the influence of potential systemic effects on the results (e.g. via heart and circulation)
must be excluded.
This type of recording is shown here for APB, a glutamate analogue,
which is known to selectively block the retinal ON-pathway [33] at the postsynaptic mGluR6 receptor of the synapse between photoreceptors and bipolar
cells [34]. After application of APB, the successive loss of the b-wave could
be observed (Figure 5B) similar to an in vitro study in superfused amphibian
eyecups [35], which was quite different from the deterioration of the ERG
after a circulatory arrest (Figure 5C).
A survey of recent electrophysiological work on zebrafish visual system
development [6] and mutant analysis [14, 21,22] disclosed that a variety of
set-ups and conditions were used by the different groups, which makes it
very difficult to compare results. In humans, the need for worldwide comparability of results has led to the formulation of an international standard
for the specification of the equipment and the measurement protocol by the
International Society for Clinical Electrophysiology in Vision and Ophthalmology (ISCEV) [20]. For the characterization of phenotypes in the increasing number of mutants with eye-related defects, it would be desirable to have
standardized measurement procedures.
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Conclusions
As the early development of the zebrafish retina is paralleled by a fast functional maturation, both standard and extended electroretinographic tests can
be performed at 5 dpf, making the zebrafish a valuable model for the functional analysis of mutations disrupting the visual system.
Acknowledgements
The authors thank Dr. Hartmut Schwahn and Stefan Beuel for initial support,
and Karin Mai and Susanne Scholz for technical assistance.
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Address for correspondence: M. Seeliger, Retinal Electrodiagnostics Research Group, University Eye Hospital, Dept. II, Schleichstr. 12–16, D-72076 Tübingen, Germany
Phone: +49-7071-298-0718; Fax: +49-7071-29-4789; E-mail: [email protected]