Ocean acidification slows retinal function in a damselfish through interference with GABA(A) receptors

Abstract

Vision is one of the most efficient senses used by animals to catch prey and avoid predators. Therefore, any deficiency in the visual system could have important consequences for individual performance. We examined the effect of CO2 levels projected to occur by the end of this century on retinal responses in a damselfish, by determining the threshold of its flicker electroretinogram (fERG). The maximal flicker frequency of the retina was reduced by continuous exposure to elevated CO2, potentially impairing the capacity of fish to react to fast events. This effect was rapidly counteracted by treatment with a GABA antagonist (gabazine), indicating that GABAA receptor function is disrupted by elevated CO2. In addition to demonstrating the effects of elevated CO2 on fast flicker fusion of marine fishes, our results show that the fish retina could be a model system to study the effects of high CO2 on neural processing.

Full text

The Journal of Experimental Biology
© 2014. Published by The Company of Biologists Ltd | The Journal of Experimental Biology (2014) 217, 323-326 doi:10.1242/jeb.092478
323
ABSTRACT
Vision is one of the most efficient senses used by animals to catch
prey and avoid predators. Therefore, any deficiency in the visual
system could have important consequences for individual
performance. We examined the effect of CO2levels projected to
occur by the end of this century on retinal responses in a damselfish,
by determining the threshold of its flicker electroretinogram (fERG).
The maximal flicker frequency of the retina was reduced by
continuous exposure to elevated CO2, potentially impairing the
capacity of fish to react to fast events. This effect was rapidly
counteracted by treatment with a GABA antagonist (gabazine),
indicating that GABAAreceptor function is disrupted by elevated CO2.
In addition to demonstrating the effects of elevated CO2on fast flicker
fusion of marine fishes, our results show that the fish retina could be
a model system to study the effects of high CO2on neural
processing.
KEY WORDS: Flicker fusion frequency, Electroretinogram, Carbon
dioxide, Vision, Coral reef
INTRODUCTION
CO2levels in the surface ocean are rising in line with rising
atmospheric CO2(Doney, 2010). It has recently been shown that
projected near-future CO2levels can impair sensory systems and
alter the behaviour of marine fishes (Munday et al., 2009; Munday
et al., 2012; Jutfelt et al., 2013). Behavioural changes include
increased boldness and activity (Munday et al., 2010; Munday et al.,
2013; Jutfelt et al., 2013), loss of behavioural lateralization
(Domenici et al., 2012; Jutfelt et al., 2013), altered auditory
preferences (Simpson et al., 2011) and impaired olfactory function
(Munday et al., 2009; Dixson et al., 2010; Ferrari et al., 2011). The
underlying reason for sensory impairment and behavioural changes
in fish exposed to elevated CO2appears to be a disruption to
neurotransmitter function, probably caused by changes to ion
gradients over neuronal membranes (Nilsson et al., 2012). Fish
regulate acid–base relevant ions, primarily bicarbonate (HCO3–) and
chloride (Cl–), to maintain blood and tissue pH when exposed to
high CO2(Ishimatsu et al., 2008). Experimental evidence suggests
that this leads to a disruption of GABAAreceptors, which are Cl–
and HCO3–channels gated by the neurotransmitter GABA (gamma-
amino butyric acid). Indeed, the sensory and behavioural alterations
caused by high-CO2exposure are virtually abolished by a moderate
SHORT COMMUNICATION
1Queensland Brain Institute, University of Queensland, Brisbane 4072, Australia.
2ARC Centre of Excellence for Coral Reef Studies, James Cook University,
Townsville, QLD 4811, Australia. 3School of Marine and Tropical Biology, James
Cook University, Townsville, QLD 4811, Australia. 4Depar tment of Biosciences,
University of Oslo, Oslo 0316, Norway.
*Author for correspondence (g.e.nilsson@imbv.uio.no)
Received 14 June 2013; Accepted 23 September 2013
dose of the GABAAreceptor blocker gabazine (Nilsson et al., 2012).
Normally, GABAAreceptors act by hyperpolarizing neuronal
membranes due to the inflow of Cl–, causing neuronal inhibition. It
has been hypothesized that during high-CO2exposure, the
transmembrane gradients of Cl–and HCO3–are altered in some
neurons, thereby affecting GABAAfunction. Given the ubiquity of
GABAAreceptors in animal nervous systems, it is likely that
exposure to elevated CO2could affect a wide variety of behavioural
functions and activities in marine organisms.
To date, research on sensory impairment of fishes at elevated CO2
levels has concentrated mainly on the effects to olfactory
discrimination (e.g. Munday et al., 2009; Dixson et al., 2010; Ferrari
et al., 2011), and to some extent on auditory preferences (Simpson
et al., 2011). Recently, Ferrari and colleagues (Ferrari et al., 2012)
found that visual risk assessment was altered in juvenile fish
exposed to 850 μatm CO2. When presented with the sight of a large
novel reef fish, of sufficient size to be a predator, juvenile
damselfish that had been reared at high CO2exhibited reduced
antipredator responses and lacked the typical signalling behaviour
(bobbing) seen in juvenile damselfishes exposed to a threatening
situation (Ferrari et al., 2012). This suggests that the function of the
visual system is affected by high CO2. Such alterations to vision-
mediated behaviour could involve processing at the retinal level or
in higher brain centres.
In this study, we focused on the possibility that visual function at
the retinal level is affected by high-CO2exposure. The rapidity of
the response of animals to visual stimuli may be correlated with fast
flicker fusion (FFF). A visual system viewing a flickering light
source has a critical flicker fusion (CFF) threshold, above which the
flicker becomes too fast for the system to follow (Fritsches et al.,
2005), and the light appears continuous to the animal, and not
flashing. The CFF threshold varies between animals, and is often
related to lifestyle and illumination level, being fast in rapidly
moving organisms in bright light and slow in nocturnal slow-movers
(Horodysky et al., 2008; Smolka et al., 2013). It is therefore likely
that reduced CFF could impair the capacity to react to fast events
such as prey capture and predator avoidance.
Here we examined the effect of elevated CO2on retinal function
in the spiny damselfish, Acanthochromis polyacanthus (Bleeker
1855) by determining the CFF threshold of its electroretinogram
(ERG). This method records the electrical light response of the
retina using a non-invasive electrode placed on the eye. We show
that continuous exposure to the CO2level projected to occur in the
surface ocean by the end of this century (944 μatm) reduces the CFF
threshold (i.e. reduces the speed of the light response) and that this
effect can be effectively counteracted by gabazine treatment,
indicating an involvement of GABAAreceptor function.
RESULTS AND DISCUSSION
The CFF of damselfish exposed to elevated CO2(78.6±3.9 Hz, mean
± s.e.m.) was significantly decreased compared with that of the
Ocean acidification slows retinal function in a damselfish through
interference with GABAAreceptors
Wen-Sung Chung1, N. Justin Marshall1, Sue-Ann Watson2,3, Philip L. Munday2,3 and Göran E. Nilsson4,*

The Journal of Experimental Biology
324
control group (89.0±1.6 Hz) (t10=5.28, P<0.0004). There was a
significant interaction between time interval and CO2treatment in
gabazine-treated fish (F4,20=35.64, P<0.0001). The CFF values of
fish from the elevated-CO2group were significantly less than those
of fish from the control group at 0, 5 and 10 min; however, the value
increased through time to reach approximately the same level as the
control group after 15–20 min (Fig. 1). The CFF of the control group
did not change significantly through time. There appeared to be a
slight increase in CFF of the control group after the first 5 min, when
gabazine was first administered, but there was no further increase
through time, indicating that gabazine itself had negligible effect on
CFF.
Our results show that the ability or the fish retina to react to fast
visual stimuli is reduced after exposure to CO2levels projected to
occur in the ocean by the end of this century. Moreover, the
underlying mechanism appears to involve altered GABAAreceptor
function, as the CFF threshold could be restored by treatment with
the GABAAreceptor antagonist gabazine, at a dose that has
previously been found to restore impaired olfactory preference and
lateralization in reef fish exposed to high CO2(Nilsson et al., 2012).
GABAAreceptors have been found to be intimately coupled to
retinal signal processing and direction selectivity at the ganglion cell
level, including the control of the fast flicker response in the fish
retina (Mora-Ferrer and Neumeyer, 2009). As GABAAreceptors
have been linked to neural dysfunction of high-CO2exposed fish, it
is not unexpected that retinal function is affected by elevated CO2
levels.
The CFF threshold of fish correlates with their lifestyle
(Horodysky et al., 2008) and a high CFF is likely to reflect the need
to react to fast events in their habitat, at the cost of reduced
performance at low light conditions. Pelagic fish CFF varies from
around 25 Hz in species living in deeper water, to 80 Hz in the
surface-dwelling yellow-fin tuna or the dolphin fish (Fritsches et al.,
2005). This fits well with the CFF of about 90 Hz we found in A.
polyacanthus, which lives in a well-lit complex environment in close
proximity to predators.
Predatory events in the ocean may also be very rapid; it is therefore
of concern that spiny damselfish exposed to a near-future CO2level
slow their retinal response, with CFF dropping from around 90 Hz to
less than 80 Hz. This decrease in visual speed might result in reduced
reaction times, for example to a rapidly approaching predator, a
possibility supported by experiments showing that prey fish exposed
to similar levels of CO2to those used in our experiments have a
reduced perception and response to predation threat (Ferrari et al.,
2012; Allan et al., 2013). While behavioural responses to visual
stimuli may be different to those measured electrophysiologically at
the level of the retina, behavioural responses would probably be
slower than the initial physiological signal, and in the light of the
ubiquitous presence of GABAAreceptors in the nervous system, it is
possible that CO2exposure may lead to additional reductions in
reaction time due to disturbances at higher levels of neural processing
(Domenici et al., 2012).
Our results add to the increasing evidence that elevated CO2can
affect critical sensory processes in marine fishes and that the
underlying mechanism is associated with the function of GABAA
receptors. An important aspect of the present result is that the fish
retina could be used as a relatively simple model system to study the
effects of high CO2on neural processing. Indeed, isolated fish retina
preparations have long been used as models for neurophysiological
research (e.g. Hankins and Ruddock, 1984). A better understanding
of how and why high-CO2exposure affects GABAAreceptor
function, and possibly other neural components, would increase the
power to predict which physiological processes are likely to be
affected, and which organisms are most at risk, from future rises in
ocean CO2levels.
MATERIALS AND METHODS
Animals and experimental treatments
Small A. polyacanthus, between 55 and 80 mm standard length (SL), were
collected from the lagoon at Lizard Island (14°40′08″S; 145°27′34″E), Great
Barrier Reef, Australia, using barrier nets. Fish were distributed among eight
aquaria supplied with a constant flow of seawater at ambient summer
temperature (28–30°C) and fed to satiation twice daily with INVE
aquaculture pellets (Dendermonde, Belgium). Four of the aquaria were
supplied with seawater at present-day CO2levels (466 μatm) and four with
elevated CO2-equilibrated (944 μatm) seawater, as described below. The
elevated CO2treatment is consistent with projected CO2levels in the
atmosphere and surface ocean at year 2100 on a business-as-usual carbon
emissions trajectory (RCP 8.5) (Meinshausen et al., 2011). Fish were
maintained at control and elevated CO2for 6–7 days prior to
experimentation, which is sufficient to induce the full range of sensory and
behavioural impairment in reef fish (Munday et al., 2010; Munday et al.,
2012; Ferrari et al., 2012). All animal care and experimental protocols
complied with ethics regulations of James Cook University and University
of Queensland.
Seawater manipulation
Elevated CO2levels were achieved by CO2-dosing seawater in a 60 l header
tank to a set pHNBS (pH calibrated in National Bureau of Standards buffers)
to match the required CO2level. A pH controller (Aqua Medic GmbH,
Bissendorf, Germany) delivered CO2into a power-head pump at the bottom
of the header tank if the pH rose above the set point. Individual aquaria
received CO2-equilibrated seawater from the header tank at ~1000 ml min−1.
The pHNBS of each aquarium was monitored regularly to ensure it remained
within ±0.05 of the desired level. Control aquaria received seawater from a
60 l header tank diffused with ambient air. The temperature in each aquarium
was measured twice daily. Seawater total alkalinity and pHNBS for CO2
calculations were measured from replicate water samples of control and high
CO2water taken throughout the experiment. Total alkalinity was estimated
by Gran titration using certified reference materials (Dr A. Dickson, Scripps
Institution of Oceanography). Carbonate chemistry values are shown in
Table 1.
SHORT COMMUNICATION The Journal of Experimental Biology (2014) doi:10.1242/jeb.092478
CFF (Hz)
Time (min)
100
90
80
70
** ** *
0 5 10 15 20
Fig. 1. The suppressed critical flicker fusion (CFF) threshold in
elevated-CO2treated fish is restored by gabazine treatment. Graph
shows CFF of control (triangles) and elevated-CO2(squares) treated
Acanthochromis polyacanthus. Gabazine was introduced into the respiratory
water of both groups at time zero. The CFF of the control group was
unaffected by gabazine, resulting in a stable phase at 93.5±1.4 Hz. The
resolution of the high-CO2fishes recovered to the control level after 15 min of
gabazine treatment. Student’s t-test, *P<0.05, **P<0.01. Error bars are s.e.m.

The Journal of Experimental Biology
Experiments
Flicker ERG (fERG) was used to test the temporal visual resolution of
control and elevated-CO2-exposed fish (SL 63.0±3.4 mm, mean ± s.d.). Fish
that had been in experimental treatments for 6–7 days were anaesthetized
using 20 ppm clove oil and pithed prior to the fERG measurement. ERGs
were recorded from the pithed fish, which was restrained in a horizontal
position on a sponge attached to a plastic board and held firmly in place
using silicone bandages. The fish was placed in a seawater bath maintained
at 29±1°C. Moistened tissue paper was placed on the upper side of the fish
and only one eye was exposed into the air for ERG recording, as described
below. Fish were maintained with constant gill irrigation using seawater at
the same CO2level as their experimental treatment (control or elevated CO2)
flowing at 0.1 l min−1. Five elevated CO2individuals and five control
individuals were tested.
A second experiment tested the potential role of GABAAreceptors in the
temporal visual resolution of high-CO2-exposed fish. The procedure
described above was used, except control and high-CO2-exposed damselfish
(SL 74.3±5.8 mm) were treated with gabazine (Sigma Chemical Co., St
Louis, MO, USA) throughout the fERG procedure. Gabazine is a fast-acting
and highly selective antagonist to the GABAAreceptor (Ueno et al., 1997).
From the start of the measurements the fish were ventilated with seawater
containing 4 mg l−1gabazine at a flow rate of 0.1 l min−1. The visual
resolution of fish from the two treatment groups was tested with fERG every
5 min for a total of 20 min. Five individuals from the elevated-CO2group
and three control individuals were tested in this experiment.
ERG setup
A white LED lamp was placed 30 cm above the test eye. The LED was
connected to a PowerLab ML 866 module (ADinstruments, Colorado
Springs, CO, USA) from which stimulus presentations were controlled by
the built-in functional generator to produce flickering stimuli (30 square
pulses) using the software LabChart Pro 7 (v7.2.5; ADinstruments). Each
pulse possessed 5 ms power-on duration, rendering a constant photon flux
per flash. The irradiance of the lamp was calibrated with a USB4000
spectrometer (Ocean Optics, Dunedin, FL, USA). The light intensity was
controlled to emit 1014 photons cm−2ms−1. Teflon-coated chlorided 0.5 mm
silver wire (Ag–AgCl2) electrodes were used to record the whole-eyeball
corneal ERGs. The recording electrode was placed on the corneal surface so
that it had contact at the edge of the pupil. The reference electrode was
placed on fatty tissue inside the orbit. Liquid conductive gel (EcoGel 200,
Mississauga, ON, Canada) was added to the tip of the recording electrode.
The system was grounded to the water of the experimental chamber. ERG
signals were amplified with a DP301 amplifier (Warner Instruments,
Hamden, CT, USA) using a 1000 gain passed through a 1 Hz high-pass and
1 kHz low-pass filter. The amplified ERG signals were further filtered with
the software’s electronic notch filter to remove periodic electrical noise
using LabChart Pro 7 with the Powerlab ML 866 module. The sampling
frequency was set at 4 kHz.
Electrophysiological procedure
The electrical response of the whole eye was measured as the frequency of
a flickering light was increased. The threshold of the FFF was determined
by a standardized method (Fritsches et al., 2005), and in physiological terms
is when the eye’s response no longer follows the modulation of the light
(supplementary material Fig. S1). Measurements were carried out during the
daytime on light-adapted fish immobilized as described elsewhere
(Horodysky et al., 2008).
The flicker rate was started at 10 Hz and the flash frequency gradually
increased until CFF. CFF is defined as the point where the modulated ERG
wavelets are no longer following the flickering light. The flickering stimuli
were presented for 3–10 s (depending on the number and length of sweeps
used). Data were recorded across 30–100 sweeps for every flicker rate. Two
methods were used to determine the CFF threshold. First, the ERG waveforms
were visually inspected to determine whether they remained in phase with the
flickering stimuli. This process was restricted to the lower frequency test (less
than 65 Hz). When the flicker frequency approached the CFF threshold, visual
inspection was insufficient to determine whether the wavelets remained in
phase with the flickering light. The CFF threshold was therefore determined
by analysing the power spectrum as described by Fritsches and colleagues
(Fritsches et al., 2005). The power at the stimulus frequency was compared
with the standard deviation of the power of a neighbouring frequency section.
The criterion for CFF was defined as the highest frequency (tested in 1 Hz
steps) at which the power of the signal was at least five times larger than the
power of the noise. At higher frequencies the power of the response signal was
indistinct compared with the power of the noise.
Statistics
A t-test was used to determine whether CFF values differed between control
fish and those in the elevated-CO2group. Repeated measures ANOVA was
used to compare CFF values of individuals in the elevated CO2group
against the control group over the 20 min duration of the gabazine
experiment. Dunn’s multiple comparisons were then used to test the time
intervals at which the mean CFF of the two groups differed.
Acknowledgements
We thank Lizard Island Research Station for excellent logistical support.
Competing interests
The authors declare no competing financial interests.
Author contributions
The study was conceived and designed by P.L.M., N.J.M. and G.E.N. Experiments
and measurements were executed by W.-S.C., G.E.N., S.-A.W. and P.L.M. All
authors participated in the analysis, interpretation and writing process.
Funding
This research was financed by the Australian Research Council (to P.L.M. and
N.J.M.), the ARC Centre of Excellence for Coral Reef Studies (to P.L.M.) and the
University of Oslo (to G.E.N.).
Supplementary material
Supplementary material available online at
http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.092478/-/DC1
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