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OPEN
received: 11 April 2016 accepted: 23 August 2016 Published: 13 September 2016
Altered brain ion gradients following compensation for elevated CO2 are linked to behavioural alterations in a coral reef fish R. M. Heuer1,2, M. J. Welch3,4, J. L. Rummer3, P. L. Munday3 & M. Grosell1 Neurosensory and behavioural disruptions are some of the most consistently reported responses upon exposure to ocean acidification-relevant CO2 levels, especially in coral reef fishes. The underlying cause of these disruptions is thought to be altered current across the GABAA receptor in neuronal cells due to changes in ion gradients (HCO3− and/or Cl−) that occur in the body following compensation for elevated ambient CO2. Despite these widely-documented behavioural disruptions, the present study is the first to pair a behavioural assay with measurements of relevant intracellular and extracellular acid-base parameters in a coral reef fish exposed to elevated CO2. Spiny damselfish (Acanthochromis polyacanthus) exposed to 1900 μatm CO2 for 4 days exhibited significantly increased intracellular and extracellular HCO3− concentrations and elevated brain pHi compared to control fish, providing evidence of CO2 compensation. As expected, high CO2 exposed damselfish spent significantly more time in a chemical alarm cue (CAC) than control fish, supporting a potential link between behavioural disruption and CO2 compensation. Using HCO3− measurements from the damselfish, the reversal potential for GABAA (EGABA) was calculated, illustrating that biophysical properties of the brain during CO2 compensation could change GABAA receptor function and account for the behavioural disturbances noted during exposure to elevated CO2. Concerns about the impact of ocean acidification on marine ecosystems has led to a growing number of studies examining the effects of elevated CO2 exposure on fish1. While some investigated endpoints such as survival and growth appear to be relatively insensitive to projected future CO2 levels2,3, significant effects of elevated CO2 include alterations to mitochondrial function4,5, metabolic rate6, otolith growth7,8, reproduction9,10, and acid-base balance11,12. Perhaps the most frequently reported and consistently adverse response to elevated CO2 exposure in fish is disruption to sensory or cognitive function. Impairments to olfaction13–15, hearing16, vision17,18, lateralization19–21, and learning22,23 in fish at ocean acidification relevant CO2 levels demonstrate that CO2 broadly affects central neuronal processing. Neurosensory impacts are particularly concerning since these traits appear to show limited capacity for acclimation13. Furthermore, fish living near highly acidic natural CO2 vent systems that presumably experience high CO2 on a regular basis also exhibit abnormal behavioural responses24. Considering the rapid rate of acidification25 and the low CO2 threshold level needed to induce sensory and neurological responses (~600–800 μatm CO2)26, understanding the physiological mechanism underlying these responses is crucial for assessing risk to fish populations and could aid in predicting adaptive capacity. Most studies to date suggest that significant effects of CO2, including behavioural disturbances, result from compensation that fish perform in response to a CO2-induced respiratory acidosis. Following exposure to elevated CO2, fish correct plasma and tissue pH by sustaining elevated HCO3− levels in intracellular and extracellular 1
University of Miami, RSMAS, 4600 Rickenbacker Causeway, Miami, FL 33149, USA. 2University of North Texas, 1511 West Sycamore, Denton, TX 76203, USA. 3Australian Research Council Centre of Excellence for Coral Reef Studies, James Cook University, Townsville, Queensland, 4811, Australia. 4College of Marine and Environmental Sciences, James Cook University, Townsville, QLD 4811, Australia. Correspondence and requests for materials should be addressed to R.M.H. (email: [email protected]) Scientific Reports | 6:33216 | DOI: 10.1038/srep33216
1
www.nature.com/scientificreports/ fluids12,27–29. Although pH is corrected to pre-exposure levels, HCO3− and PCO2 consequently remain elevated throughout high CO2 exposure. Increased plasma HCO3− concentrations are often paired with a corresponding decline in Cl− 27,30,31. Surprisingly, examination of CO2 acid-base balance disturbances and associated compensatory mechanisms have only been performed at ocean acidification relevant scenarios in a limited number of studies5,12,32,33. In 2012, Nilsson and colleagues reported a series of seminal experiments on fish, suggesting compensation for elevated CO2 affects olfaction and lateralization by disrupting the function of the GABAA receptor34. Under most circumstances, the GABAA receptor and its associated neurotransmitter (GABA) are thought to be largely responsible for inhibitory responses throughout the vertebrate nervous system. In the model proposed by Nilsson and colleagues, following stimulation by GABA, HCO3− and/or Cl− ions enter the cell through the GABAA receptor under control conditions, leading to cellular hyperpolarization, and a concomitant inhibitory response that is associated with a normal behavioural phenotype in fish. However, expected changes in extracellular and/or intracellular HCO3− and Cl− that occur during CO2 compensation are thought to reverse ion movement through the GABAA receptor, leading to a depolarizing excitatory response and a disrupted behavioural phenotype34. Alleviation of olfactory and lateralization disturbances in CO2-exposed fish upon treatment with gabazine, a competitive GABAA receptor antagonist that presumably closes the GABAA receptor, implicated GABAA receptor involvement in the impaired behavioural responses induced by elevated CO2. Since this initial study, the apparent link between CO2-induced behavioural disturbances and the GABAA receptor has been supported by several other studies examining a variety of species (tropical and temperate, marine and freshwater), utilizing an array of sensory and behavioural assays as well as different GABAA receptor antagonists and agonists17,21,22,35–38. Similar behavioural effects of high CO2 exposure that are restored by GABAA receptor antagonists have also been observed in marine invertebrates39. Further support for the role of the GABAA receptor in abnormal behaviour during CO2 exposure has been provided by theoretical calculations of the GABAA receptor equilibrium potential (EGABA)1 using HCO3− values estimated from the Gulf toadfish12. However, it is important to keep in mind that altered ion gradients due to high CO2 exposure would not necessarily have to cause a complete reversal of current to invoke a behavioural change. Even an attenuation of the normal inhibitory response of the GABAA receptor due to changes in ion gradients could alter the function of neurons and account for noted behavioural disruptions. Behavioural assays paired with GABAA-targeted drug treatments have strongly supported the argument that altered ion gradients in a high CO2 environment change the function of the GABAA receptor; however, adjustments to acid-base parameters that would reverse or attenuate the current through the GABAA receptor have yet to be measured in a marine fish showing a behavioural disruption. Accordingly, the aim of this study was to test the hypothesis that altered intracellular and extracellular HCO3− due to CO2 compensation occurs in a species that also exhibits a behavioural disturbance when exposed to elevated CO2. The first objective of this study was to measure intracellular whole-brain HCO3− and pH (pHi) and extracellular HCO3− levels in blood plasma of the spiny damselfish (Acanthochromis polyacanthus) exposed to either control or 1900 μatm CO2. A second objective was to confirm that spiny damselfish exposed to the applied CO2 level displayed altered behavioural responses to olfactory cues as previously reported13,15. The third and final objective was to apply the measured values in an assessment of GABAA receptor function by calculating EGABA in control and CO2-exposed fish. To our knowledge, this is the first study to report direct measurements of both intracellular and extracellular HCO3− and intracellular pHi in a coral reef fish species.
Results
Physiological measurements: Brain and plasma analyses. As expected, brain HCO3− (mmol/kg)
and brain pHi (Fig. 1a,b) were both significantly higher in damselfish exposed to 1900 μatm CO2 for 4 days when compared to controls (Fig. 1a,b; brain HCO3−, P
OPEN
received: 11 April 2016 accepted: 23 August 2016 Published: 13 September 2016
Altered brain ion gradients following compensation for elevated CO2 are linked to behavioural alterations in a coral reef fish R. M. Heuer1,2, M. J. Welch3,4, J. L. Rummer3, P. L. Munday3 & M. Grosell1 Neurosensory and behavioural disruptions are some of the most consistently reported responses upon exposure to ocean acidification-relevant CO2 levels, especially in coral reef fishes. The underlying cause of these disruptions is thought to be altered current across the GABAA receptor in neuronal cells due to changes in ion gradients (HCO3− and/or Cl−) that occur in the body following compensation for elevated ambient CO2. Despite these widely-documented behavioural disruptions, the present study is the first to pair a behavioural assay with measurements of relevant intracellular and extracellular acid-base parameters in a coral reef fish exposed to elevated CO2. Spiny damselfish (Acanthochromis polyacanthus) exposed to 1900 μatm CO2 for 4 days exhibited significantly increased intracellular and extracellular HCO3− concentrations and elevated brain pHi compared to control fish, providing evidence of CO2 compensation. As expected, high CO2 exposed damselfish spent significantly more time in a chemical alarm cue (CAC) than control fish, supporting a potential link between behavioural disruption and CO2 compensation. Using HCO3− measurements from the damselfish, the reversal potential for GABAA (EGABA) was calculated, illustrating that biophysical properties of the brain during CO2 compensation could change GABAA receptor function and account for the behavioural disturbances noted during exposure to elevated CO2. Concerns about the impact of ocean acidification on marine ecosystems has led to a growing number of studies examining the effects of elevated CO2 exposure on fish1. While some investigated endpoints such as survival and growth appear to be relatively insensitive to projected future CO2 levels2,3, significant effects of elevated CO2 include alterations to mitochondrial function4,5, metabolic rate6, otolith growth7,8, reproduction9,10, and acid-base balance11,12. Perhaps the most frequently reported and consistently adverse response to elevated CO2 exposure in fish is disruption to sensory or cognitive function. Impairments to olfaction13–15, hearing16, vision17,18, lateralization19–21, and learning22,23 in fish at ocean acidification relevant CO2 levels demonstrate that CO2 broadly affects central neuronal processing. Neurosensory impacts are particularly concerning since these traits appear to show limited capacity for acclimation13. Furthermore, fish living near highly acidic natural CO2 vent systems that presumably experience high CO2 on a regular basis also exhibit abnormal behavioural responses24. Considering the rapid rate of acidification25 and the low CO2 threshold level needed to induce sensory and neurological responses (~600–800 μatm CO2)26, understanding the physiological mechanism underlying these responses is crucial for assessing risk to fish populations and could aid in predicting adaptive capacity. Most studies to date suggest that significant effects of CO2, including behavioural disturbances, result from compensation that fish perform in response to a CO2-induced respiratory acidosis. Following exposure to elevated CO2, fish correct plasma and tissue pH by sustaining elevated HCO3− levels in intracellular and extracellular 1
University of Miami, RSMAS, 4600 Rickenbacker Causeway, Miami, FL 33149, USA. 2University of North Texas, 1511 West Sycamore, Denton, TX 76203, USA. 3Australian Research Council Centre of Excellence for Coral Reef Studies, James Cook University, Townsville, Queensland, 4811, Australia. 4College of Marine and Environmental Sciences, James Cook University, Townsville, QLD 4811, Australia. Correspondence and requests for materials should be addressed to R.M.H. (email: [email protected]) Scientific Reports | 6:33216 | DOI: 10.1038/srep33216
1
www.nature.com/scientificreports/ fluids12,27–29. Although pH is corrected to pre-exposure levels, HCO3− and PCO2 consequently remain elevated throughout high CO2 exposure. Increased plasma HCO3− concentrations are often paired with a corresponding decline in Cl− 27,30,31. Surprisingly, examination of CO2 acid-base balance disturbances and associated compensatory mechanisms have only been performed at ocean acidification relevant scenarios in a limited number of studies5,12,32,33. In 2012, Nilsson and colleagues reported a series of seminal experiments on fish, suggesting compensation for elevated CO2 affects olfaction and lateralization by disrupting the function of the GABAA receptor34. Under most circumstances, the GABAA receptor and its associated neurotransmitter (GABA) are thought to be largely responsible for inhibitory responses throughout the vertebrate nervous system. In the model proposed by Nilsson and colleagues, following stimulation by GABA, HCO3− and/or Cl− ions enter the cell through the GABAA receptor under control conditions, leading to cellular hyperpolarization, and a concomitant inhibitory response that is associated with a normal behavioural phenotype in fish. However, expected changes in extracellular and/or intracellular HCO3− and Cl− that occur during CO2 compensation are thought to reverse ion movement through the GABAA receptor, leading to a depolarizing excitatory response and a disrupted behavioural phenotype34. Alleviation of olfactory and lateralization disturbances in CO2-exposed fish upon treatment with gabazine, a competitive GABAA receptor antagonist that presumably closes the GABAA receptor, implicated GABAA receptor involvement in the impaired behavioural responses induced by elevated CO2. Since this initial study, the apparent link between CO2-induced behavioural disturbances and the GABAA receptor has been supported by several other studies examining a variety of species (tropical and temperate, marine and freshwater), utilizing an array of sensory and behavioural assays as well as different GABAA receptor antagonists and agonists17,21,22,35–38. Similar behavioural effects of high CO2 exposure that are restored by GABAA receptor antagonists have also been observed in marine invertebrates39. Further support for the role of the GABAA receptor in abnormal behaviour during CO2 exposure has been provided by theoretical calculations of the GABAA receptor equilibrium potential (EGABA)1 using HCO3− values estimated from the Gulf toadfish12. However, it is important to keep in mind that altered ion gradients due to high CO2 exposure would not necessarily have to cause a complete reversal of current to invoke a behavioural change. Even an attenuation of the normal inhibitory response of the GABAA receptor due to changes in ion gradients could alter the function of neurons and account for noted behavioural disruptions. Behavioural assays paired with GABAA-targeted drug treatments have strongly supported the argument that altered ion gradients in a high CO2 environment change the function of the GABAA receptor; however, adjustments to acid-base parameters that would reverse or attenuate the current through the GABAA receptor have yet to be measured in a marine fish showing a behavioural disruption. Accordingly, the aim of this study was to test the hypothesis that altered intracellular and extracellular HCO3− due to CO2 compensation occurs in a species that also exhibits a behavioural disturbance when exposed to elevated CO2. The first objective of this study was to measure intracellular whole-brain HCO3− and pH (pHi) and extracellular HCO3− levels in blood plasma of the spiny damselfish (Acanthochromis polyacanthus) exposed to either control or 1900 μatm CO2. A second objective was to confirm that spiny damselfish exposed to the applied CO2 level displayed altered behavioural responses to olfactory cues as previously reported13,15. The third and final objective was to apply the measured values in an assessment of GABAA receptor function by calculating EGABA in control and CO2-exposed fish. To our knowledge, this is the first study to report direct measurements of both intracellular and extracellular HCO3− and intracellular pHi in a coral reef fish species.
Results
Physiological measurements: Brain and plasma analyses. As expected, brain HCO3− (mmol/kg)
and brain pHi (Fig. 1a,b) were both significantly higher in damselfish exposed to 1900 μatm CO2 for 4 days when compared to controls (Fig. 1a,b; brain HCO3−, P
