A Behavioral and Neuropharmacological Study of Zebrafish as Model for Ocean Acidification by Investigating GABAA Receptor Subunits
Md Abdul Alim *1, Svante Winberg 2
Corresponding Author: Md. Abdul Alim, PhD,
Copy Right: © 2023 Md. Abdul Alim, This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Received Date: May 24, 2023
Published Date: June 01, 2023
Abstract
The function, signal transduction mechanisms and dynamics of neurotransmitters and their receptors, including the gamma-aminobutyric acid A (GABAA) receptor, are highly conserved across the vertebrate subphylum. GABAA receptors are Cl-channels that have inhibitory, hyperpolarizing, effects in most neural circuits. Elevated ocean CO2 levels have been reported to cause drastic behavioral alterations in marine fish. In teleosts, acidosis induced by hypercapnia results in an uptake of HCO3- in exchange for Cl-. This results in a decrease in extracellular [Cl] and a simultaneous increase in [HCO3-]. Previous results suggest that these changes in extracellular [Cl-] and [HCO3-] will reverse the action of the GABAA receptor, making it excitatory instead of inhibitory. The aim of this study was to investigate if zebrafish (Danio rerio) could be applied as a model for studies on ocean acidification. Adult zebrafish were reared at elevated pCO2 (1700 ppm) for 40 days. Behavioral effects were assessed using an open-field test. An automatic video-tracking system (Noldus Ethovision) was applied to quantify fish locomotion, pattern, swimming distance, and activity. In addition, the behavioral effects of the GABA-A receptor antagonist, gabazine, were studied using the behavioral assay. The effects of elevated pCO2 on the expression of the gabra1, gabra2, gabra3, gabra4, gabra5 gabra6a, gabra6b genes were analyzed using quantitative RT-qPCR. We found that elevated pCO2-exposed animals are quite stressed and anxious by measuring their swimming activity, velocity (cm/s), movement(s) and distance travelled to the center point (cm). We also found that GABAA receptor antagonist can restore their swimming behavior by reducing the velocity and distance moved but increased their immobility time. Thus, animal showed clear preference into a particular area of the tank as their “safe zone” where animal feel more secure and spent more time. The study has also demonstrated that gabra2, gabra3, gabra5 gabra6a, and gabra6b genes are relatively expressed higher than in control fish. Importantly, this preliminary data could be in line with better observations that can report for the study of ocean acidification and serve as a key model for the study of therapeutic effects of drugs on anxiety and stress like-phenotypes. In addition, this finding can also be promising for quantifying new target genes of GABAA receptor and its subunits for further gene study. Subsequently, studies can be performed to determine GABA-activated currents and membrane potential changes in neurons by high throughput patch-clamp methodology.
Keywords: Zebrafish, GABAA receptor, open filed test, qPCR, ocean acidification, anxiety and stress response, gabazine.
Introduction
As a result of anthropogenic emissions of CO2 to the atmosphere, ocean pCO2 increases (Doney et al., 2012). Studies have reported that teleost fish are highly tolerable to the effects of ocean acidification (Ruckelshaus et al., 2012 and Ishimatsu et al., 2008). In fish the gill is the major organ which regulates the pH by excreting H+ and absorbing HCO3− (Melzner et al., 2009). This regulatory mechanism effectively buffers the blood pH at the transient period of high pCO2, making fish tolerant to modest CO2-challenges by ocean acidification (Brauner et al., 2009, Melzner et al., 2009). Blood pH is maintain regardless of CO2 exposure, but results in alterations of blood ion concentrations, including increased HCO3−, decreased Cl−, (Brauner et al., 2009; Foss et.al. 2003). In a near future ocean CO2 concentrations will have increased from today’s 395 ppm (µatm) to 700-900 ppm. During the last few years, researchers have found that many tropical marine fish species change their behavior dramatically when subjected to elevated CO2 concentrations. Clownfish (Amphiprion percula) change their olfactory ability and display other abnormal responses when subjected to elevated CO2 concentrations (about 700-900 ppm) (Munday et al., 2009 and 2010). In addition to disturbance of the olfactory system, clownfish reared at elevated pCO2 display reduced auditory response (Simpson et al., 2011), impaired visual risk assessment and learning (Ferrari et. al., 2012). Similarly, brown dottyback (Pseudochromis fuscus) exposed to elevated pCO2 show decreased olfactory prey detection and feeding, but increased activity (Cripps et. al., 2011). In addition, reduced behavioral lateralization and prey detection has been observed in larval Neopomacentrus azysron, as well as other coral reef species at elevated pCO2 (Domenici et.al 2012; Nilsson et.al 2012. Recently, it has been showed that several European fish species including three-spined stickleback (Gasterosteus aculeatus), European plaice (Pleuonectes platessa) as well as few species of goby (Gobiusculus flavescens) also demonstrated similar behavioral disturbances in response to elevated pCO2 (Jutfelt et.al., 2013).
Zebrafish (Danio rerio), is a small (30 mm standard length, Ls) cyprinid fish which mainly originates from the river basins of Ganges and Brahmaputra in north-eastern India, Bangladesh, Nepal and few parts of northern Myanmar (Laale 1977; Barman 1991; Rowena et.al., 2008). Zebrafish is becoming a more widely used animal model for studies on the molecular basis of neurobiology, including applications in neuropharmacology and neurotoxicology (Linney et al., 2004; Teraoka et al., 2003). Recently, zebrafish has also been used for studies of complex behavioral functions, like memory, learning and anxiety responses, and the central neural circuits mediating these functions (Xue et al., 2007). Thus, this fish model is becoming an efficient and complementary model for the study of anxiety and stress responses in vertebrates.
Open field test (Kulikov et al., 2008, Choleris et al., 2001, Prut and Belzung, 2003) is an important and well-studied behavioral test for zebrafish. The open field test (OFT) is a relatively simple, and unconditioned test that can easily asses the natural and spontaneous behavior of animals during their exploration of a novel environment (Sousa et.al., 2006). In general, OFT measure the exploring behavior of animals at the novel environment (to find food, mating partner, escape routes) and fear of about the large unknown place (Prut et al., 2003; Sousa et.al., 2006). At the novel environment, the animals those are habituated by exploring them by several text trials, got experienced, and have the aversive properties like reduced or inhibit exploratory behavior and promote thigmotaxis in rodent (Sousa et.al. 2006). However, most recently a version of OFT as referred to drive test (novel tank driving test) has been developed for zebrafish (Levin et al., 2007; Bencan et al., 2007; Egan et al., 2009). OFT is the most widely used behavioral assay for monitoring anxiety-like behavior. As for example, avoidance of the center zone of the arena is the key marker of anxiety in rodents (Levin et al., 2006). In OFT test, zebrafish show a strong avoidance of the center zone and fish mostly swim in the periphery of the arena (Kulikov et al., 2008 and Lamprea et al., 2008). This behavior is referred as thigmotaxis (Sousa et al., 2006). Anti-anxiety drugs reduce thigmotaxis in zebrafish.
Gabazine is a selective GABAA receptor antagonist. Basically, Gabazine binds to the GABA binding site of the receptor-channel complex, inhibiting opening of the ion channel (Yeung et al., 2003). GABAA receptors function as the major inhibitory input in most neural communication. However, Gabazine restored normal behavior in fish reared at elevated pCO2. A recent study has also showed that the effect of GABAA receptor antagonist, Gabazine has the ability to restore the olfactory and behavioral abnormalities which were caused by high CO2 (900 ppm) (Nilsson et al., 2012). GABAA receptors have high conductivity for chloride ion (Cl-) but lower conductivity for HCO3-. After activation of GABAA receptor the ion gradient across the neural membrane will cause inflow of the Cl- into the cell by hyperpolarization and thereby inhibition of the neuron signal (Fig.1).
Figure 1. A proposed mechanism of GABAA receptor functions by increased level of CO2 concentration in sea water. In the normal mood the ion gradients are such that help to open the channel results in Cl- and HCO3- inflow, causing the neural inhibition through hyperpolarizing current. On the other hand, elevated sea water pCO2 leads ion-gradient and regulates the adjustment of ion balance by making neural excitation and behavioral changes (Fig. is edited from Nilsson et al. 2012).
The main purpose of this project was to investigate the behavioral changes of zebrafish fish as a model for studies on ocean acidification. This finding also aimed to investigate the behavioral alteration of fish by drug administration. Furthermore, the study was aiming to investigate GABAA receptor and its subunits.
Materials and Methods
Animals and housing
Adult zebrafish (AB-strain), bred in captivity was used in this experiment. The use of animals in this study was permitted by Uppsala Ethical Committee (permit number C55/13. The fish were housed in the laboratory at Uppsala University Biomedical Center (BMC). Fish were kept in tanks with municipal tap water (pH≈7.3) of which 15% were exchanged daily and kept at a temperature of 27?C. Fluorescent ceiling light tubes illuminating at 14h:10h light: dark cycle was provided. Fish were fed once or twice per day with Tropical flake food (Sera San) and frozen Artemia naupli. A total of 20 fish were used ((3–5-month-old; 50:50 male: female ratio)). Due to a limited number of equally sized tanks two different kinds were used to isolate and house the fish; one with the capacity of 2.8 L (measured according to 15 cm height X 27 cm top X 22 cm bottom X 7 cm width) and one with the capacity of 1.8 L (14 cm height X 26 cm top X 23 cm bottom X 5 cm width). Basically, all fish were made in two groups according to control group (500 ppm) and high CO2 exposure group (1700 ppm).
CO2 exposure system
The pCO2 of all aquaria, was measured regularly by using direct pCO2 measurements with an infra-red CO2 probe (GMT 222, Vaisala, Finland), connected to a submerged gas-permeable silicone membrane as well as the air inside the membrane, was distributed in a closed loop to equilibrate with dissolved pCO2 (Munday et al., 2012). Accurate factory calibration of the probe was established on several occasions throughout the experiment using water systematically bubbled with a gas mixture of 1010±10 ppm CO2 in air (AGA, Sweden). The pCO2 of the two CO2 treatment heading tanks were well-maintained at the target value of 1700 µatm using pH stat Computers (Aqua Medic, Bissendorf, Germany) connected to solenoid valves regulating management of 100% CO2 gas (AGA, Sweden). The pCO2 of control aquaria was 333 µatm±30 SD and the pCO2 of the CO2 treatment aquaria was 991 µatm (57±SD). The variance in pCO2 between aquaria within each header tank system was below the detection limit (<10 µatm), while the variance between header tank systems within treatment was measurable. The tap water carbonate system speciation was calculated on the basis of salinity, temperature, pCO2, and alkalinity in CO2calc (Hansen, USGS, USA); (Roy et al., 1993; Dickson et al.,1990).
Apparatus and Behavioral testing Experimental setup
The open field test arena used in our experiment mainly consisted of a rectangular white glass box (34 cm length X 29 cm width X 14 cm height) filled by system water (6 cm in depth). Before introducing the fish to the test arena, fish were immersed for 30 minutes in treatment beakers (20 cm length X 9 cm width X 7 cm height) with lids to reduce the anxiety and stress. Open field boxes were mainly made opaque by using masking tape. Subsequently, these boxes were placed on a white infrared table with a camera detector attached in the ceiling above. OFT were based on the following endpoints: time spent in the top of the tank (sec), transitions number (entries) to the top, swimming velocity, number of erratic movements, and count of the duration of freezing bouts, and distance moved to the center point.
Computer-Aided Analysis
The OFT apparatus were connected and recorded by commercially available video-tracking software named Ethovision XT7 (Noldus Information Technology, Netherlands). According to the video tracking software OFT were setups before starting the trial text.
Acclimation, pre-treatment and treatment
The tested animals were transported from the CO2 exposure tank to the experimental room for acclimation 30 minutes prior to test. The water used in the OFT followed by rake system tap water and temperature must be same as the holding room. However, different water temperature might evoke unwanted stress in animals. Therefore, adjusted temperature is required during the treatment and pre-treatment condition. We mainly had two groups of fish as control and high CO2-exposed. Firstly, we have treated both groups of fish by systematic tap water, after that we performed OFT testing to see the effect of CO2 on fish behavior. Then, we kept fish groups separately at same exposure system with same conditions (500 ppm for control group and 1700 ppm for high CO2 group) for a day. Next day, the same group of fish as control and high CO2-exposed fish were treated with water and the drug Gabazine (8.1 mg/L)) respectively, to evaluate the drug effect on fish behavior. Notably, all treatment duration was 30 minutes for both cases (water and Gabazine) and the water temperature were kept at 27°C (monitored with a thermometer).
OFT testing
At the same time four OFT apparatus were performed with the adjusted video tracking system. The OFT tanks were filled with rake system tapwater (room temperature) with 6 cm depth. Video recording started by gently placing the fish into the centre of the OFT to begin the trial and avoid to enter the arena, otherwise it may interrupt the fish tracking. The OFTs were performed for 30 minutes but the trial duration may be modified as researcher’s needs. After 30 minutes experiment the animals were kept back at the holding room. Basically, behavioral testing took place between 11-16 h. Subsequently, by using Ethovision XT software (Noldus, the Netherlands) we measured different types of movement pattern (distance moved, duration in the center zone, mobility and immobility time and zone transitions etc.). However, the center zone was defined as half of the area of the arena.
Reverse Transcriptase Quantitative PCR
To investigate the potential gene expression of GABAA receptor from the AB zebrafish line, we have used a sensitive reverse transcriptase quantitative polymerase chain reaction (RT- qPCR). After the behavioral performance all the fish were sacrificed to isolate RNA samples. Firstly, body weight and length of fish were measured and found to be statistically indistinguishable from other strains (not shown). Thus, the fish were sacrificed by decapitation and the total brain of the fish (except the eyes) was quickly removed from the skull and immediately put into RNAlater and subsequently saved at − 80 °C until RNA extraction. Total RNA was extracted from the brains according to the manufacturer's protocol GenElute™ Mammalian Total RNA Miniprep Kit (Sigma Aldrich, Sweden AB). After isolating total RNA samples, RNA quantity and quality were determined by using the Spectrophotometer Nano Drop ND-1000 (Nano Drop Technologies, Inc. Wilmington, DE, USA). For most genes RNA quality was determined as being excellent but a minority of genes had to be excluded from this study. On the basis of RNA purity and concentration, 500 ng RNA was used for semi-quantitative RT-qPCR and cDNA was synthesized according to Maxima First Strand cDNA Synthesis Kits for RT-qPCR protocol (Fermentas Life sciences). Several genes were chosen and selected as targets for investigation of potential gene expression level of the AB line zebrafish. The selected genes used during this study were gabra1, gabra2, gabra3, gabra4, gabra5 gabra6a, gabra6b. In addition, ACTB1and UBC genes were regarded as a house keeping genes to use as endogenous control. Eventually, the intensity of bands obtained for the target genes were normalized by using the ACTB1 and UBC. PCR primer pairs were designed by our PhD collaborator using the Primer3 software application and sequence data were acquired from GenBank. In general, primers having 40- 60% GC content and temperature about 55-60 °C were preferred. The primer sequences for target and control gene (s) were used for quantitative RT-qPCR are reported in Table 1 and Table 2 respectively.
Statistical Analysis
All behavioral data were analyzed with one or two-way analysis of variance (ANOVA) and post hoc comparisons between the groups were made with the Fisher’s post hoc test. Student’s test with equal variances was used to analyze experiments with two groups.
Results
Elevated CO2 induces an anxiety or stress-like phenotype in zebrafish
In order to see the effect of high CO2 on fish behaviors, we have tested our control and high CO2-exposed fish by placing on OFT paradigm. We have found that high CO2-exposed adult zebrafish changed their swimming behaviors compared to control fish during a 30 mins test period. According to the OFT results, affected animals increased their velocity (cm/s), distance moved to the center point (cm) and mobility time (s) compared to control fish. However, animals showed erratic movement (rapidly changed their swimming pattern and increased velocity), high number of entities to the center point of the tank which correlate with high stress or anxiety like phenotype ((Fig. 2, panel A and B). The effect of the high CO2 on the time spent in the different zones of the apparatus was statistically analyzed by a 1-way ANOVA and showed a significant interaction “zone” x “group” interaction [F(2,80)=13.109, p<0.0001]. The following Fisher post-hoc comparison confirmed the difference between the two groups in the time spent in both the center and corner of the tank (± SEM. *** p< 0.001) and (± SEM. * P< 0.05).
Figure 2. Anxiety/stress-like phenotype in high CO2-exposed fish. OFT test is performed in control (n=8) and high CO2-exposed (n=8) fish after water treatment. The graph shows A) velocity (measured in cm/s), B) distance moved to the center point (measured by cm) and C) movement (mobility and immobility times measured by sec) by the animals in the center as well as in the corner or near the wall of the apparatus, during 30 min test. Data are expressed as mean ± SEM. *** p< 0.0001, *p <0.05 vs control fish, same zone.
Thus, we found that our animals are more stressed and aggressive by the effect of high CO2 which leads anxiety/stress-like phenotype.
Anxiety and stress-like behaviors are reversed by gabazine treatment
To investigate the involvement of GABA neurotransmission systems in anxiety- and stress - like response in fish, we verified the signaling ability of GABAA receptor by administration of Gabazine. Furthermore, we subjected the control (water-treated) and highly CO2-exposed (Gabazine-treated) fish in OFT to monitor their behavioral alteration. During these experiments, we saw that our highly CO2-exposed fish dramatically reduced their velocity (cm/s), distance moved to the center point (cm) rather than water treated control fish (Fig. 3 panel A and B). Interestingly, we also found that Gabazine increased the immobility time (s) but decreased mobility of the highly CO2-exposed fish compared to control fish (see Fig. 3 panel C).
Figure 3. Gabazine showed opposite effect on high CO2-exposed fish. After water and Gabazine treatment, an OFT test was performed in control (n=8) and high CO2-exposed (n=8) fish, respectively. The graph shows the A) velocity (measured in cm/s), B) distance moved to the center point (measured by cm) and C) movement (mobility and immobility times measured by sec) by the animals in the center as well as in the corner or near the wall of the apparatus, during 30 min OFT test. Data are expressed as mean ± SEM. ** p< 0.01, *p <0.05 vs control fish.
The 1-way ANOVA analysis showed a significant “zone” x “group” interaction [F(3,29)=5.863, p=0.0034], The following Fisher post-hoc comparison confirmed the difference between the groups in the time spent in both at corner and center of the OFT apparatus. Thus, the 1-way ANOVA revealed a significant treatment effect [F(3,29)=5.863, p=0.0034], and the following Fisher post-hoc indicated that in fact highly CO2-exposed fish totalized a lower immobility time compared to the control fish, and Gabazine-treated fish showed higher immobility time compared to control fish which was treated by water.
However, previous results in our study showed that elevated CO2 induced anxiety or stress- like behaviors in our fish model, but after Gabazine treatment these behaviors could reversed.
Elevated CO2-exposed fish showed higher expression level of GABAA receptor and its subunits by relative quantification.
The relative expression of GABAA receptor and its subunits were analyzed with RT-qPCR by using the CT values of target (GABAA subunits) and control genes (ACTB1 and UBC). As endogenous control, ACTB1 and UBC were used for normalization of the target genes and the expression levels of GABAA receptor subunits were monitored. To normalize the genes of interest the following formula were used to calculate ΔCT.
DCT = CT Target gene - CT Endogenous control--------------------------------------------------------------------------------- (1)
Target gene and reference gene are required to normalize the sample to calibrator sample
DDCT, and the given formula is:
DDCT = (DCT target) – (DCT reference)--------------------------------------------------------------------------------- (2)
To find relative quantity of the target gene, the following formula is used, Relative quantity (fold expression) = 2-DDCT-------------------------(3)
Figure 4. Relative quantification of GABAA receptor subunits in control and high CO2-exposed zebrafish. The RT-qPCR analysis showed that the level of expression of gabra2, gabra3, gabra5 gabra6a, and gabra6b genes are higher than in control fish. Elevated CO2 induced higher expression of target subunits which mimics that the animals are anxious and stressful rather than control. Data are shown as mean ± SEM. *** p < 0.001, ± SEM. ** p< 0.01, ± SEM. * p < 0.05 1 vs control; NS: gabra1 is not showed any significance with control.
All together these results demonstrated that the anxiety- and stress-like phenotype induced by ocean acidification in our zebrafish model, can be restore by the selective drug antagonist Gabazine. Thus, Gabazine showed a significant effect on highly exposed fish by altering their swimming behaviors.
Discussion
In this study, OFT test is scored and discussed on the basis of fish time spent in the top of the tank (sec), transitions number (entries) to the top, number of erratic movements, and count the duration of freezing bouts. Erratic movements are known as sharp changes in direction and/or velocity, which represented the rapid anxiety-like behaviors (Egan RJ, et al). Freezing is defined as a total absence of movement, without the gills and eyes movement for 2 s or longer. In general, many studies have revealed that reduced exploration (fewer entries to the top, more freezing) and increased erratic movements correlate with high stress or anxiety-like phenotype. According to OFT, fish scored their behavioral performance and showed the significant results in figure 2 panel A and panel B (p < 0.001) and these behavioral altertations were compaired with control fish. However, statistically we found significant differences between control and high CO2-exposed fish. In addition, the control group exploired their swimming performance as like in their home (safe zone) where animals thaught to be more seciure at near and/or corner of the wall of the OFT aparatus, but high CO2-exposed fish showed a quite different swiming pattern where they expolored over the whole tank in a similar way as described by Eilam and Golani, 1989. Our study also reaveled that high CO2-exposed fish significantly [p < 0.0001] moved more distance than non-stressed control fish (Fig. 2). Thus, the affected fish are anxious and stressed by changing their normal swimming behaviors and locomotion. Two-way ANOVA analysis for repeated measures and Fisher post hoc analysis revealed the main effect of exposure [p < 0.0001] indicating that stressor-exposed zebrafish crossed significantly (all p < 0.001 and p < 0.05) more zones than non-stressed controls during the entire duration of the test. At OFT, the definition of several distinct zones, allowed to analysis of the patern of locomotor and zone preference. The study found that zebrafish displayed general behavioral hyperactivity at the initilal period (first 2 min) to a novel enviroment as suggested by the greater distance moved and high velocity. However, these behaviors subsequently attenuated towards the end of 30 mintutes test period. As a result, zebrafish had the capacity for habituation which mimic non-associtative learning. The study investigated the impact of stress and anxiety response in animals by characterizing behavioral performance of fish which is traditionaly monitored as increase in the time, speed and distance moved to the centre point of the arena. Therefore, the findings suggested that acute stress may fleetingly relieve anxiety in zebrafish compared to others study (Belzung et. al., 2007; Prut et. al., 2003; Markou et. al., 2009). Another study has reported oposite effects and suggested that acute stress can temporarily increase swimming activity/exploration in the OFT test. Furthermore, acute stress can also reduce anxiety-like phenotype (Quartermain et. al., 1996). However, the reason underlying this phenomenon are unkown and uncertain. According to our finding it can be suggested that stressed zebrafish seem to adopt less coping style that is not suited for successful escape and thefore be the reason of less adaptive in nature (increase risk for predator attacks) and mimic imparied cognitive defecits.
Gabazine is a highly specific antagonist on its mode of action to GABAA receptor (Steinbach et al., 1997). Our results clearly indicated that anxiety and stress-related behaviors are altered by the treatment of Gabazine (8.1 mg/l). Gabazine reduced swimming velocity (cm/s), total distance travel to the centre point but incresed immobility time in the open field test (all p < 0.001 and p < 0.05). However, these types of alteration might become changed in Cl- and/or HCO3- gradients over neural membranes that is caused by acid-base balance. Accumulating evedences suggested that Gabazine binds to the GABA binding site of the receptor-channel complex and that it acts as an allosteric inhibitor for opening the ion channel (Yeung et al., 2003). Subsequently, elevated CO2 level of sea water decreased the behavioral abnormalities of marine fish by changing the function of GABAA receptor. Thus, the net effect is to reduce GABA-mediated synaptic inhibition by inhibiting chloride flux across the cell membrane resulting in neuronal hyperpolarization. A recent study was able to show that the treatment with Gabazine completely restored the olfactory and behavioral abnormalities that were caused by high CO2 (900 ppm) (Nilsson et al., 2012). GABAA receptor has high conductivity for Cl- but lower conductivity for HCO3-. By activation of GABAA receptor with Gabazine, the ion gradient across the neural membrane will cause inflow of Cl- into the cell, hyperpolarization, and inhibition of neuron signaling. Reversal of GABAA receptor function showed a mechanistic link between high pCO2 and brain functions in zebrafish. The influence of elevated CO2 levels on GABAA receptor function may have widespread and unrecognized effects on fish behaviors and ecosystems. The potential problem is underscored by the occurrence of GABAA receptor in both vertebrates and primitive invertebrate and serving as ubiquitous inhibitory neurotransmitter receptor during neural circuits’ formation (Akerman et al., and Tsang et al., 2007). A previous study has predicted that GABAergic effects of elevated CO2 are more likely to follow in the aquatic phase rather than in the terrestrial, because water breathers are exposed to much lower CO2 and HCO3- level than air breathers (Heisler et al., 1986). Thus, we suggested that the effect of high CO2 on GABAA receptor activity modulates swimming behavior in different fish species, and this alteration might be possible to restore the behavior by selective drug agonist and/or antagonist. Although, this idea wouldn’t be more realistic for the treatment of fish species one by one at sea but would be more helpful to understand the molecular mechanism of GABA, GABA-associated pathways and drug-receptors interaction. In addition, study can reveals that which CO2 concentrations might be reasonable for ocean acidification and changing the normal behaviors of fish species.
Eventually, selected seven target genes (gabra1, gabra2, gabra3, gabra4, gabra5 gabra6a, gabra6b) were studied and we measured their expression levels. These genes were chosen because of their known involvement in high CO2 level-induced functional changes in the brain of mammals as well as zebrafish (Y. Pan et al., 2011). Our study revealed through RT- qPCR analysis that expression of gabra2, gabra3, gabra5 gabra6a, and gabra6b were significantly higher (± SEM *** p < 0.001; ** p< 0.01, ± SEM. * p < 0.05) than control genes. Possibly, elevated CO2 level might have higher impact on expression of GABAA subunits that contributes to the anxiety- and stress like fish behaviors.
Conclusion
The findings presented here confirm that highly CO2-exposed zebrafish in open fieled test can exhibit anxiety- or stress-like behavioral responses. Furthermore, the study revealed that the GABAA receptor-selective antagonist Gabazine has effect on behavioral alteration of zebrafish by reducing swimming velocity, total distance moved to the center and increases/decreases the mobility time. Moreover, this study has found that the targeted genes gabra2, gabra3, gabra5 gabra6a, and gabra6b are highly expressed due to elevated CO2 level compared to control gene. However, data from this study shows the effectiveness of the antagonist Gabazine to significantly reduce the stress-induced alterations in behavioral strategies and supports the value of this assay to disclose disturbances of the anxiety or stress- related system using zebrafish models for the study of ocean acidification. A single neuroactive drug able to restore anxiety and stress responses is certainly needed. In the future, the zebrafish model could be an important research tools for improving pre-clinical drug screening approaches towards the goal of uncover novel and neuroeffective drug. In addition, this finding can also be more auspicious for computing new target genes of GABAA receptor and its subunits for further gene study. Subsequently, future studies can explore GABA- activated currents and action potential changes in neurons by high throughput patch-clamp methodology.
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