The authors have declared that no competing interests exist.
Conceived and designed the experiments: SS NO SC. Performed the experiments: SS PK WNWI NO MU SN MDT. Analyzed the data: SS SN. Contributed reagents/materials/analysis tools: LN SN SC. Wrote the paper: SS SC.
Current address: Department of Veterinary Basic Sciences, PMAS Arid Agriculture University, Rawalpindi, Pakistan
Perfluorooctane sulfonate (PFOS) is a widely spread environmental contaminant. It accumulates in the brain and has potential neurotoxic effects. The exposure to PFOS has been associated with higher impulsivity and increased ADHD prevalence. We investigated the effects of developmental exposure to PFOS in zebrafish larvae, focusing on the modulation of activity by the dopaminergic system. We exposed zebrafish embryos to 0.1 or 1 mg/L PFOS (0.186 or 1.858 µM, respectively) and assessed swimming activity at 6 dpf. We analyzed the structure of spontaneous activity, the hyperactivity and the habituation during a brief dark period (visual motor response), and the vibrational startle response. The findings in zebrafish larvae were compared with historical data from 3 months old male mice exposed to 0.3 or 3 mg/kg/day PFOS throughout gestation. Finally, we investigated the effects of dexamfetamine on the alterations in spontaneous activity and startle response in zebrafish larvae. We found that zebrafish larvae exposed to 0.1 mg/L PFOS habituate faster than controls during a dark pulse, while the larvae exposed to 1 mg/L PFOS display a disorganized pattern of spontaneous activity and persistent hyperactivity. Similarly, mice exposed to 0.3 mg/kg/day PFOS habituated faster than controls to a new environment, while mice exposed to 3 mg/kg/day PFOS displayed more intense and disorganized spontaneous activity. Dexamfetamine partly corrected the hyperactive phenotype in zebrafish larvae. In conclusion, developmental exposure to PFOS in zebrafish induces spontaneous hyperactivity mediated by a dopaminergic deficit, which can be partially reversed by dexamfetamine in zebrafish larvae.
Perfluorinated compounds (PFCs) are a family of chemical compounds that are very stable in the environment due to the carbon-fluorine bonds (reviewed in
In rodents, developmental toxicity studies on the effects of PFOS have revealed reduction of fetal weight, reduced neonatal survival, defects in the peripheral nervous system, and behavioral alterations
Most developmental neurotoxicity data available on PFCs were generated using rodents as animal models. Zebrafish (
PFOS (heptadecafluorooctanesulfonic acid potassium salt, CAS 2795-39-3, purity >98%) was purchased from Sigma-Aldrich, dissolved in DMSO (1 mg/mL), and stored at 4°C as stock solution for exposure in zebrafish embryos. For exposing pregnant mouse females, PFOS was dissolved in pure ethanol immediately before administration on palatable pieces of food (cookie bits that were readily consumed by the animals).
All experimental procedures were performed in agreement with the Swedish animal protection legislation and European regulations, and were approved by the local Animal Ethics Committee (Stockholms Norra djurförsöksetiska nämnd).
Wildtype AB zebrafish embryos were obtained from the zebrafish core facility at Karolinska Institutet. Breeding groups of adult fish (3 males and 2 females) were housed together overnight in 10 L spawning tanks containing environmental enrichment (commercially available aquaria decoration made of non-toxic plastic). Thirty minutes after turning the light on (9:00 a.m.), the fertilized eggs were collected and stored at 28.5°C until further processing. The eggs were washed twice with fresh E3 water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4; 0.05% methylene blue; pH 7.4) in order to remove all debris. This procedure was performed on a clean open lab bench, at room temperature, under constant illumination (approximately 500 lx). Stock PFOS was further diluted in DMSO to yield a final concentration of 0.1% DMSO in the rearing water. The zebrafish embryos were exposed to PFOS concentrations of either 0.1, or 1 mg/L (0.186 or 1.858 µM, respectively). The doses were selected based on our experience and earlier reports
(A) Representative sequence of spontaneous activity (20 s) recorded at 6 dpf in one control and one larva exposed to 1 mg/L PFOS. In the control larva, the spontaneous activity consists of short, evenly spread bouts of swimming. In the larva exposed to 1 mg/L PFOS, the spontaneous activity consists of clusters of intense activity separated by extended periods of inactivity. Background noise (displacement below 0.2 mm/frame) shaded in gray; inset in top panel – magnification of a representative bout of spontaneous activity. (B) Quantification of frequency of spontaneous bouts of activity. Note the dramatic decrease in bout frequency induced by exposure to 1 mg/L PFOS. (C) Average distance moved during one spontaneous bout of activity. Zebrafish larvae exposed to 1 mg/L PFOS have a hyperactive phenotype characterized by a 2.5 fold higher distance swum during spontaneous bouts of activity. (D) Illustrative sequence of spontaneous locomotion during the active phase of the circadian cycle in one control and one mouse exposed to 3 mg/kg/dy PFOS during gestation. The spontaneous locomotor activity (visits) in the homecage is integrated over consecutive, non-overlapping 10 min bins and spline-interpolated for clarity. (E, F) Similar to the pattern found in zebrafish larvae, the mice exposed to 3 mg/kg PFOS display less frequent (E), but more intense (F) bouts of activity. B, C, E, F – one-way ANOVA followed by Dunnett's post-hoc test; * p<0.05 PFOS exposed vs. control. The number of independent observations is indicated at the bottom of each column in B, C, E and F.
To characterize the hyperactivity induced by the dark pulse, we used two orthogonal parameters derived from the cumulative activity curve: the total locomotor activity and the index of curvature (IOC). IOC was calculated according to the formula below, as described by Fry et al.
In light of the phenotypical alterations found in zebrafish, as well as to strengthen the relevance of zebrafish larvae as experimental model for developmental neurotoxicity, we further analyzed historical behavioral data from mice. The developmental exposure to PFOS in mice has been described earlier
All statistical analyses were performed using Statistica 10 software package (StatSoft Inc., Uppsala, Sweden). The data were analyzed by ANOVA (one-way, factorial, or repeated measures) as appropriate. Between-group differences were tested using post-hoc analysis: Dunnett's test for comparison against a single control group, as in one-way or repeated measures ANOVA design; and unequal N HSD test for comparisons against multiple controls as in simple factorial (“exposure” and “treatment” as factors) or between-group with replication factorial designs (“within-group” and “treatment” as factors). The ANOVA tables are shown in
The average concentration of PFOS measured in the zebrafish larvae was 21.6±5.4 ng/mg wet tissue and 213.5±62.7 ng/mg wet tissue in larvae exposed to 0.1 or 1 mg/L PFOS respectively. We did not find any effect of PFOS exposure on the viability, time of hatching, or incidence of developmental abnormalities (
The spontaneous swimming activity in zebrafish is characterized by discrete bouts of activity separated by inactive intervals. This pattern, also described as “beat-and glide”, is consistently displayed after the age of 4 dpf, and marks the developmental switch to increased spontaneous locomotion to support foraging
The visual motor response (VMR) in zebrafish larvae describes the transient hyperactivity induced by a dark pulse during the light phase
(A) The dark pulse induces a similar pattern of fluctuations in the frequency of spontaneous bouts in larvae exposed to 0.1 mg/L PFOS as in controls. Larvae exposed to 1 mg/L PFOS respond with an increase in frequency that does not vary over time. In addition, the frequency of spontaneous bouts is restored directly to baseline level after the dark pulse. (B) Hyperactivity and habituation in zebrafish larvae during the dark pulse; 30 s timebins. (C) Quantification of total distance and IOC in zebrafish larvae. (D) Novelty-induced hyperactivity in mice. (E) Quantification of total distance moved and habituation rate (estimated by IOC) in mice. Note the similarity between the dose-response curves of the effect of exposure to PFOS on habituation in zebrafish and mice. (F) Swimming activity in zebrafish larvae at the transition between light and dark (gray shaded areas). The larvae exposed to 1 mg/L PFOS display hyperactive episodes of higher magnitude that last considerably longer than in controls. * p<0.05 PFOS exposed vs. control; ANOVA followed by Dunnett's post-hoc test. The number of independent observations is indicated at the bottom of each column in C and E. Graphs in A and B are based on the same number of observations reported in C; graphs in D are based on the same number of observations as in E.
The VMR experimental setting also allows for investigating the effects of the transitions between light and dark phases
The spontaneous activity of 6 dpf larvae in baseline conditions is characterized by bouts occurring at a rate of around 50/min, which yields a sub-unit probability to record 1 bout of activity within each second. Therefore, we inferred that measuring the distance moved by individual larvae in 1 s long timebins would accurately isolate the startle bout from the background spontaneous activity in control larvae. We found that the startle response in control larvae typically consists of a single bout of activity (
(A) Typical vibrational startle response in a 6 dpf zebrafish larvae. Control larvae display a single bout of swimming activity after triggering the stimulus (arrowhead), followed by a prolonged silent period. Note that in control larvae, the swimming bout induced by the vibrational stimulus is considerably more robust than the spontaneous bouts (analysis shown in B), and is followed by a period of inactivity. In contrast, in larvae exposed to 1 mg/L PFOS, the vibrational stimulus is followed by a prolonged sequence of swimming bouts that have the amplitude similar to spontaneous bouts. The time interval between triggering the stimulus and the initiation of the bout is defined as latency to startle (analysis shown in D). The delay between the end of the startle bout and the following bout (analysis shown in E) can be defined as the latency to resume spontaneous swimming activity in control, but not in larvae exposed to 1 mg/L PFOS. (B) Comparison of the distance moved within spontaneous vs. stimulation-induced swimming bouts. Control larvae and larvae exposed to 0.1 mg/L PFOS consistently display a robust increase in the amplitude of activity bout in response to vibrational stimulation. In contrast, zebrafish larvae exposed to 1 mg/L PFOS swam significantly longer distances within spontaneous bout, but do not increase the distance moved in response to the vibrational stimulation. (C) Average distance moved integrated over 1s timebins. The increase in distance moved is accounted for by a single, more robust bout in controls (see A). In contrast, the amplitude of the startle response in larvae exposed to 1 mg/L PFOS is significantly larger than in controls, and it is presumably accounted for by more than 1 bout. In addition, the larvae exposed to 1 mg/L PFOS remain hyperactive for about 4 s after stimulation. The higher variability before in spontaneous activity before and after the startle response can be explained by irregularity in occurrence of spontaneous bouts. Note also that the larvae exposed to 1 mg/L PFOS display an increase in activity before the stimulation. (D, E) Analysis of the latency to startle (D) and inactive period (E). Zebrafish larvae exposed to 1 mg/L PFOS have longer latency to startle, and have shorter inactive period than controls or larvae exposed to 0.1 mg/L PFOS. B - repeated measures ANOVA followed by unequal N HSD post-hoc test; * p<0.05 startle vs. spontaneous; § p<0.05 PFOS exposed vs. controls. C –repeated measures ANOVA followed by unequal N HSD, or Dunnett's post-hoc test, respectively; § p<0.05 PFOS exposed vs. control; * p<0.05 vs. baseline. D, E - ANOVA followed by Dunnett's post-hoc test; * p<0.05 PFOS exposed vs. controls. The number of independent observations is indicated at the bottom of each column in D and E. The graphs in B and C are based on the same number of observation as reported in D and E.
To summarize, the developmental exposure to 1 mg/L PFOS induced changes in spontaneous swimming activity at baseline characterized by less frequent, but more intense bouts of activity as compared to controls. In addition, stimuli that normally induce only a brief hyperactive episode in control larvae, in PFOS exposed larvae induce abnormally prolonged hyperactive episodes consisting of series of bouts of constant amplitude. This results in a pattern of spontaneous activity that is fragmented and highly variable over time (
We recorded the startle response in 6 dpf zebrafish larvae 30 min after spiking the rearing water with a catecholamine reuptake inhibitor, dexamfetamine (1 or 10 µM). The concentrations of dexamfetamine were selected based on the dose-response curves reported earlier
(A) Dexamfetamine displays a bell-shaped dose-dependence of spontaneous bout frequency, but does not alter the distance swam per bout in controls and in larvae exposed to 0.1 mg/L PFOS. In contrast, dexamfetamine monotonically increases the frequency of spontaneous bouts of activity, and reduces the distance moved per bout in larvae exposed to 1 mg/L PFOS. (B) Average rate of response to vibrational stimulation. At baseline, the rate of response is significantly lower in the larvae exposed to 1 mg/L PFOS than in controls. Upon administration of dexamfetamine, the rate of response is increased only at 1 µM in controls, and at both doses in the larvae exposed to 1 mg/L PFOS. (C) Acute dexamfetamine administration alters the spontaneous activity before and after stimulation (presumably by altering the frequency of spontaneous bouts; see also A), but does not influence the amplitude of the startle response (accounted for by a single bout; see also D) in controls and larvae exposed to 0.1 mg/L PFOS. In larvae exposed to 1 mg/L PFOS, the amplitude of the startle response is not altered (presumably accounted for by more than one bout; see also D and E), but the duration of hyperactivity following the vibrational stimulation is shortened by both 1 and 10 µM dexamfetamine (see also
To confirm the dopamine signaling deficiency, and investigate the effects of dopamine receptor agonists on specific phenotypes, we treated the zebrafish larvae with non-specific (apomorphine), and specific (quinpirole for D2, and SKF-81297 for D1) dopamine receptor agonists. In zebrafish larvae exposed to 1 mg/L PFOS we found that quinpirole increased the frequency of spontaneous swimming bouts, while SKF-81297 reduced the within-bout activity (
In controls, dexamfetamine increased the response rate (at 1 µM;
The expected facilitating effect of dopamine receptor agonists on movement in the vibrational startle experiments manifested as a decrease in the latency to startle (induced by apomorphine), and in the inactive period (induced by apomorphine and quinpirole) in controls and larvae exposed to 0.1 mg/L PFOS (
When we analyzed the amplitude of the activity bout induced by the vibrational stimulus in relation to the spontaneous activity bouts, we found that dexamfetamine treatment restored the amplitude modulation of the startle response in larvae exposed to 1 mg/L PFOS essentially by reducing the amplitude of spontaneous bouts (
In this study we found that zebrafish larvae exposed to PFOS in the rearing water from 2 hpf display dose-dependent behavioural alterations: zebrafish larvae exposed to 0.1 mg/L PFOS habituate faster than controls during a dark pulse, while 1 mg/L PFOS induces a disorganized pattern of spontaneous activity and persistent hyperactivity. Similarly, mice exposed to 0.3 mg/kg/day PFOS habituated faster than controls to a new environment, while mice exposed to 3 mg/kg/day PFOS displayed more intense and disorganized spontaneous activity. To further investigate the hyperactivity induced by PFOS, we analysed the startle response to a vibrational stimulus and found that larvae exposed to 1 mg/L PFOS display sustained hyperactivity after stimulation. Both spontaneous and startle-induced hyperactivity were partly reversed by acute administration of dexamfetamine.
In this study we found that zebrafish larvae exposed to PFOS in the rearing water from 2 to 144 hpf accumulated the chemical in the body, in agreement with earlier reported pharmacokinetics data
We found that the larvae exposed to 1 mg/L PFOS displayed disorganized spontaneous activity, characterized by less frequent, but more intense bouts. Similarly, mice prenatally exposed to 3 mg/kg/day PFOS displayed a decreased frequency associated with an increased intensity of spontaneous activity bouts. This pattern of alterations is suggestive of disturbances in the modulation of locomotion. To further investigate these alterations in zebrafish larvae, we induced sustained hyperactivity over a limited period by exposure to a dark pulse. The resulting fluctuations in distance moved was consistent with earlier reports and theoretical models
The larvae exposed to 1 mg/L PFOS displayed stereotypical adaptive changes in spontaneous activity with virtually no amplitude modulation of the activity bouts as compared to baseline, and very little variation in bout frequency during the dark pulse. The exaggerated response to the transition from light to dark was in agreement with the report by Huang et al
The developmental exposure to 1 mg/L PFOS induced complex phenotypical alterations consisting of a hypokinetic component associated to hyperactivity. The hypokinetic phenotype is illustrated by the reduced frequency of spontaneous activity bouts, while the hyperactivity is illustrated by the increase in distance moved within spontaneous activity bouts and by the persistent hyperactivity following the startle response. In light of the dopamine deficiency hypothesis, we tested whether dopamine receptors agonists can correct the behavioral alterations. Indeed, we found that the hypokinetic component was partly reversed by acute administration of D2 agonists (apomorphine and quinpirole), which also facilitated the initiation of movement in control larvae. The hyperactive phenotype was corrected by the D1 agonists (apomorphine and SKF-81297), which in turn had no effect in controls at the doses applied in our study. Importantly, the effects of dexamfetamine were more extensive than the effects of the dopamine receptor agonists, and corrected the hyperactive phenotype in a monotonic dose-dependent fashion. This suggests that the phenotype is caused by alterations in several diffuse neurotransmitter systems, such as noradrenaline and serotonin, in addition to the core defects in dopaminergic signaling. In zebrafish, the dopaminergic system is highly conserved
Several lines of evidence support the association of ADHD with alterations in dopamine signaling (reviewed in
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We thank Prof. Gilberto Fisone and Prof. Sven-Ove Ögren for their support in designing the pharmacological probing of the dopaminergic system; Prof. Abdel ElManira and Dr. Stefan Örn for the fruitful discussions on the zebrafish model.