The effects of developmental exposure to PFOS on startle response in 6 dpf zebrafish larvae.

<p>(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.</p

The structure of spontaneous locomotion in zebrafish larvae and mice during the active phase of the circadian cycle (light for zebrafish larvae, dark for mice).

<p>(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.</p

The effects of acute administration of dexamfetamine on spontaneous swimming and on startle response.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094227#pone.0094227.s005" target="_blank">Figure S5</a>). (D) Dexamfetamine restores the amplitude-modulation of the activity bouts in larvae exposed to 1 mg/L PFOS by decreasing the distance moved for spontaneous bouts. (E) Dexamfetamine shortens the inactive period only in controls and in the larvae exposed to 0.1 mg/L PFOS. A, D - factorial ANOVA followed by unequal N HSD post-hoc test; § p<0.05 PFOS exposed vs. control; * p<0.05 vs. baseline. B- repeated measures ANOVA, followed by Dunnett's post hoc test with the first 3 samples as control (baseline); * p<0.05 vs. baseline. E - repeated measures ANOVA followed by unequal N HSD post-hoc test; * p<0.05 startle vs. spontaneous; § p<0.05 PFOS exposed vs. controls. The number of independent observations is indicated at the bottom of each column in A, B and E. Graphs in C and D are based on the same number of observation as reported in B and E.</p

VMR in zebrafish larvae and novelty-induced hyperactivity in mice.

<p>(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.</p