Persistent behavioral effects following early life exposure to retinoic acid or valproic acid in zebrafish

Abstract

Background: Moderate to severe dysregulation in retinoid signaling during early development is associated with a constellation of physical malformations and/or neural tube defects, including spina bifida. It is thought that more subtle dysregulation of this system, which might be achievable via dietary (i.e. hypervitaminosis A) or pharmacological (i.e. valproic acid) exposure in humans, will manifest on behavioral domains including sociability, without overt physical abnormalities.

Methods: During early life, zebrafish were exposed to low doses of two chemicals that disrupt retinoid signaling. From 0 to 5dpf, larvae were reared in aqueous solutions containing retinoic acid (0, 0.02, 0.2 or 2nM) or valproic acid (0, 0.5, 5.0 or 50μM). One cohort of zebrafish was assessed using a locomotor activity screen at 6-dpf; another was reared to adulthood and assessed using a neurobehavioral test battery (startle habituation, novel tank exploration, shoaling, and predator escape/avoidance).

Results: There was no significant increase in the incidence of physical malformation among exposed fish compared to controls. Both retinoic acid and valproic acid exposures during development disrupted larval activity with persisting behavioral alterations later in life, primarily manifesting as decreased social affiliation.

Conclusions: Social behavior and some aspects of motor function were altered in exposed fish; the importance of examining emotional or psychological consequences of early life exposure to retinoid acting chemicals is discussed.

Keywords: Behavior; Cognition; Development; Retinoic acid; Social behavior; Vitamin A valproic acid; Zebrafish.

Figure 1

The proportion of zebrafish larvae remaining after unfertilized eggs, embryos with arrested development and dead or malformed zebrafish were removed from the RA (0-5 dpf) treated group (Panel A) and from the VPA (0-5 dpf) treated group (Panel B).

Figure 2

Larval activity data are plotted as a function of time, across 10-min condition blocks, for each group of RA treated fish (Panel A) and VPA treated fish (Panel B). Panel C displays the mean activity during the light and dark portions of the test in the follow-up study with additional doses of RA and VPA. The lines below the asterisks across the light and dark bars indicate significant comparisons of average activity across lighting conditions of the exposed groups to control to convey the Dunnett’s comparisons within the significant main effect of exposure. Asterisks directly over individual light and dark bars without the lines indicate significant comparisons of that particular lighting condition back to control performance for that lighting condition to convey the Dunnett’s comparisons within the significant interaction of exposure x lighting condition. Error bars represent SEM.

Figure 3

Distance traveled following the tap stimulus presentation is plotted for each RA (Panel A) and VPA (Panel B) dose condition (grouped bars). Each bar represents the mean of two tap presentations. Error bars represent SEM.

Figure 4

Mean distance traveled for each min of the trial is plotted by RA dose (Panel A) and VPA dose (Panel C) condition (grouped bars). Mean distance from the tank floor (a measure of tank location) is plotted by RA dose (Panel B) and VPA dose (Panel D) condition (grouped bars). Error bars represent SEM.

Figure 5

Mean distance from the shoal (cm) for each min of the trial is plotted by RA dose (Panel A) and VPA dose (Panel C) condition (grouped bars), inset graphs plot the mean distance from the shoal (cm) for first one minute of the session, which highlights the initial difference among groups for RA (Panel B) and VPA (Panel D) conditions. Error bars represent SEM. An “*” denotes p<0.05, difference from control (Dunnett’s post-hoc).

Figure 6

Mean distance to predator stimulus is plotted by RA dose (Panel A) and VPA dose (Panel B) condition (grouped bars). Error bars represent SEM.