Sex-specific effects of perinatal FireMaster® 550 (FM 550) exposure on socioemotional behavior in prairie voles

Abstract

The rapidly rising incidence of neurodevelopmental disorders with social deficits is raising concern that developmental exposure to environmental contaminants may be contributory. Firemaster 550 (FM 550) is one of the most prevalent flame-retardant (FR) mixtures used in foam-based furniture and baby products and contains both brominated and organophosphate components. We and others have published evidence of developmental neurotoxicity and sex specific effects of FM 550 on anxiety-like and exploratory behaviors. Using a prosocial animal model, we investigated the impact of perinatal FM 550 exposure on a range of socioemotional behaviors including anxiety, attachment, and memory. Virtually unknown to toxicologists, but widely used in the behavioral neurosciences, the prairie vole (Microtus ochrogaster) is a uniquely valuable model organism for examining environmental factors on sociality because this species is spontaneously prosocial, biparental, and displays attachment behaviors including pair bonding. Dams were exposed to 0, 500, 1000, or 2000 μg of FM 550 via subcutaneous (sc) injections throughout gestation, and pups were directly exposed beginning the day after birth until weaning. Adult offspring of both sexes were then subjected to multiple tasks including open field, novel object recognition, and partner preference. Effects were dose responsive and sex-specific, with females more greatly affected. Exposure-related outcomes in females included elevated anxiety, decreased social interaction, decreased exploratory motivation, and aversion to novelty. Exposed males also had social deficits, with males in all three dose groups failing to show a partner preference. Our studies demonstrate the utility of the prairie vole for investigating the impact of chemical exposures on social behavior and support the hypothesis that developmental FR exposure impacts the social brain. Future studies will probe the possible mechanisms by which these effects arise.

Keywords: Anxiety; Brain; Endocrine disrupting chemicals; Endocrine disruptors; Flame retardants; Neural; Neurodevelopment; Social.

Figure 1.

Study design.

Figure 2.

Litter outcomes. (A) Number of total, male, and female pups per litter. Dams exposed to 5MT had significantly larger litters and more male pups. (B) Dams exposed to 2000 μg FM 550 had a significantly greater percentage of males per litter compared to control dams and the expected, equal sex ratio (50%). Bar graphs depict mean ± SEM; box and whiskers plots depict mean ± 95 % CI; individual litters are represented by circles. For each dose (n = 11–20); *significant effect of FM 550; Φ significant effect of 5MT; (* and ^Φp ≤ .05). Significant difference from equal sex ratio (50% dotted line) is indicated by λ (^λλp ≤ .01).

Figure 3.

Open field outcomes were sex specific with effects most profound in females. (A) Representative activity traces from a control and FM 550 exposed female superimposed on the arena digital layout used for analysis. (B) Total distance traveled was sexually dimorphic with females traveling more than males. There was a significant, dose-responsive effect of FM 550 exposure in females, with exploration decreasing with dose. (C) In females, there was a main effect of FM 550 exposure on center entries with entries significantly decreased at the lowest and highest dose levels and suggestive at the mid-level dose. (D) Time in the center was also significantly lower in the lowest and highest female dose groups. (E) Distance traveled and (F) duration in center was binned into 5-min intervals to evaluate activity and exploration patterns across the 30-min test. Significant group differences at individual time points are depicted by letters (a: 500 μg; b: 1000 μg; c: 2000 ug FM 550; d: 5MT). Graphs depict mean ± SEM; circles represent individual animals (B-D); circles = control, triangles = 500 μg, hexagons = 1000 μg, squares = 2000 μg, diamonds = 5MT (E-F). For each dose (n= 8–16), * denotes statistically significant exposure effects and # denotes a statistically significant dose-response effect, within sex, while ψ denotes significant sex differences between controls. For binned data (E-F), letters identify group differences from control at individual time intervals: a = 500 μg, b = 1000 μg, c = 2000 μg, d = 5MT. A single symbol represents p ≤ .05 and a double symbol represents p ≤ .01.

Figure 4.

Sociability test outcomes were sex specific, with effects primarily in females. (A) Arena schematic depicting placement of each element and the zone around each cup in which the test animal was considered to be in contact with the cup. (B) Number of individuals per group that failed to interact with either the stranger or empty cup. The 500 μg FM 550 males had a marginally higher incidence rate of single interaction compared to same sex controls. (C) Sociability index revealed no preference for either cup in the unexposed controls but greater preference for the empty cup in the 1000 μg FM 550 group of both sexes and the 2000 μg FM 550 female dose group. (D) FM 550 dose-dependently reduced time spent with either the stranger or empty cup in females with the 1000 μg and 2000 μg dose groups spending significantly less time in contact with the stranger. A main effect of exposure was not found for males but the 1000 μg FM 550 group spent significantly less time with the stranger. In both sexes, the 5MT group spent significantly more time with the empty cup. (E) Total investigation time in females dose-dependently decreased with FM 550 exposure and was significantly lower in the 1000 μg and 2000 μg dose groups. Conversely, investigation time was higher in the 5MT exposed females. (F) In FM 550 exposed females, total distance traveled was dose-dependently reduced. The 1000 μg FM 550 group was statistically different from same sex controls and decreases were suggestive in the other two dose groups. The serotonin agonist 5MT significantly increased distance traveled only in males. Bar graphs depict mean ± SEM; box and whisker plots depict mean ± 95 % CI; individual animals are depicted by circles. For each dose (n= 9–19); *significant effects of FM 550; Φ significant effect of 5MT; # significant dose-response effect within sex (*^{, #,} and ^Φp ≤ .05; ** and ^##p ≤ .01; ^###p ≤ .001). In the sociability index, a significant difference from chance (SI= 0, dotted line) is indicated by λ (^λp ≤ .05, ^λλλp ≤ .001). A sociability index of 1.0 indicates preference for stranger animal and an index of −1.0 indicates preference for the empty cup.

Figure 5.

Social preference test. (A) Social preference arena schematics showing the placement of each element. FM 550 exposure affected social preference in both sexes. (B) Exposure did not impact the number of animals failing to interact with either the stranger or empty cup. (C) Preference for the familiar animal was not displayed by the 500 and 1000 μg groups of both sexes. (D) FM 550 exposure impacted time with the stranger, with exposed females spending less time with the stranger animal. (E) Total investigation time in females decreased with FM 550 exposure. (F) Total distance traveled was decreased by FM 550 exposure in females. Social preference behavior was also affected by 5MT, with exposed males traveling more compared to controls. Both males and females exposed to 5MT displayed a social preference for the familiar animal over the stranger. Bar graphs depict mean ± SEM; box and whisker plots depict mean ± 95 % CI; individual animals are depicted by circles. For each dose (n= 9–19), significant differences due to FM 550 exposure denoted by * and Φ for 5MT. # denotes a dose-response effect within sex (*^{, #,} and ^Φp ≤ .05; **p ≤ .01; ^###p ≤ .001). Significant difference from chance (SI= 0, dotted line) for social preference indicated by λ (^λp ≤ .05, ^λλp ≤ .01, ^λλλp ≤ .001). A social preference index of 1.0 indicates maximal preference for familiar animal while an index of −1.0 indicates maximal preference for the stranger.

Figure 6.

Effects on novel object recognition were sex specific and more pronounced in females. (A) Schematics depicting the placement of the objects and the circular zones in which investigation was measured during the familiarization and subsequent short-term (30 min) and long-term (24 hr) memory test sessions (B). In females, FM 550 dose-dependently increased total investigation time in the 24 hr recollection session. In males, a main effect of FM 550 was found for both the 30 min and 24 hr session with investigation decreased in exposed animals. 5MT had no effect in either sex (C). Novel object recognition was impaired at both time points in the 2000 μg FM 550 females. Males exposed to 1000 and 2000 μg FM 550 did not display recognition during the short-term memory test and none of the male groups appeared to achieve novel object recognition in the long-term memory test. None of the 5MT groups displayed a preference for the novel object. (D) In females, total distance traveled significantly decreased with dose in both sessions. For each dose (n= 7–15), significant differences due to exposure of FM 550 denoted by *, while # denotes a dose-response effect within sex (* and ^#p ≤ .05; ^##p ≤.01; ***p ≤ .001). Significant difference from chance (IR= 0.5, dotted line) is indicated by λ (^λp ≤ .05, ^λλλp ≤ .001). An investigation ratio of 1.0 indicates maximal preference for novel object while a ratio of 0 indicates maximal preference for the familiar object.

Figure 7.

Effects on partner preference were male specific. (A) Schematics depicting the placement of the animals and the circular zones where contact between the test animal and the cups was recorded. (B) Control males, but not control females, displayed a significant partner preference. In females, the 500 μg and 2000 μg FM 550 groups displayed preference for the partner. By contrast no partner preference was observed in any male FM 550 group and the 5MT males preferred the stranger. (C-D). Time with the partner (solid line) and stranger (dotted line) at 1 min intervals across the test revealed rapidly emerging partner preference in 500 μg and 2000 μg FM 550 females as well as control males. 5MT males developed a rapid preference for the stranger. Control females only developed a preference for their partner toward the end of the test. For each dose (n = 6–15), significant difference from equal preference (PPI= 0, dotted line) is indicated by λ (^λp ≤ .05). A partner preference index of 1.0 indicates maximal preference for partner while an index of −1.0 indicates maximal preference for the stranger. Significant differences in time spent with the stranger and familiar animal is indicated by δ at each minute interval. A single symbol represents p ≤ .05, double symbol represents p ≤ .01 and triple symbol represents p ≤ .001.