Developmental exposure to near roadway pollution produces behavioral phenotypes relevant to neurodevelopmental disorders in juvenile rats

Abstract

Epidemiological studies consistently implicate traffic-related air pollution (TRAP) and/or proximity to heavily trafficked roads as risk factors for developmental delays and neurodevelopmental disorders (NDDs); however, there are limited preclinical data demonstrating a causal relationship. To test the effects of TRAP, pregnant rat dams were transported to a vivarium adjacent to a major freeway tunnel system in northern California where they were exposed to TRAP drawn directly from the face of the tunnel or filtered air (FA). Offspring remained housed under the exposure condition into which they were born and were tested in a variety of behavioral assays between postnatal day 4 and 50. To assess the effects of near roadway exposure, offspring of dams housed in a standard research vivarium were tested at the laboratory. An additional group of dams was transported halfway to the facility and then back to the laboratory to control for the effect of potential transport stress. Near roadway exposure delayed growth and development of psychomotor reflexes and elicited abnormal activity in open field locomotion. Near roadway exposure also reduced isolation-induced 40-kHz pup ultrasonic vocalizations, with the TRAP group having the lowest number of call emissions. TRAP affected some components of social communication, evidenced by reduced neonatal pup ultrasonic calling and altered juvenile reciprocal social interactions. These findings confirm that living in close proximity to highly trafficked roadways during early life alters neurodevelopment.

Fig. 1. Timeline and quantification of roadside TRAP exposure.

a Pregnant female rats were transported to the roadside exposure facility at approximately gestational day (GD) 14 and were randomly assigned to be housed in either the TRAP or filtered air (FA) exposure chambers. Offspring, remained in the exposure condition into which they were born, were tested on a variety of developmental milestone assays between four days after birth (postnatal day (PND) 4) and weaning at PND 21 and then a battery of standardized behavioral assays between PND 21 and 50. b, c Particulate matter (PM) concentrations of the TRAP air and FA were quantified on 19 days. Both b PM_2.5 and c PM₁₀ concentrations of the TRAP air were significantly higher than those of the FA. *p < 0.05, paired t-test.

Fig. 2. Reduced isolation-induced ultrasonic vocalizations (USV) of TRAP-exposed pups at PND 5.

a Male pups exposed to TRAP emitted significantly fewer USV during the three min isolation compared to lab controls. b Exposure did not affect USV emission in females, although the trend indicated reduced numbers of calls in the TRAP group compared to lab controls. *p < 0.05, one-way ANOVA followed by Tukey’s multiple comparisons test.

Fig. 3. Delayed growth and milestone achievement of roadside TRAP- and FA-exposed pups.

a Male pups exposed to TRAP or FA had significantly reduced body length and b body weight throughout early development compared to lab controls. Males of both roadside groups exhibited a significant delay in the development of c rooting and d forelimb grasping reflexes. e Female pups exposed to TRAP or FA also had reduced body length and f body weight and developed g rooting and h forelimb grasping reflexes later than lab controls. a, b, e, f *p < 0.05, repeated measures ANOVA followed by Tukey’s multiple comparisons test. c, d, g, h *p < 0.05, Log-Rank (Mantel-Cox) test.

Fig. 4. Roadside TRAP- and FA-exposed rats differed from lab controls during juvenile reciprocal social interactions on several key parameters.

a Roadside exposures did not affect levels of exploration during the social interaction assay for males, however b TRAP-exposed males spent significantly more time following or chasing the stimulus animal than did FA-exposed or lab controls. c Roadside-reared males showed typical levels of social sniffing but d there was a significant effect of group on anogenital sniffing, with post hoc trends suggesting that both roadside exposure groups spent more time anogenital sniffing compared to lab controls. e Both TRAP- and FA-exposed males spent more time self-grooming than lab controls. f Females of all groups exhibited comparable levels of exploration, but g TRAP-exposed females spent more time following or chasing than lab controls. h FA-exposed females spent significantly less time social sniffing relative to lab controls and i both roadside groups had elevated levels of anogenital sniffing. j Females of all groups displayed similar levels of self-grooming. *p < 0.05, one-way ANOVA followed by Tukey’s multiple comparisons test.

Fig. 5. Atypical exploratory activity in a novel open field in rats exposed to roadside TRAP and FA.

a Roadside exposures did not affect males’ gross locomotion or b time spent in the center during a 30-min exploration of a novel arena. c In females, there was a significant effect of group on distance moved, with trends suggesting that FA-exposed females covered more distance during the assay compared to lab controls. d TRAP-exposed females spent less time in the center than did FA-exposed females, and both TRAP- and FA-exposed females displayed significantly reduced center time relative to lab controls. *p < 0.05, repeated measures ANOVA followed by Tukey’s multiple comparisons test.

Fig. 6. Learning and memory in roadside exposed rats.

a Males exposed to TRAP or FA displayed intact novel object recognition as evidenced by spending significantly more time sniffing the novel object than the familiar object. b Exposure to TRAP did not affect contextual or cued fear memory in males and both TRAP and FA groups displayed high levels freezing day 1 post-training and to the cue presentation on day 3. c Both groups of roadside exposed females spent significantly more time investigating the novel object compared to the familiar object and d no group differences were observed in percent time freezing during the test of contextual and cued memory. a, c *p < 0.05, paired t-test, familiar vs. novel. b, d *p < 0.05, Day 1, 3: repeated measures ANOVA; Day 2: Student’s t-test, TRAP vs. FA.

Fig. 7. No effect of gestational transport alone on offspring development and behavior.

Pups born to dams that experienced a transport event at approximately GD 14 exhibited no physical or behavioral abnormalities. a Body length and b body weight were typical throughout early life, as was the timing of the development of c rooting and d forelimb grasping reflexes. e Gestational transport did not affect the number of isolation-induced pup ultrasonic vocalizations at PND 5 and f juveniles exhibited similar exploratory activity in a novel open field as indicated by total distance moved and g time spent in the center. a, b, f, g *p < 0.05, repeated measures ANOVA. c, d *p < 0.05, Log-Rank (Mantel-Cox) test. e *p < 0.05, Student’s t-test.