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

The study addresses whether developmental exposure to traffic-related air pollution (TRAP) and near-roadway environmental stressors causally contributes to neurodevelopmental disorder (NDD)-relevant behavioral phenotypes. NDDs, including ASD and ADHD, are prevalent, often co-occur, and impose significant societal burden. Epidemiological studies have associated maternal residence near major roadways and TRAP exposure—especially during late gestation and early life—with increased risk of NDDs, but causality is uncertain due to confounding and exposure quantification challenges. The authors aim to model real-world near-roadway exposures (air pollution, noise, vibration) during gestation and early postnatal development in rats, and to evaluate effects on growth, psychomotor reflex development, communication (ultrasonic vocalizations), social interaction, activity, and learning/memory. The work is intended to complement human studies and enhance translational relevance by replicating complex pollutant mixtures and exposure timing linked to higher risk in epidemiology (e.g., third trimester).

Multiple independent epidemiological investigations across regions and methods implicate TRAP or proximity to heavily trafficked roads as risk factors for NDDs and developmental delays, with associations strongest for late gestation and/or early life exposure. The CHARGE study reported increased autism risk for residences within 309 m of freeways during each trimester, with the third trimester showing the strongest association, informing exposure timing in this study. Prior preclinical exposure methods often lack translational fidelity due to factors such as repeated anesthesia or failure to replicate real-world emission complexity and concentrations. This study leverages a real-world exposure paradigm to address these limitations.

Design and cohorts: - Species/strain: Sprague-Dawley rats. Breeders paired at PND 80–90; females singly housed around GD14. - Roadside facility exposures: Dams transported to a facility adjacent to a major freeway tunnel in Northern California; randomly assigned to TRAP (tunnel-face air) or filtered air (FA). Offspring remained under the same exposure condition postnatally. Two males and two females per litter tested from 20 dams. Behavioral timeline: developmental milestones (PND 4, 6, 7, 9, 10, 12), pup USV (PND 5), juvenile reciprocal social interaction (PND 32–34), open field (PND 39–41), novel object recognition (PND 40–42), fear conditioning (PND 44–48). - Laboratory control cohort: Dams and offspring housed at UC Davis vivarium (7 litters). Testing: developmental milestones (PND 4, 6, 7, 9, 10, 12), pup USV (PND 5), social interaction (PND 34–36), open field (PND 42–43). - Transport-stress control: Half of dams driven ~45 min (halfway to roadside site) and returned; the other half remained at UC Davis. All housed at UC Davis thereafter (11 dams). Testing: developmental milestones (PND 4, 6, 7, 9, 10, 12), pup USV (PND 5), open field (PND 38–41). - Additional laboratory cohorts for learning/memory controls: separate groups for novel object recognition (5 litters, PND 45–53) and fear conditioning (7 litters, PND 42–44) at UC Davis. Exposure facility and air handling: - TRAP group air: drawn directly from the eastern face of a freeway tunnel via flexible ducting to exposure chambers. - FA group air: drawn from outside the facility and passed through a series of controls: pre-filter (large debris/coarse PM), activated carbon (volatile/semi-volatile organics), barium oxide-based catalytic converters (NO removal), ultrahigh efficiency Teflon-bound glass microfiber filters (fine/ultrafine PM removal); flow/temperature conditioned; chambers maintained at slight negative pressure. - Environmental stressors: Building insulated to reduce noise; vibration isolators under chambers; noise limited to <85 dB in facility; UC Davis vivarium ambient noise lower (64 dB; testing rooms 43–47 dB). - Chambers: Two exposure chambers (12.8 ft L x 3 ft W x 7.8 ft H), up to 108 cages (filter tops removed). Pumps/blowers outside to minimize noise. Behavioral assays: - Developmental milestones: body length/weight; rooting reflex (head turn to whisker stimulation); forelimb grasping (bar grasp). - Isolation-induced pup USV (PND 5): 3-min isolation recordings using Avisoft systems; spectrogram counting by blinded observer. - Juvenile reciprocal social interaction: 10-min dyadic interactions (strain-, age-, sex-matched unfamiliar stimulus rat housed in FA at roadside); behaviors scored (exploring, following/chasing, social sniffing, anogenital sniffing, self-grooming) by blinded observer; ~30 lux illumination. - Open field (30 min): total distance and center time measured via EthoVision or Digiscan; ~30 lux. - Novel object recognition: Day -1 30-min habituation; Day 0 30-min habituation + 10-min familiarization to two identical objects; 60-min delay; 5-min test with one familiar and one novel object (identity/side counterbalanced); sniffing time via EthoVision; objects: orange plastic cones and glass bell jars; ~30 lux. - Contextual and cued fear conditioning: Day 1 training with three 30-s white noise (80 dB) cues co-terminating with 0.7 mA, 2-s footshock; context with specific visual/odor/tactile cues (~100 lux, vanilla odor). Day 2 contextual test (5 min, no CS/US). Day 3 altered context (dark, smooth floor, black angled walls, lemon odor): 3-min exploration + 3-min CS presentation; freezing quantified by VideoFreeze. Animal handling and blinding: - Pups identified by paw tattoos (PND 2) and tail marks at weaning; coding enabled blinded testing/scoring. - Two males and two females per litter selected; most animals tested across assays except 16 tested only as pups; at least 48 h between tests, ordered least to most stressful. Statistics: - PM comparisons: paired t-test. - USV: unpaired t-test (two groups) or one-way ANOVA with Tukey post hoc (three groups). - Developmental milestones and open field: two-way repeated measures ANOVA (exposure between-subjects; time within-subjects) with Tukey post hoc. - Developmental milestone attainment rates: Log-Rank (Mantel-Cox) test. - Social interaction: one-way ANOVA with Tukey post hoc. - Novel object recognition: within-group paired t-test (novel vs familiar sniff time). - Fear conditioning: repeated measures ANOVA for training/cued; unpaired t-test for contextual. - Significance p < 0.05, two-tailed tests where applicable; data checked for normality; results reported as mean ± SEM; multiple comparisons corrected with Tukey.

- Near-roadway exposure during gestation and early life delayed offspring physical growth and the development of psychomotor reflexes. - Both roadside-reared groups (TRAP and FA), which experienced near-roadway noise/vibration context, exhibited altered social interactions and abnormal locomotor activity in the open field relative to laboratory controls. - Isolation-induced 40-kHz pup ultrasonic vocalizations (PND 5) were reduced by near-roadway exposure, with the TRAP group emitting the fewest calls, indicating additional TRAP-specific effects on early social communication. - TRAP exposure affected components of social communication, including reduced neonatal pup USVs and altered juvenile reciprocal social interaction patterns. - Transport stress control showed no evidence that brief dam transport alone accounted for offspring behavioral outcomes. - Reproductive metrics: no group effect on litter size or sex ratio; most dams across groups carried to term (as reported in Results). - Overall, findings support that early-life proximity to highly trafficked roadways alters neurodevelopment with added deficits attributable to TRAP constituents beyond environmental context alone.

The study directly tests and supports a causal link between early-life near-roadway exposures and NDD-relevant behavioral alterations. By modeling real-world TRAP mixtures and environmental context (noise, vibration) during late gestation through early postnatal periods, the findings align with epidemiological associations of increased NDD risk near major roadways, particularly in late pregnancy. The observation that both TRAP and FA roadside groups showed delays in growth/reflex development, abnormal activity, and altered social behaviors indicates that aspects of the near-roadway environment beyond airborne pollutants (e.g., noise/vibration and possibly residual low-level pollutants) can adversely affect neurodevelopment. Additional decrements in social communication (reduced USVs) and social interaction in the TRAP group point to pollutant-specific effects, suggesting that certain TRAP constituents further compromise neurodevelopmental trajectories. The absence of effects from transport stress isolates exposure at the facility as the key factor. These results underscore the neurodevelopmental relevance of early-life environmental exposures common to populations living near busy roadways and provide translational preclinical evidence complementing human studies.

This work introduces and validates a real-world near-roadway exposure model demonstrating that gestational and early-life exposures elicit NDD-relevant behavioral phenotypes in juvenile rats. Both the near-roadway environment and TRAP itself contribute to adverse outcomes, with TRAP adding specific deficits in social communication. The study strengthens causal inference suggested by epidemiology and provides a platform for mechanistic investigations. Future research should: (1) dissect contributions of specific TRAP components and non-chemical stressors (noise, vibration), (2) examine dose-response and critical windows of susceptibility, (3) assess persistence into adolescence/adulthood and cognitive domains, and (4) integrate neurobiological endpoints (e.g., histopathology, neuroinflammation, circuit function) to link exposure with behavioral outcomes.

- Attribution of effects to specific environmental factors is incomplete: both TRAP and FA groups at the roadside facility shared contextual exposures (noise/vibration), making it difficult to fully disentangle non-chemical stressors from residual air constituents, despite mitigation measures. - Affiliations of some authors and full exposure composition details are deferred to companion/supplementary materials; mechanistic endpoints are reported in a companion manuscript. - Behavioral assessments focused on early life to juvenile stages (up to PND ~50); long-term persistence into adulthood and comprehensive cognitive phenotyping were limited in the presented data. - Generalizability from rats to humans requires caution despite enhanced translational design. - The excerpted results provide qualitative/relative findings without detailed statistics for each endpoint in this text segment.