Developmental CPF exposure reduced isolation-induced pup ultrasonic vocalizations (USV).
Pup ultrasonic vocalizations (USV) of infant rats measure an early communicative behavior between pups and mother. Isolation-induced USV were collected for 3 min as social communication signals in rat pups, as previously described (Berg et al. 2018). CPF-exposed pups emitted significantly fewer USV across early development, as expected (Fig. 1Amales F(2, 90) = 286.5, p < 0.001; Fig. 1Bfemales F(2, 90) = 267.7, p < 0.001). As pups grow, they learn to temperature regulate and open their eyes and are less reliant on maternal care which is why USV decrease in number over developmental days. There was a significant main effect of experimental group on USV emission (Fmales (3, 45) = 3.048, p < 0.05). Holm-Sidak corrected post hoc analysis for multiple comparisons highlighted significant differences on PND 12, when fewer USV were emitted in the 1.0 mg/kg CPF-exposed male pups, and on PND 16 in all CPF dose groups compared to vehicle. CPF-exposed female pups also emitted significantly fewer USV (Ffemales (3, 37) = 2.949, p < 0.05). Holm-Sidak corrected post hoc analysis for multiple comparisons highlighted strong trending differences on PND 8, as fewer USV were emitted in the 0.3 mg/kg CPF-exposed female pups (p = 0.061), and significant differences at PND 12 and PND 16 in the 0.3 mg/kg CPF-exposed female pups compared to vehicle.
Body weight and temperature were also collected to assure the reduced USV were not the result of being physically smaller as body weight is known to alter pup USV emission (Hofer 1996; Hofer et al. 2002). Body temperature did not differ between CPF exposure groups and vehicle (Fig. 1Cmales F(3, 46) = 0.5381, p > 0.05; Fig. 1Dfemales F(3, 46) = 0.67, p > 0.05). Weight did not differ between CPF exposure groups and vehicle (Fig. 1Emales F(3, 46) = 0.2745, p > 0.05; Fig. 1Ffemales F(3, 46) = 0.5234, p > 0.05), indicating typical growth and ability to thrive. In addition to being important control metrics for the pup USV assay, the lack of observation that overall growth and health was not impacted by CPF exposure confirms the lack of systemic toxicity that has been reported with higher CPF doses using a functional observation battery (Bushnell et al. 2001; Moser 1995).
Analysis of typical early neurological reflexes did not reveal any significant differences between CPF-exposed pups and vehicle controls (Supplementary Fig S2). Specifically, there were no significant differences between exposure groups in latencies to navigate upright in negative geotaxis and circle traverse, simple metrics for motoric, postural, and proprioceptive processes that underlie the ability of infant rodents to navigate on an inclined plane or to the outer rim from the center of circle (Fig. S2Amales F(3, 46) = 0.4776, p > 0.05; Fig S2Bfemales F(3, 46) = 1.098, p > 0.05; Fig. S2Cmales F(3, 46) = 1.224, p > 0.05; Fig S2Dfemales F(3, 46) = 1.1319, p > 0.05).
Normal locomotion and exploratory activity following developmental CPF exposure.
Normal motor function following early life exposure to low doses of CPF was confirmed by lack of an effect of CPF on motor abilities in the open field exploratory locomotion task across a 30-min session. No CPF effect was observed in activity metrics of horizontal activity (Fig. 2Amales F(3, 46) = 0.2303, p > 0.05; Fig. 2Bfemales F(3, 46) = 0.3341, p > 0.05), vertical activity (Fig. 2Cmales F(3, 46) = 0.2278, p > 0.05; Fig. 2Dfemales F(3, 46) = 0.2562, p > 0.05), nor time spent in the center of the arena (Fig. 2Emales F(3, 46) = 0.7749, p > 0.05; Fig. 2Ffemales F(3, 46) = 2.150, p > 0.05).
Reduced social exploration to affiliative 50-kHz ultrasonic calls (USV) in female CPF-exposed juveniles.
Social exploratory behavior displayed by the male (Fig. 3C t (1, 13) = 3.576, p < 0.005) and female vehicle control groups (Fig. 3D t (1, 13) = 3.509, p < 0.005) control groups was directed towards playback of pro-social 50-kHz USV, as reflected in the parameter of time spent on the arms proximal to the sound source emitting 50-kHz USV as compared to the distal arms of the radial maze. All groups of male juvenile rats (vehicle and each dose of CPF) spent significantly longer on the arms proximal to the speaker emitting the 50-kHz USV upon playback (Fig. 3C0.1 dose t (1, 13) = 2.738, p < 0.02; Fig. 3C0.3 dose t (1, 13) = 4.587, p < 0.001; Fig. 3C1.0 dose t (1, 13) = 4.502, p < 0.001). In contrast, the 0.3 mg/kg and 1.0 mg/kg CPF-exposed females rats failed to spend significantly more time on the proximal arms (Fig. 3D0.1 dose t (1, 13) = 3.001, p < 0.005; Fig. 3D0.3 dose t (1, 13) = 1.373, p > 0.05; Fig. 3D1.0 dose t (1, 13) = 0.7127, p > 0.05).
Distance traveled in response to the white noise control stimulus did not differ between exposure groups, and all groups exhibited no locomotor response and/or behavioral inhibition (i.e., a reduction in motion following the noise control) (Fig. 3Emales F(3, 46) = 1.276, p > 0.05 and Fig. 3Ffemales F(3, 46) = 0.2981, p > 0.05). These findings rule out the possibility of a confounding hearing deficit in the CPF-exposed groups.
CPF-exposed rats demonstrated intact contextual and cued fear memory.
Learning and memory was initially evaluated using two measures of Pavlovian fear conditioning with a 24-hr contextual component and a tone cued fear conditioning. High levels of freezing were observed, subsequent to the conditioned stimulus (CS) – unconditioned stimulus (UCS) pairings, on the training day, in both exposed groups (Fig. 4Amales no exposure group difference in post-training freeze scores F(3, 46) = 0.3342, p = 0.801 and Fig. 4B females no exposure group difference in post-training freeze scores F(3, 46) = 0.2033, p = 0.894), indicating no confounds and no deficits in the learning of the associations between the context stimuli and tone cues. No exposure group difference in freezing was observed 24-hrs following CS-UCS training (Fig. 4Cmales F(3, 46) = 0.02571, p = 0.994 and Fig. 4Dfemales F(3, 46) = 0.2045, p = 0.893), when placed in the context chamber from conditioning training with identical stimulus cues. Levels of freezing, pre- and post-cue presentation 48-hrs after training, showed no effect of exposure (Fig. 4Emales,pre−cue F(3, 46) = 0.1365, p = 0.312, Fig. 4Emales,cue F(3, 46) = 0.6103, p = 0.612 and Fig. 4Ffemales,pre−cue F(3, 46) = 0.3858, p = 0.764, Fig. 4Ffemales,cue F(3, 46) = 0.2999, p = 0.825).
Neuroanatomical pathology at PND35 following developmental CPF exposure.
Overall, the total brain volumes were not observed to be different between groups (1683 ± 101 mm3 for vehicle, 1649 ± 51 mm3 for a CPF dosage of 0.1 mg/kg, 1675 ± 123 mm3 for 0.3 mg/kg, and 1662 ± 68 mm3 for 1.0 mg/kg). An unignorable difference in total brain volume between vehicle and exposure at 0.3 mg/kg was − 2.27% observed in the females, as this was a mere one one hundredth from significance (p = 0.06, q = 0.22). There were no significant findings for any CPF exposure group nor for any sex when correcting for multiple comparisons. There was a trend towards a decrease in the hippocampal region (-3.29%, p = 0.03, q = 0.22), which appeared to be localized to Ammon’s Horn (-3.52%, p = 0.02, q = 0.22). Additional trends towards a loss in volume were found in the fiber tracts (-2.61%, p = 0.03, q = 0.22), with the strongest trends found in the fimbria (-3.63%, p = 0.02, q = 0.22) and the cortical spinal tract (-5.11%, p = 0.01, q = 0.22). Voxelwise comparisons also revealed no significant differences found, but again interesting trends were seen in the female rats at a dosage of 0.3 mg/kg (Fig. 5). Interestingly, at the 0.3 mg/kg dosage, opposite effects are seen in males versus females with males showing positive effect size differences and females showing negative effect size differences (Fig. 5).
Normal Brain and Blood AChE Activity Following CPF Exposure
None of the three doses of CPF significantly altered the enzymatic activity of AChE in the brain (Fig. 6A F(3,35) = 0.1252, p = 0.9446) or in the blood (Fig. 6B F(3,34) = 0.2137, p = 0.8862).