Representative SERT expression in different cortical areas after CTM treatment at three different developmental periods (E11–E19, P1–P7, and P8–P21). Note the rather thick, rod-like SERT-ir fibers (indicated by arrows in A2, B2, C2, D2, E2, and F2) in drug-exposed males compared with the thinner, more evenly distributed fibers in saline-exposed (SAL) or no treatment (NT) rats (A1, B1, C1, D1, E1, and F1). (Scale bar: 50 μm.) CA1, hippocampus; mPFC, medial prefrontal cortex; SI, primary somatosensory cortex.

Representative photomicrographs of Fluoro-Gold retrogradely labeled callosal cells in the supragranular layer of the barrel cortex. Images were obtained from adult male rats that received CTM at different developmental periods. Note the reduction of callosal cells in the treatment groups [B, E11–E19; C, P1–P7; and D, P8–P21 (10 mg/kg)] compared with the normal subject, A. (Scale bar: 100 μm.) (E) Bar graph of data from both male and female subjects shows that the loss of retrogradely labeled callosal neurons is restricted to the supragranular layer. Values represent the mean ± SEM of 11 (CTRL), 4 (E11–E19; 20 mg/kg maternal dose), 5 (P1–P7; 10 mg/kg dose), 3 (P8–P21 Lo; 5 mg/kg dose), and 3 (P8–P21 Hi; 20 mg/kg dose) subjects per group. Repeated-measures ANOVA revealed a main effect of layer (F_1, 21 = 38.791, P < 0.001) and CTM exposure (F_4, 21 = 3.423, P = 0.026) but no interaction between layer and exposure. *P < 0.05 vs. control, a priori univariate F test. Supra- differed from infragranular under all exposure conditions (P < 0.05, a priori univariate F test).

Effect of neonatal (P8–P21) CTM exposure (20 mg/kg per d) on behavior. (A) Neonatal exposure preferentially decreases response (distance traveled) to a novel tone in male rats at P25. Data represent the mean ± SEM for 15–17 subjects. Baseline and tone conditions were presented for 120 s each. (B) In comparison with females, male CTM-exposed rats show a clear reduction in the exploration of a novel object at P39. Exploration of a novel object for 10 min was tested after a 10-min acclimation period. Data represent the mean ± SEM of 15–17 subjects. (C) Male rat pups that were exposed to CTM show virtually no participation in juvenile play in comparison with female CTM-exposed animals at P31. Data represent the mean ± SEM of 16–18 subjects. *P < 0.05, Bonferroni-corrected t test.

Early-life CTM exposure on corpus callosum axons and OLs. (A–H) Electron photomicrographs of corpus callosum axons and OLs obtained from adult males treated at different developmental periods. Note the morphology of myelinated axons and OLs in controls (A and E) vs. subjects treated at E11–E19 (B and F), P1–P7 (C and G), and P8–P21 (D and H). B, C, and D demonstrate distortions in myelin sheathing, and F, G, and H illustrate abnormal OL morphologies. Multinucleated cells (indicated by asterisks) with inclusions (possibly enveloped myelinated axons, indicated by arrows) were observed in CTM-treated animals. (Scale bars: 1.56 μm (A–D); 4 μm (E–H). (I) Relative frequency of abnormal axons in three treatment groups (control, gestational, and postnatal). Data represent mean ± SEM of six to nine subjects per group (males and females combined). Gestational CTM treatment: maternal treatment from E11–E19 with 20 mg/kg per d CTM; postnatal CTM treatment: exposure of pups from P1 to P7 with 10 mg/kg per d CTM and from P8 to P21 with 20 mg/kg per d CTM. (J and K) Data are illustrated by sex. Where n = 2, values represent mean ± SD. For I, J, and K, analysis of covariance (ANCOVA) revealed a main effect of treatment period (F_2, 18 = 13.332, P < 0.001) and a covariate effect of sex (F_1, 18 = 5.262, P = 0.034). *P < 0.001 vs. control, Bonferroni-corrected t test.

Degraded A1 frequency representation caused by CTM. (A) Representative A1 characteristic-frequency map from a control P22 rat (Left) and an age-matched 10 mg/kg CTM-treated rat (Right). Hatched areas represent cortical sites with an irregularity index (Materials and Methods) above 4. (Scale bar: 0.75 mm.) X, unresponsive cortical site; O, non-A1 cortical site (Materials and Methods). (B) Representative A1 receptive fields obtained in controls (I and II in characteristic-frequency map shown in A) and CTM-treated rats (III, IV, and V). (C) A threefold increase in irregularly shaped tuning curves (average irregularity index >4) was observed in CTM-treated vs. control subjects. (D) Distribution of all recorded characteristic frequencies in controls and CTM-treated rats plotted against a normalized tonotopic axis (Materials and Methods). The increased scatter of characteristic frequencies around the ideal tonotopic axis (black diagonal line) was quantified with the tonotopic index (TI), which was significantly higher in the CTM-treated group. (E) Distribution of tuning curve BW20 separated by characteristic frequency for controls and CTM-treated rats. (Controls: n = 14, 406 recorded sites; CTM-treated rats: n = 9, 407 recorded sites). Values shown are mean ± SEM. **P < 0.001, t test.