Developmental exposure to polychlorinated biphenyls interferes with experience-dependent dendritic plasticity and ryanodine receptor expression in weanling rats

Abstract

Background: Neurodevelopmental disorders are associated with altered patterns of neuronal connectivity. A critical determinant of neuronal connectivity is the dendritic morphology of individual neurons, which is shaped by experience. The identification of environmental exposures that interfere with dendritic growth and plasticity may, therefore, provide insight into environmental risk factors for neurodevelopmental disorders.

Objective: We tested the hypothesis that polychlorinated biphenyls (PCBs) alter dendritic growth and/or plasticity by promoting the activity of ryanodine receptors (RyRs).

Methods and results: The Morris water maze was used to induce experience-dependent neural plasticity in weanling rats exposed to either vehicle or Aroclor 1254 (A1254) in the maternal diet throughout gestation and lactation. Developmental A1254 exposure promoted dendritic growth in cerebellar Purkinje cells and neocortical pyramidal neurons among untrained animals but attenuated or reversed experience-dependent dendritic growth among maze-trained littermates. These structural changes coincided with subtle deficits in spatial learning and memory, increased [3H]-ryanodine binding sites and RyR expression in the cerebellum of untrained animals, and inhibition of training-induced RyR upregulation. A congener with potent RyR activity, PCB95, but not a congener with negligible RyR activity, PCB66, promoted dendritic growth in primary cortical neuron cultures and this effect was blocked by pharmacologic antagonism of RyR activity.

Conclusions: Developmental exposure to PCBs interferes with normal patterns of dendritic growth and plasticity, and these effects may be linked to changes in RyR expression and function. These findings identify PCBs as candidate environmental risk factors for neurodevelopmental disorders, especially in children with heritable deficits in calcium signaling.

Keywords: PCBs; dendrite; developmental neurotoxicity; neurodevelopmental disorders; plasticity; ryanodine receptor.

Figure 1

Developmental A1254 exposure decreases serum thyroid hormone levels. Developmental A1254 exposure caused significant dose- dependent decreases in total serum T₄ and T₃ at P21. This effect persisted in the 6 mg but not the 1 mg/kg/day A1254 treatment group at P31. No sex differences were observed. Data are presented as mean ± SEM (n = 7–9/group). *p < 0.05 (two-way ANOVA, with treatment and sex as main effects; Fisher’s LSD post hoc).

Figure 2

Developmental A1254 exposure at 1 mg but not 6 mg/kg/day impairs performance in the Morris water maze. (A) Escape latency as a function of trial. (B ) Percentage of animals that reached criterion (escape latency ≤ 10 sec) during the 7-day training period. (C) Probe tests conducted on day 4. Dotted line indicates predicted time spent in the training quadrant by chance alone. (D) Performance in visual cue test and swimming speed. (E) Representative swim paths from probe tests on day 4. Data are presented as mean ± SEM. *p< 0.05, **p < 0.01 ( A, repeated measures ANOVA; B, Fisher’s exact test; C and D, ANOVA; Newman-Keuls post hoc).

Figure 3

Developmental A1254 exposure interferes with normal dendritic growth and experience-dependent dendritic plasticity. Dendritic morphology was analyzed among P31 rats trained in the Morris water maze (Maze) and among littermates identically housed and exposed but not trained (Nonmaze). (A) Total dendritic length of cerebellar Purkinje cells in nonmaze-trained animals. (B) Significant effect of maze training on total dendritic length of Purkinje cells. (C, D) Effects of maze training and developmental A1254 exposure on dendritic growth in cortical neurons. Data are presented as mean ± SEM (n = 17–21 neurons/group). Percent change in dendritic length as a function of maze training was calculated as the difference in dendritic length of neurons in maze-trained animals versus nonmaze-trained animals divided by dendritic length of neurons in maze-trained animals multiplied by 100. *p < 0.05, **p < 0.01, #p < 0.001 (A and B, ANOVA followed by Newman-Keuls; C and D, Wilcoxon test).

Figure 4

Developmental A1254 exposure increases specific [³H]-ryanodine (Ry) binding to cerebellar membranes. Binding constants (K_d and B_max) were determined from a [³H]-ryanodine–binding curve measured in cerebellar membranes from P21 pups (A) using Scatchard analysis (B). (C) Measurements of specific binding of [³H]-ryanodine (5 nM) to cerebellar membranes from P21 and maze-trained P31 rats. The percent change in ryanodine binding between P21 and P31 was calculated as the difference in B_max between P21 and P31 divided by B_max at P21 multiplied by 100. Data are presented as mean ± SD (n = 4). *p< 0.05, **p < 0.01, #p < 0.001 (ANOVA; Newman-Keuls post hoc).

Figure 5

Developmental A1254 exposure alters RyR expression in the cerebellum. (A) Representative blot of cerebellar membrane samples from P21 and maze-trained P31 rats probed with RyR antibodies. (B) Densitometric analyses of RyR1 and RyR2 expression in the cerebellum at P21. Band densities of samples from A1254-treated animals are plotted as percentage of mean control band densities. (C) Cerebellar expression of RyR1 and RyR2 as a function of age in nonmaze-trained animals. Data are presented as percent change in mean pixel density from P21 to P31 within each treatment group. (D) Effects of maze training and developmental A1254 exposure on RyR1 and RyR2 expression in the cerebellum. Data are presented as mean ± SEM (n ≥ 4). Asterisks associated with individual bars indicate statistically significant differences between P21 and maze-trained P31 rats within a group; asterisks above horizontal lines indicate statistically significant difference between groups. *p< 0.05, **p < 0.01, #p < 0.001 (ANOVA; Bonferroni post hoc).

Figure 6

PCB95, but not PCB66, promotes dendritic growth in cultured neocortical neurons. Neurons dissociated from embryonic rat cortices were plated at high density and transfected at 6-DIV with MAP2-GFP, which labels the somatodendritic compartment of 0.5–2% of neurons in the culture. At 7-DIV, cultures were treated with vehicle (0.1% DMSO), PCB66, or PCB95. Photomicrographs of GFP-positive neurons treated with vehicle (A), PCB95 (B), or PCB66 (C) at 250 nM. PCB95, a congener with potent RyR activity, significantly enhanced dendritic growth in cultured cortical neurons in a nonmonotonic fashion (B, D). In contrast, PCB66, a congener that lacks RyR activity, had no effect on dendritic growth in cultured cortical neurons (C, D). (E) PCB95–induced dendritic growth is blocked by the ryanodine receptor antagonist FLA365 (10 μM). Data are presented as mean ± SEM (n = 30 neurons/condition). **p < 0.01, #p < 0.001 (ANOVA; Newman-Keuls post hoc).

Figure 7

A1254 induction of CYP expression and activity in the liver is dose and age dependent. (A) Microsomal CYP content and (B) EROD or (C) PROD activities were measured in liver at P21 and P31. EROD and PROD were chosen as biomarkers of exposure to coplanar and noncoplanar PCBs, respectively. Liver CYP content was significantly increased relative to controls in the 6 mg/kg treatment group on P21. Developmental A1254 exposure dose-dependently increased EROD and PROD activity at P21 and P31; the increase in PROD activity was significantly different from control only in the 6 mg/kg/day A1254 group. Data are presented as mean ± SEM (n = 5–7/group at P21; n = 10–13/group at P31). *p < 0.05, **p < 0.01, #p < 0.001 (ANOVA; Newman-Keuls post hoc).