Developmental Exposure to Polychlorinated Biphenyls Interferes with Experience-Dependent Dendritic Plasticity and Ryanodine Receptor Expression in Weanling Rats

1Polychlorinated biphenyls (PCBs) are a structurally related group of stable and highly lipophilic chemicals with widespread distribution in the environment. Despite being banned in 1977, PCBs persist in the environment, and high residue levels are still detected in human tissues. Epidemiologic data indicate that PCBs negatively impact neuropsychologic function in exposed children, and experimental animal studies confirm that developmental PCB exposure causes cognitive and psycho-motor deficits. However, the cellular and molecular mechanisms mediating these effects remain speculative.

2PCBs interfere with endocrine functions, specifically those mediated by thyroid hormone and estrogen, and increase neuronal Ca2+ levels via several mechanisms, including ryanodine receptor (RyR) activation. It is not clear, however, how these molecular effects relate to PCB developmental neurotoxicity. Thyroid hormone, estrogen, and Ca2+ influence neuronal connectivity via dynamic control of dendritic structure. Altered patterns of dendritic growth and plasticity are associated with impaired behavior in experimental models and are thought to contribute to diverse neurodevelopmental disorders, suggesting the possibility that PCBs elicit developmental neurotoxic effects by interfering with neuronal connectivity.

3Consistent with this hypothesis, we recently observed that developmental PCB exposure disrupts the balance of neuronal inhibition to excitation in the developing rat auditory cortex and accelerates dendritic growth in the hippocampus and cerebellum of weanling rats. Questions yet to be addressed include the relationship between PCB interference with neuronal connectivity and known molecular targets of PCBs and whether developmental exposures to PCBs interfere with experience-dependent dendritic plasticity, a phenomenon critical to associative learning and memory. In this study, we used the Morris water maze as a tool for inducing experience-dependent neural plasticity in weanling rats exposed to Aroclor 1254 (A1254) in the maternal diet throughout gestation and lactation. In addition to assessing dendritic morphology in these animals, we also quantified RyR expression and function, thyroid hormone levels, and developmental end points regulated by sex steroids. Our findings suggest that developmental PCB exposure interferes with dendritic growth and plasticity coincident with delayed spatial learning and that perturbation of RyR expression and function contributes to these effects.

4Detailed descriptions of all methods are available online in Supplemental Material (http://www.ehponline.org/members/2008/11771/suppl.pdf).

5Animals were treated humanely and with regard for alleviation of suffering according to protocols approved by the Institutional Animal Care and Use Committees of the Johns Hopkins University and Oregon Health & Science University. Adult Long Evans rats were purchased from Charles River Laboratories (Hollister, CA) and housed individually, except during breeding, in standard plastic cages with Alpha-Dri bedding (Shepherd Specialty Papers, Watertown, TN) in a temperature-controlled (22 ± 2°) room on a 12-hr reverse light–dark cycle. Food and water were provided ad libitum. Dams used in the study delivered litters of 10–15 pups (n = 11 dams per treatment group). By postnatal day 2 (P2), litters were culled to five males and five females. Pups were weaned on P21.

6Dams were dosed with the commercial PCB mixture Aroclor 1254 (A1254, lot #124-191; AccuStandard, New Haven, CT) at 1 mg or 6 mg/kg/day beginning 2 weeks prior to breeding and continuing until P21. A1254 was diluted in corn oil and pipetted onto one-half of a Keebler Golden Vanilla Wafer (Kellogg Company, Battle Creek, MI). Control animals received wafers dosed with an equal volume (500 μL) of vehicle. Doses were adjusted daily to account for changes in body weight of the dams. Dams were fed the wafers in a separate cage to prevent the pups from accessing the wafers and were watched carefully to ensure that the entire wafer was consumed (typically within 5 min).

7High-density cultures of dissociated neocortical neurons (105 cells/cm2) were prepared from P1 Sprague Dawley rats (Charles River Laboratories) and maintained in Neurobasal-A (Invitrogen, Carlsbad, CA) supplemented with B27 (Invitrogen) as previously described. On day 6 in vitro (6-DIV) cultures were transfected with plasmid encoding a microtubule-associated protein-2 (MAP2)-enhanced green fluorescent protein (GFP) fusion construct using Lipofectamine-2000 (Invitrogen) according to the manufacturer’s protocol. On 7-DIV, cultures were treated for 48 hr with vehicle (DMSO at 1:1,000 dilution), PCB95 (2,2′,3,5′,6-pentachlorobiphenyl, > 95% purity; AccuStandard), or PCB66 (2,3′,4,4′-tetrachlorobiphenyl, > 95% purity; AccuStandard).

8Total thyroxine (T4) and triiodothyronine (T3) levels were determined in serum samples by radioimmunoassay (Diagnostic Products Corp, Los Angeles, CA) as previously described.

9At P2, litter size, sex ratio, and pup body mass were measured. Anogenital distance (AGD) was measured at P2, P10, and P21. At P21, litters were weaned and housed with same-sex siblings. At P40 (puberty), body mass was measured, and the presence of preputial separation or vaginal opening was recorded in males and females, respectively. At P70, blood samples collected from the retro-orbital sinus were analyzed for serum levels of testosterone in males and estradiol in females by radioimmunoassay per the manufacturer’s protocol [ICN Biochemicals, Inc. (MP Biomedical, Solon, OH)]. Samples were assayed in triplicate and cross-reactivity with other steroids was < 0.1%. After blood collection, animals were euthanized, and reproductive organs were removed and weighed. The tunica was then stripped from paired testes, and the seminiferous tubules were homogenized in 0.5% Triton-X 100 with 0.01% thimerosal to determine sperm concentration using a Newbauer chamber.

10Spatial learning and memory was assessed on P24 in one male and one female from 11 different litters within each treatment group, using the Morris water maze as previously described. Rats were tested in one trial per day, except on the first day, when two trials were administered. This modification increases difficulty such that relatively small differences between treatment groups can be detected, yet the task is not too difficult for rats to learn quickly. An escape latency of 10 sec was chosen as the criterion that animals had learned the task, based on previous studies using rats of comparable age in a similar size pool. To test spatial memory, a probe test was administered 30 min after the spatial training trials on the first day that the mean escape latency of any treatment group reached criterion.

11On P31, animals were euthanized and perfused with 4% paraformaldehyde. To visualize Purkinje cell dendritic arbors, parasagittal cryosections (12 μm) were cut from both cerebellar hemispheres, starting 1 mm from the midline, and reacted with antibody specific for calbindin-D28K (Sigma, St. Louis, MO), which specifically labels Purkinje cells. Dendritic arbors of neocortical neurons were visualized by Golgi staining as previously described. Dendritic arbors were quantified in cultured neocortical neurons transfected with MAP2-eGFP as previously described. An average of 10 neurons per culture from three cultures per group was analyzed, and results were confirmed in two independent dissections.

12Specific [3H]-ryanodine (5 nM) binding to whole particulate cerebellar membranes was measured at P21 and P31, as previously described. Western blot analyses were used to quantify RyR expression as previously described.

13Hepatic CYP content was determined as previously described. 7-Ethyoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depentylase (PROD) activities were analyzed in hepatic microsomes according to the method of Lubet as modified by Kennedy and Kono. Enzyme activities were normalized to protein concentration as determined using the BCA Protein Assay (Pierce, Rockford, IL).

14Whole brains from P31 rats were stored at –80°C and thawed immediately before extraction, cleanup, and fractionation using gel permeation chromatography as previously described.

15Consistent with previous reports, dietary exposure of dams to A1254 at 1 mg or 6 mg/kg/day, starting 2 weeks before conception and continuing throughout gestation and lactation, did not negatively impact developmental outcomes or cause overt signs of intoxication in dams or pups as determined by lack of treatment-related changes in maternal weight gain during gestation, maternal body weight during lactation, length of gestation, litter size, and weight gain of offspring during lactation [see Supplemental Material (http://www.ehponline.org/members/2008/11771/suppl.pdf), Figure 1].

16Developmental exposure to A1254 at 1 mg or 6 mg/kg/day significantly decreased serum concentrations of T4 and T3 at P21 (Figure 1). By P31, serum T3 and T4 had recovered to control levels among pups in the 1 mg/kg/day A1254 treatment group, but were still significantly depressed in the 6 mg/kg/day treatment group. No sex differences were observed.

17As shown in Table 1, developmental A1254 exposure differentially altered a subset of reproductive developmental end points regulated by estrogens and androgens including: a) male to female ratio of litters, which was increased in the 6 mg/kg/day A1254 treatment group; b) absolute but not relative AGD among female offspring, which was longer than control in both A1254 treatment groups; c) vaginal opening, which was significantly delayed among females in the 6 mg/kg/day A1254 treatment group; and d) both absolute and relative prostate mass, which was increased in P70 males exposed to A1254 at 1 mg/kg/day but not 6 mg/kg/day, in the maternal diet. A number of other sex steroid–dependent developmental end points were not affected by developmental exposure to either dose of A1254, including AGD (absolute or relative) in males, preputial separation in males, the size of female reproductive organs, the size of male reproductive organs other than the prostate, sperm counts or plasma concentrations of estradiol in females and testosterone in males.

18The Morris water maze has been shown to detect subtle but significant changes in cognitive function in weanling rats exposed to developmental neurotoxicants, and training in this task induces experience-dependent dendritic growth. Although considered a test of hippocampal function, performance in the Morris water maze is also dependent on the function of the cerebellum and neocortex, which is particularly relevant to the current studies because a) the vulnerability of the cerebellum to developmental hypothyroidism is proposed as a primary mechanism for PCB developmental neurotoxicity; and b) our previous studies of molecular bio-markers of dendritic growth and development of excitatory to inhibitory balance in neurotransmission indicated that neuronal connectivity in the neocortex may be particularly susceptible to modulation by PCBs.

19Training in the Morris water maze was initiated on P24 and concluded on P30 (Figure 2). Repeated-measures two-way ANOVA, with sex and treatment as the between-subjects factors and trial day as the repeated-measures factor, identified a significant interaction between treatment and trial (p < 0.001) but a lack of interaction of sex with treatment or trial. There was no three-way interaction among trial, treatment, and sex. Subsequent post hoc Newman-Keuls multiple comparisons on each trial day revealed a significant difference in escape latency between the 1 mg/kg/day treatment group and both vehicle and 6 mg/kg/day treatment groups on day 4. Although significant differences between treatment groups were not detected on other trial days, it is clear from the plot of escape latency that from day 3 through day 7, the 1 mg/kg/day treatment group took longer to find the platform than the control and 6 mg/kg/day treatment group (Figure 2A). The percentage of animals within each treatment group that reached criterion by the end of the training period was significantly reduced in the 1 mg but not 6 mg/kg/day treatment group relative to controls (Figure 2B), further suggesting that weanling rats exposed developmentally to A1254 at 1 mg/kg/day were not as proficient at spatial learning as animals in either the control or 6 mg/kg/day A1254 group. Spatial memory was assessed during the probe test on days 4 and 7, the first day that controls and 1 mg/kg/day–treated animals reached criterion, respectively. On day 4, the 1 mg/kg A1254 rats spent significantly less time in the training quadrant than those from the control or 6 mg/kg/day A1254 groups (Figures 2C and 2E). On day 7, however, there were no significant differences between groups (data not shown), suggesting that although learning and memory were impaired in the 1 mg/kg/day A1254 group, with additional training, rats in this group did acquire the task. Developmental exposure to A1254 at either dose had no effect on escape latency in the visual cue test or on swimming speed (Figure 2D), indicating that learning and memory deficits observed in the training trials and probe tests were not due to negative impacts of A1254 on vision, motivation, or swim speed.

20Morphometric analyses of Nissl-stained sections indicated no overt treatment-related effects on development of the cerebellum or neocortex [see Supplemental Material (http://www.ehponline.org/members/2008/11771/suppl.pdf), Figure 2]. To assess effects of developmental A1254 exposure on cellular indices of neural circuitry, dendritic length was quantified in individual cerebellar Purkinje cells and neocortical pyramidal neurons at P31. Because no sex differences were observed in PCB effects on performance in the Morris water maze, morphometric studies were restricted to males.

21Morphometric analyses of cerebellar neurons immunopositive for calbindin, which is a specific marker of Purkinje cells, indicated that among untrained animals, developmental exposure to A1254 at 1 mg but not 6 mg/kg/day significantly increased total dendritic length relative to vehicle controls (Figure 3A). Analysis of the percent change in dendritic length of cerebellar Purkinje cells as a function of maze training within groups revealed that Morris water maze training significantly increased total dendritic length in Purkinje cells of controls, caused significant dendritic retraction in the 1 mg/kg/day A1254 group, and had no significant effect on dendritic length among rats in the 6 mg/kg/day A1254 group (Figure 3B). Comparison between groups indicated that training-induced dendritic growth observed among controls was significantly attenuated by developmental exposure to A1254 at 6 mg/kg/day and actually reversed by A1254 at 1 mg/kg/day (Figure 3B).

22Representative camera lucida drawings of the basilar dendritic arbor of neocortical pyramidal neurons from untrained and maze-trained littermates within each group (Figure 3C) demonstrate effects similar to those observed in cerebellar Purkinje cells. Quantification of dendritic length by Sholl analysis indicated that in untrained animals, developmental exposure to A1254 increased dendritic length in neocortical pyramidal neurons by 20% and 17% in the 1 mg and 6 mg/kg/day groups, respectively, relative to controls. Maze training increased dendritic length of neocortical neurons among controls by 22% but caused dendritic length to decrease by 17% in the 1 mg/kg/day A1254 treatment group (Figures 3C and 3D). In animals exposed developmentally to A1254 at 6 mg/kg/day, maze training caused neither significant expansion nor retraction of the dendritic arbor relative to untrained littermates (Figures 3C and 3D). Comparison between groups of the percent change in dendritic length of neocortical pyramidal neurons as a function of maze training indicated that the training-induced dendritic growth observed in controls was inhibited by developmental exposure to A1254, with significantly more pronounced effects observed in the 1 mg versus 6 mg/kg/day A1254 group (Figure 3D).

23One of the most sensitive molecular targets of PCBs is RyR activation. All three RyR isoforms are expressed in the brain, and RyR activity influences use-dependent synaptic plasticity. These observations suggest that PCBs may interfere with dendritic growth and plasticity via RyR-mediated mechanisms. To test this hypothesis, we first determined whether developmental A1254 exposure influenced RyR function and expression. Because the effects of developmental A1254 exposure on dendritic growth and plasticity were similar between cerebellar Purkinje cells and neocortical pyramidal neurons, these studies focused on the cerebellum. To measure the density and level of functional activation of RyR channels, we analyzed specific high-affinity binding of [3H]-ryanodine to membranes prepared from cerebella of males within each group. Scatchard analysis of binding data obtained from cerebellar membranes of P21 animals indicated that developmental A1254 dose-dependently increased the [3H]-ryanodine binding density (Bmax) without significantly changing apparent affinities (KD) (Figures 4A and 4B). Comparison of these values to those obtained from [3H]-ryanodine binding to cerebellar membranes isolated from maze-trained P31 rats indicated that maze training did not affect the density of [3H]-ryanodine binding sites measured in controls, but significantly increased the density of functional RyR channels among animals exposed developmentally to A1254 at 1 mg or 6 mg/kg/day, with significantly more pronounced effects observed at the higher dose (Figure 4C).

24[3H]-ryanodine binds with high affinity and specificity to all three RyR isoforms and is a measure of the expression levels of functional RyR proteins as well as the stability of the open state of these channels. To evaluate the effects of developmental A1254 exposure on specific RyR isoforms, we examined expression levels by Western blot using monoclonal antibodies (mAbs) that selectively bind to RyR1 and RyR3 (mAb 34C) or to RyR2 (mAb C3-33). Both RyR1 and RyR2 were observed in cerebellar membranes isolated from P21 and P31 rats, but bands corresponding to the molecular weight of RyR3 were not detected in any sample (Figure 5A). Developmental A1254 exposure increased RyR1 and RyR2 expression levels in the cerebellum of P21 rats (Figure 5B). Although the effect on RyR2 expression was similar between A1254 groups, the effect on RyR1 expression was significantly greater in the 1 mg/kg/day A1254 group relative to the 6 mg/kg/day A1254 group. Comparison of cerebellar expression levels of RyR1 and RyR2 between P21 and untrained P31 rats within controls indicated that expression of these RyR isoforms did not increase with age (Figure 5C). As previously reported for RyR2, maze training significantly increased RyR1 and RyR2 expression in controls (Figure 5D). In rats exposed developmentally to A1254, maze training also significantly increased RyR2 expression but significantly decreased RyR1 expression (Figure 5D). Comparison between groups of the percent change in RyR1 and RyR2 expression as a function of maze training suggested that developmental A1254 exposure reversed the effects of maze training on RyR1 expression and attenuated the effects of maze training on RyR2 expression; these responses were more pronounced in the 1 mg relative to the 6 mg/kg/day A1254 group.

25The observation of similar inverted dose-related effects and training-dependent biphasic responses of dendritic morphology and RyR expression in A1254-exposed animals suggested a causal relationship between these effects. We previously demonstrated that noncoplanar PCBs possessing 2–3 chlorine ortho substitutions are the most potent RyR activators, consistent with findings from other laboratories that noncoplanar, but not coplanar, PCBs increase intra-cellular Ca2+ in neurons. Noncoplanar PCBs at nanomolar concentrations interact with RyRs to dramatically increase their sensitivity to activation by nanomolar Ca2+ and attenuate their sensitivity to inhibitory feedback by millimolar Ca2+ and Mg2+. To further probe the relationship between PCB effects on dendritic growth and RyRs, we quantified dendritic growth in primary cultures of neocortical neurons exposed to individual PCB congeners with differential effects on RyR activity at concentrations that did not adversely influence cell viability in cultured neocortical neurons. A 48-hr exposure of cultured neocortical neurons (7–9 DIV) to nanomolar concentrations of PCB95, a congener that potently activates RyRs, significantly enhanced dendritic growth, whereas exposure to PCB66, a congener with little activity at the RyR, had no effect on dendritic growth (Figure 6A–D). Interestingly, micromolar concentrations of PCB95 had no net effect on dendritic growth compared with controls, recapitulating the inverted dose-related effects of developmental A1254 exposure on dendritic growth in vivo. PCB-95–induced dendritic growth was completely blocked in the presence of the selective RyR antagonist FLA365 (Figure 6E).

26Our in vitro observations strongly suggest that noncoplanar PCB congeners mediated the effects of A1254 in vivo. As an indirect test of this hypothesis, we measured CYP activities in hepatic microsomes and quantified levels of individual PCB congeners in the whole brain obtained at P31 from male and female littermates of animals trained in the Morris water maze. Total CYP content was significantly increased by developmental A1254 exposure at P21, but only in the 6 mg/kg/day treatment group, and this effect was no longer evident at P31 (Figure 7A). EROD and PROD activity represent CYP isozymes differentially up-regulated by coplanar and noncoplanar PCBs, respectively. Developmental A1254 exposure dose-dependently increased EROD (Figure 7B) and PROD (Figure 7C), and these effects persisted until P31, although the absolute levels of EROD and PROD activity decreased in all treatment groups with increasing age (Figure 7C).

27Of the 32 congeners chosen for analysis based on their toxicity, presence in A1254, abundance in environmental samples, and analytical capability, 30 were below the detection limit in brains of controls; the two congeners that were detected, PCB158 and PCB187, were found in only one of four samples (Table 2). In contrast, 14 congeners were detectable in brains from animals in the 1 mg/kg/day A1254 group and 16 in brains from the 6 mg/kg/day A1254 group. These were predominantly ortho-substituted, non-coplanar PCBs, and levels were significantly higher in the 6 mg relative to the 1 mg/kg/day A1254 group.

28The major findings of this study are that developmental PCB exposure enhanced basal dendritic growth but decreased experience-dependent dendritic plasticity, and that these effects correlated better with altered RyR expression than with endocrine disruption.

29Developmental A1254 exposure significantly enhanced dendritic growth in cerebellar Purkinje cells and neocortical pyramidal neurons among P31 rats not trained in the Morris water maze, which is consistent with our previous observations that similar exposures accelerated dendritic growth in Purkinje cells and hippocampal CA1 pyramidal neurons between P21 and P60. In Purkinje cells, this effect was observed among animals in the 1 mg but not 6 mg/kg/day A1254 group, whereas in neocortical neurons, responses were comparable between A1254 groups. The reason for the different dose–response relationship in different brain regions is not known. Possibilities include regional differences in RyR regulation or differential upregulation of cytochrome P450 enzymes by AhR ligands in the cerebellum versus neo-cortex, which could result in regional differences in PCB toxicodynamics and toxicokinetics, respectively.

30Previous studies have shown that experience increases dendritic complexity, and we observed that among controls, training in the Morris water maze significantly increased dendritic length in both Purkinje cells and neocortical neurons. However, maze training caused no change in dendritic length in the 6 mg/kg/day A1254 group and significant dendritic retraction in the 1 mg/kg/day A1254 treatment group. Structural plasticity of dendrites is considered the cellular substrate of learning and memory, and we observed that developmental A1254 exposure caused subtle but statistically significant delays in learning and memory that exhibited an inverted dose-related response similar to that observed for experience-dependent plasticity in A1254-treated animals. That these behavioral effects may be of biological significance is suggested by comparison with the human literature, which similarly demonstrates an association between developmental PCB exposures and subtle effects on cognitive function that may be overcome by training or increasing age. Such subtle effects may have significant biological and social costs when considered at the population level.

31It is widely postulated that PCB developmental neurotoxicity is mediated by endocrine disruption. Developmental PCB exposure is reported to modulate systemic estrogen levels, increase estrogen sensitivity, and compete for binding to the estrogen receptor, yet we observed no A1254-related effects on plasma levels of estradiol or testosterone and only minor effects on estrogen- and androgen-dependent developmental end points. The discrepancies probably reflect differences in doses, which were generally much lower in our study, or congener profiles. As previously reported, developmental A1254 exposure significantly decreased serum thyroid hormone levels. However, it seems unlikely that PCB effects on dendritic growth were due to hypothyroxinemia, because a) neonatal hypothyroidism decreases basal dendritic growth, whereas developmental PCB exposure significantly enhanced basal dendritic growth; and b) PCB effects on dendritic growth were recapitulated in cultured neocortical neurons removed from systemic thyroid hormone influence. We recently demonstrated that PCB95 significantly disrupts the normal balance of excitatory and inhibitory neurotransmission in the auditory cortex at a dose that has no measurable effect on auditory brain stem responses (ABRs), which is a confirmed T4-dependent target of PCB. Although we cannot preclude the possibility that PCBs influence thyroid hormone signaling downstream of cognate receptors, our data suggest that endocrine disruption is not the sole mechanism underlying PCB effects on neuronal connectivity. Another critical determinant of dendritic morphology is Ca2+ signaling. Kodavanti and colleagues demonstrated that noncoplanar, but not coplanar, PCBs increase intracellular Ca2+ in neurons. Several mechanisms have been shown to mediate this response, but one of the most sensitive is RyR sensitization. RyR-mediated signals influence neuronal excitability, regulate synaptic plasticity, and activate cytosolic and nuclear transcriptional events implicated in activity-dependent dendritic growth.

32Here we show that developmental A1254 exposure dose-dependently increased RyR activity in the cerebellum as determined by [3H]-ryanodine receptor-binding analysis. This is most likely the result of dose-dependent accumulation of PCBs in the brain. The most abundant congeners found in the brain were noncoplanar, which are potent sensitizers of RyR channels. One possible consequence of chronic RyR sensitization is altered fidelity of Ca2+ signaling. In our study, elevated [3H]-ryanodine binding was closely associated with differential expression of RyR1 and RyR2 isoforms within the cerebellum, and changes in expression of isoforms were highly dependent on A1254 dose and training status. Interestingly, effects of dose and training on RyR expression closely paralleled their effects on dendritic morphology. Given the fundamental role of RyR in Ca2+ signaling and the critical influence of Ca2+ signaling on basal and activity-dependent dendritic growth, these data suggest that PCB effects on RyR could be largely responsible for the effects of developmental A1254 exposure on neuronal connectivity observed in weanling rats. In support of this hypothesis, nanomolar concentrations of PCB95, a congener that potently activates RyRs, enhanced dendritic growth in primary cultures of neocortical neurons, whereas similar concentrations of PCB66, a congener with negligible effects on RyR activity, had no effect on dendritic growth. Moreover, pharmacologic antagonism of RyR activity blocked PCB95–enhanced dendritic growth. That PCB-mediated RyR dysfunction modulates development of neuronal networks is further supported by our previous demonstration that developmental exposure to PCB95 significantly enhanced the ratio of excitatory to inhibitory currents within the primary auditory cortex (A1) of weanling rats, which was associated with irregularly shaped topographic organization of A1 and disruption of the critical period plasticity that underlies normal postnatal auditory system development. It is plausible that these effects reflect changes in basal and activity-driven dendritic complexity, as demonstrated in the present study.

33Based on our findings, we propose a novel model of PCB developmental neurotoxicity in which noncoplanar PCBs sensitize RyR activity and alter Ca2+-dependent signaling mechanisms that link neuronal activity to dendritic growth and plasticity. A critical role for RyRs in PCB interference with neuronal connectivity suggests several explanations for the inverted dose-related effects as well as training-dependent biphasic outcomes in dendritic morphology. First, chronic RyR sensitization alters RyR expression, and consequently, downstream Ca2+-dependent events that regulate dendritic growth in an inverted dose-related manner, perhaps because of negative feedback at the higher A1254 dose. Second, intracellular Ca2+ promotes dendritic growth in a concentration-dependent manner in early neuronal development. In later neuronal development, however, moderate increases in Ca2+ promote dendritic growth, whereas large increases cause dendritic retraction. Thus, increasing PCB doses may increase intracellular Ca2+ from concentrations that promote dendritic growth to those that cause dendritic retraction. Furthermore, training-induced increases in intracellular Ca2+ superimposed on a background of PCB exposure may push Ca2+ concentrations towards those that cause dendrite retraction. Although further studies are required to test these mechanistic hypotheses, the observation of an inverted (or non-monotonic) dose–response relationship has global regulatory implications as screening and testing programs for endocrine disruption and developmental neurotoxicity that emphasize very high doses of test chemicals with little to no acknowledgement of the importance of nonmonotonic dose–response relationships are finalized.

34The relevance of our findings to human health is suggested by several considerations. First, altered dendritic growth and impaired experience-dependent dendritic plasticity are thought to contribute to the clinical manifestations of various environmentally induced neurodevelopmental disorders in humans. Second, congener-specific analyses of brains from weanling rats in the 1 mg/kg/day A1254 group identified predominantly ortho-substituted congeners at concentrations ranging from 0.5 ng to 3 ng/g wet weight. Analyses of PCB levels in human brains obtained from the general adult population similarly identified predominantly ortho-substituted congeners at concentrations ranging from 0.07 ng to 12 ng/g wet weight. Ortho-substituted congeners with the highest activity towards RyRs, including PCB95, collectively represent 40–50% of total PCBs currently found in environmental and biotic samples, and their net effects are likely to be additive. Even lower levels of PCB exposure might amplify adverse effects in genetically susceptible individuals, particularly if both the genetic factor and PCBs converge to dysregulate the same developmental process. Interestingly, genes that encode Ca2+-regulated signaling proteins involved in synapse formation and dendritic growth are implicated in neurodevelopmental disorders. Considered together, these observations identify PCBs, particularly ortho-substituted PCBs with high RyR activity, as candidate environmental risk factors in neurodevelopmental disorders and provide important new clues about the possible role of RyR in contributing to environmentally triggered neurodevelopmental deficits.