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

Animals 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.

Dams 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).

High-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 (Wayman et al. 2006). 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 (Wayman et al. 2006) 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).

Total thyroxine (T₄) and triiodothyronine (T₃) levels were determined in serum samples by radioimmunoassay (Diagnostic Products Corp, Los Angeles, CA) as previously described (Kodavanti et al. 1998).

At 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.

Spatial 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 (Jett et al. 1997). 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 (Jett et al. 1997, 2001; Kuhlmann et al. 1997). 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 (Jett et al. 1997, 2001; Markwiese et al. 1998; Rudy et al. 1987). 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.

On 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-D_28K (Sigma, St. Louis, MO), which specifically labels Purkinje cells (Christakos et al. 1987). Dendritic arbors of neocortical neurons were visualized by Golgi staining as previously described (Lein et al. 2007). Dendritic arbors were quantified in cultured neocortical neurons transfected with MAP2-eGFP as previously described (Wayman et al. 2006). An average of 10 neurons per culture from three cultures per group was analyzed, and results were confirmed in two independent dissections.

Specific [³H]-ryanodine (5 nM) binding to whole particulate cerebellar membranes was measured at P21 and P31, as previously described (Wong et al. 1997). Western blot analyses were used to quantify RyR expression as previously described (Roegge et al. 2006).

Hepatic CYP content was determined as previously described (Omura and Sato 1964). 7-Ethyoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depentylase (PROD) activities were analyzed in hepatic microsomes according to the method of Lubet (Lubet et al. 1990) as modified by Kennedy and Kono (Kennedy and Jones 1994; Kono et al. 1999). Enzyme activities were normalized to protein concentration as determined using the BCA Protein Assay (Pierce, Rockford, IL).

Whole brains from P31 rats were stored at –80°C and thawed immediately before extraction, cleanup, and fractionation using gel permeation chromatography as previously described (Sethajintanin et al. 2004).

Consistent with previous reports (Roegge et al. 2004), 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].

Developmental 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.

As 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.

The Morris water maze has been shown to detect subtle but significant changes in cognitive function in weanling rats exposed to developmental neurotoxicants (Jett et al. 1997, 2001; Markwiese et al. 1998), and training in this task induces experience-dependent dendritic growth (Greenough et al. 1979). Although considered a test of hippocampal function, performance in the Morris water maze is also dependent on the function of the cerebellum and neocortex (Lalonde and Strazielle 2003; Save and Poucet 2000), which is particularly relevant to the current studies because a) the vulnerability of the cerebellum to developmental hypothyroidism (Dong et al. 2005) is proposed as a primary mechanism for PCB developmental neurotoxicity (Koibuchi and Iwasaki 2006; Roman 2007; Zoeller 2007); and b) our previous studies of molecular bio-markers of dendritic growth (Lein et al. 2007) and development of excitatory to inhibitory balance in neurotransmission (Kenet et al. 2007) indicated that neuronal connectivity in the neocortex may be particularly susceptible to modulation by PCBs.

Training 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.

Morphometric 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.

Morphometric analyses of cerebellar neurons immunopositive for calbindin, which is a specific marker of Purkinje cells (Christakos et al. 1987), 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).

Representative 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).

One of the most sensitive molecular targets of PCBs is RyR activation (Pessah and Wong 2001). All three RyR isoforms are expressed in the brain, and RyR activity influences use-dependent synaptic plasticity (Berridge 2006). 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 (B_max) without significantly changing apparent affinities (K_D) (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).

[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 (Buck et al. 1992). 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) (Airey et al. 1990; Lai et al. 1992). 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 (Cavallaro et al. 1997), 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.

The 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 (Pessah et al. 2006), consistent with findings from other laboratories that noncoplanar, but not coplanar, PCBs increase intra-cellular Ca²⁺ in neurons (Kodavanti 2005). 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⁺ (Pessah and Wong 2001). 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 (Howard et al. 2003). A 48-hr exposure of cultured neocortical neurons (7–9 DIV) to nanomolar concentrations of PCB95, a congener that potently activates RyRs (Pessah et al. 2006), significantly enhanced dendritic growth, whereas exposure to PCB66, a congener with little activity at the RyR (Pessah et al. 2006), 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 (Mack et al. 1992) (Figure 6E).

Our 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 (Hansen 1999). 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).

Of 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.