PCB-95 Promotes Dendritic Growth via Ryanodine Receptor–Dependent Mechanisms

Background: Aroclor 1254 (A1254) interferes with normal dendritic growth and plasticity in the developing rodent brain, but the mechanism(s) mediating this effect have yet to be established. Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) enhance the activity of ryanodine receptor (RyR) calcium ion (Ca2+) channels, which play a central role in regulating the spatiotemporal dynamics of intracellular Ca2+ signaling. Ca2+ signaling is a predominant factor in shaping dendritic arbors, but whether PCB potentiation of RyR activity influences dendritic growth is not known. Objective: We determined whether RyR activity is required for PCB effects on dendritic growth. Methods and Results: Golgi analysis of hippocampi from weanling rats confirmed that developmental exposure via the maternal diet to NDL PCB-95 (2,2´,3,5´6-pentachlorobiphenyl), a potent RyR potentiator, phenocopies the dendrite-promoting effects of A1254. Dendritic growth in dissociated cultures of primary hippocampal neurons and in hippocampal slice cultures is similarly enhanced by PCB-95 but not by PCB-66 (2,3,4´,4-tetrachlorobiphenyl), a congener with negligible effects on RyR activity. The dendrite-promoting effects of PCB-95 are evident at concentrations as low as 2 pM and are inhibited by either pharmacologic blockade or siRNA knockdown of RyRs. Conclusions: Our findings demonstrate that environmentally relevant levels of NDL PCBs modulate neuronal connectivity via RyR-dependent effects on dendritic arborization. In addition, these findings identify RyR channel dysregulation as a novel mechanism contributing to dysmorphic dendritogenesis associated with heritable and environmentally triggered neurodevelopmental disorders.

Dendritic architecture is a critical determinant of neuronal connectivity (Libersat and Duch 2004;Scott and Luo 2001). Abnormalities in dendritic shape are the most consistent pathologic correlate of behavioral deficits in heritable and environmentally triggered neuro developmental disorders (Bourgeron 2009;Fukuda et al. 2005;Garey 2010;Penzes et al. 2011;Svitkina et al. 2010). Polychlorinated biphenyls (PCBs) are known human develop mental neurotoxicants (Carpenter 2006;Korrick and Sagiv 2008;Schantz et al. 2003), and we previously demonstrated that develop mental exposure to the PCB mixture Aroclor 1254 (A1254) interferes with normal patterns of dendritic growth and plasticity in wean ling rats (Lein et al. 2007;Yang et al. 2009) coincident with deficits in spatial learning and memory (Yang et al. 2009).
The dynamic structural remodeling of den drites that occurs during development (Chen and Nedivi 2010;Cline 2001) is driven in large part by calcium ion (Ca 2+ )dependent signaling pathways triggered by NMDA (Nmethyldaspartate) receptor activation and extrinsic cues such as neurotrophins (Lohmann and Wong 2005;Wayman et al. 2008). Nondioxinlike (NDL) PCBs increase intr acellular Ca 2+ in neurons via several mecha nisms, the most sensitive of which is poten tiation of ryanodine receptor (RyR) activity (Pessah et al. 2010). RyRs are ion channels in the endoplasmic reticulum (ER) that regu late Ca 2+ release from the ER and modulate the gating response and signal gain of plasma membrane ion channels, including the NMDA receptor (Pessah et al. 2010). RyR activity determines the amplitude and spatiotemporal patterns of intracellular Ca 2+ fluxes (Berridge 2006). NDL PCBs interact with RyRs to dra matically alter their sensitivity to physiological modulators .
A1254 consists primarily of NDL PCBs (Kostyniak et al. 2005) with varying RyR sensitizing potencies (Pessah et al. 2006), sug gesting that effects of develop mental A1254 exposure on dendritic arborization are medi ated by RyRdependent mechanisms. In sup port of this hypothesis, we demonstrated that A1254 interference with dendritic growth and plasticity in cerebellar Purkinje cells correlated with altered RyR expression and activity in the cerebellum (Yang et al. 2009) and that nanomolar concentrations of environmentally relevant PCB95 (2,2´,3,5´,6penta chloro biphenyl), a congener that potently activates RyRs (Pessah et al. 2006), enhanced dendritic growth in primary cultures of neo cortical neurons (Yang et al. 2009). Here, we pro vide evidence of a causal relationship between PCB95-induced dendritic growth and RyR activation in hippo campal neurons. In vivo PCB exposures. Adult Long Evans rats were purchased from Charles River Laboratories (Hollister, CA), and husbandry practices were as previously described (Yang et al. 2009). Dams were dosed with A1254 (1 or 6 mg/kg/day), PCB95 (0.1, 1, or 6 mg/kg/day), or vehicle (peanut oil) as previ ously described (Yang et al. 2009) beginning 14 days before breeding and continuing until postnatal day (PD) 21. Dams delivered 10-15 pups (n = 11 dams per treatment group). By PD2, litters were culled to 10 pups. Pups were weaned on PD21 and euthanized at PD31 (A1254 studies) or PD38 (PCB95 studies). Exposure to either A1254 or PCB95 in the maternal diet throughout gestation and lacta tion did not affect the body weight of pregnant and lactating dams, litter size, sex ratios, or growth rates of the pups.

Methods
Cell culture. Hippo campal neurons (10 5 cells/cm 2 ) were dissociated from PD1 Sprague Dawley rats (Charles River Laboratories) and cultured in NeurobasalA (Invitrogen, Carlsbad, CA) supplemented with B27 (Invitrogen) as described previously (Wayman Background: Aroclor 1254 (A1254) interferes with normal dendritic growth and plasticity in the developing rodent brain, but the mechanism(s) mediating this effect have yet to be established. Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) enhance the activity of ryanodine receptor (RyR) calcium ion (Ca 2+ ) channels, which play a central role in regulating the spatiotemporal dynamics of intracellular Ca 2+ signaling. Ca 2+ signaling is a predominant factor in shaping dendritic arbors, but whether PCB potentiation of RyR activity influences dendritic growth is not known. oBjective: We determined whether RyR activity is required for PCB effects on dendritic growth. Methods and results: Golgi analysis of hippocampi from weanling rats confirmed that developmental exposure via the maternal diet to NDL PCB-95 (2,2´,3,5´6-pentachlorobiphenyl), a potent RyR potentiator, phenocopies the dendrite-promoting effects of A1254. Dendritic growth in dissociated cultures of primary hippo campal neurons and in hippocampal slice cultures is similarly enhanced by PCB-95 but not by PCB-66 (2,3,4´,4-tetrachlorobiphenyl), a congener with negligible effects on RyR activity. The dendrite-promoting effects of PCB-95 are evident at concentrations as low as 2 pM and are inhibited by either pharmacologic blockade or siRNA knockdown of RyRs. conclusions: Our findings demonstrate that environmentally relevant levels of NDL PCBs modu late neuronal connectivity via RyR-dependent effects on dendritic arborization. In addition, these findings identify RyR channel dysregulation as a novel mechanism contributing to dysmorphic dendrito genesis associated with heritable and environmentally triggered neuro developmental disorders.  et al. 2006). At 5-6 days in vitro (DIV), cul tures were transfected with plasmid encoding microtubuleassociatedprotein2B (MAP2B) fused to enhanced green fluorescent protein (EGFP), which selectively labels the somato dendritic domain (Wayman et al. 2006), using Lipofectamine2000 (Invitrogen) according to the manufacturer's protocol. At 7 DIV, cultures were treated for 48 hr with vehicle (DMSO; 1:1,000 dilution), PCB95 (2 fM-2 μM), or PCB66 (2,3,4´,4tetrachlorobiphenyl; 200 nM) diluted from 1,000× stocks. Hippo campal slices from PD5 SpragueDawley rats were cultured for 3 days as described (Lein et al. 2011). To visualize dendritic arbors, slice cultures were biolistically transfected with plasmid encoding tomato fluorescent protein (TFP), which fills the entire cell, using a Helios Gene Gun (BioRad, Hercules, CA), according to the manufacturer's protocol. DNA amounts, transfection reagent amounts, and transfec tion duration were optimized to minimize toxicity and maximize transfection efficiency. Following transfection, slices were allowed to recover for 24 hr before exposure to PCB95 for 48 hr.
Dendritic analyses. Dendritic arbors of pyramidal neurons in the CA1 hippo campus of weanling rats were Golgi stained and quan tified by Sholl analysis (Lein et al. 2007). Sholl data were evaluated using the Wilcoxon ranksign test applying a conservative alpha level based on the number of measurements (Dawson and Trapp 2004). Soma size was ana lyzed using Image J version 1.44p with the Neuron J plugin version 1.42 to trace neurons (Meijering et al. 2004), and significant differ ences were determined using Student ttest with significance set at p < 0.05. Dendritic morphology in dissociated hippo campal cul tures or hippo campal slice cultures was quanti fied from digital images of green fluorescent protein-positive (GFP + ) or TFP + neurons, respectively, using NeuronJ (Meijering et al. 2004). Dendritic length and number of den dritic termini per neuron were analyzed by oneway analysis of variation with significance set at p < 0.05. Differences between treatment groups were identified by post hoc Tukey's test. All morpho metric experi ments were repli cated in cultures derived from at least three independent dissections.

PCBs enhance dendritic growth in the developing hippo campus.
A1254 is a commercial PCB mixture that includes many of the congeners associated with human exposures (Kodavanti et al. 2001;Kostyniak et al. 2005). We have previously showed that exposure to A1254 at 1 and 6 mg/kg/day in the maternal diet through out gestation and lactation resulted in PCB levels in the brains of weanling rats compa rable to those reported in human brain tissue and also promoted dendritic growth in cere bellar Purkinje cells and neocortical pyramidal neurons (Yang et al. 2009). Using this dosing regimen, we examined the effects of develop mental A1254 exposure on hippo campal pyramidal neurons at PD31. Representative camera lucida drawings of the basilar dendritic arbor of Golgiimpregnated CA1 pyramidal neurons demonstrate that A1254 significantly increases dendritic complexity ( Figure 1A). Sholl analysis indicates that dendritic arboriza tion was significantly increased relative to vehicle controls by 25% and 29% in the 1 and 6 mg/kg/day A1254 treatment groups, respectively ( Figure 1B).
To determine the potential contribution of RyRactive NDL PCBs to the dendrite promoting activity of A1254, we examined dendritic arborization in pyramidal neurons of weanling rats exposed throughout gestation and lactation to PCB95 in the maternal diet at 0.1, 1.0, or 6.0 mg/kg/day. PCB95 is the most potent RyR activator yet identified among the NDL congeners (Pessah et al. 2006). Sholl analyses of Golgistained pyramidal neurons in PD38 weanlings indicated that develop mental PCB95 exposure increased dendritic growth by 20% and 21% in the 0.1 and 1.0 mg/kg/day PCB95 treatment groups, respectively, but that dendritic growth in the 6 mg/kg/day PCB95 treatment group did not differ significantly from vehicle controls ( Figure 1C). Consistent with previous studies (Lein et al. 2007), soma dendritic arbor of Golgi-stained CA1 pyramidal neurons from PD31 weanling rats exposed throughout gestation and lactation to vehicle or A1254 in the maternal diet (n = 45 neurons per group). (C) Sholl analyses of the basilar dendritic arbor of Golgi-stained pyramidal neurons from PD38 weanling rats exposed throughout gestation and lactation to vehicle or PCB-95 in the maternal diet (n = 60 neurons per group). *p < 0.05 relative to vehicle control.  size of pyramidal neurons was not affected by develop mental exposure to either A1254 or PCB95 (data not shown).

RyR expression in cultured hippo campal neurons.
Mechanistic studies of RyR involve ment in the dendrite promoting activity of PCB95 were studied with primary cultured hippo campal neurons. All three RyR isoforms are expressed in the brain although their rela tive expression varies as a function of develop mental age and brain region (Berridge 2006); therefore, we first determined patterns of RyR expression in cultured hippo campal neurons by Western blotting. Monoclonal antibody (mAb) 34C or C333 at concentrations that selectively bind to RyR1 and RyR3 (Airey et al. 1990) or to RyR2 (Lai et al. 1992), respec tively labeled 565 and 557 kDa bands, in 7 and 12DIV lysates, whereas only RyR1 was detectable at 2 DIV (Figure 2A). A band with a lower molecular mass corresponding to RyR3 (545 kDa) was not detected by mAb 34C from cultures at any time point examined. This is consistent with reports that RyR3 accounts for about 2% of the total RyR protein in the brain (Kim et al. 2007;Murayama and Ogawa 1996) and in hippo campal cultures not stimu lated with BDNF (brain derived neurotrophic factor) (Adasme et al. 2011). These data verify that both RyR1 and RyR2 were expressed in hippo campal neurons during the period of most robust dendritic growth in these cultures (Wayman et al. 2006).
Immunocytochemical localization of RyRs in dissociated hippo campal neuronal cell cul tures colabeled with the Factin label phal loidin to identify actinrich dendritic spines confirmed RyR immunoreactive puncta throughout the dendritic shafts of cultured hippo campal neurons ( Figure 2B).

PCB-induced dendritic growth is mediated by RyR-dependent mechanisms.
To further test the validity of our in vitro model system, we determined whether the dendrite promoting activity of PCB95 is recapitulated in cultured hippo campal neurons. Dendritic arbors of individual neurons in highdensity neuron glia cocultures were visualized by expressing a MAP2BEGFP construct under the control of the CAG promoter, which exhibits neuron specific expression (Wayman et al. 2006). Expression of MAP2BEGFP is restricted to the somato dendritic compartment in cultured hippo campal neurons and does not alter their intrinsic dendritic growth patterns (Wayman et al. 2006). Exposure to PCB95 between 7-9 DIV significantly enhanced dendritic growth of hippo campal neurons in these cul tures as observed at 9 DIV ( Figure 3A). Initial concentration rangefinding studies indicated increased dendritic length and branching at concentrations as low as 2 pM; however, these effects were no longer evident at concentra tions ≥ 2 μM ( Figure 3B). Experi ments to refine the upper range of the concentrationeffect curve indicates dendritic responses of comparable magnitude at concentrations of 2-200 nM; at concentrations of > 200 nM, PCB95 no longer elicited increased dendritic growth (data not shown). Subsequent mech anistic studies used the maximally effective concentration of 200 nM PCB95.
If PCBs (200 nM) enhance dendritic growth by potentiating RyR activation, then congeners with differential effects on RyR acti vation should differentially influence dendritic growth. To test this, we compared dendritic growth in hippo campal cultures exposed to PCB95 vs. PCB66, which exhibit potent vs. negligible effects on RyR activity, respectively (Pessah et al. 2006). Exposure to PCB95 (200 nM) during 7-9 DIV significantly increased total dendritic length and branching as observed at 9 DIV ( Figure 4A, B). In con trast, dendritic length and branching in hippo campal neurons exposed to PCB66 (200 nM) for the same period of time did not differ from that observed in vehicle controls ( Figure 4A,B). Moreover, block of RyR channels by FLA365 (4(2aminopropyl) 3,5dichloroN,N dimethylaniline) (Chiesi et al. 1988;Mack et al. 1992) suppressed the dendrite promoting activity of PCB95 but had no effect on den dritic length or branching in control cultures not exposed to PCB95 ( Figure 4A,B).
The physiological relevance of these obser vations was confirmed using hippo campal slice cultures from PD5 rats biolistically labeled with TFP at 3 DIV and then exposed to PCBs from 5-7 DIV. PCB95, but not PCB66, significantly increased the number of dendritic termini per neuron in TFP + neurons in a con centrationdependent manner, and this effect was blocked by FLA365 ( Figure 4C).
PCB95 sensitizes both RyR1 and RyR2 (Wong and Pessah 1996). Therefore, to Densitometric data are presented as the pixel intensity of the RyR-immunoreactive band normalized to the pixel intensity of the α-tubulin immunoreactive band in the same sample. Expression of RyR1 and RyR2 at 7 and 12 DIV was verified in cultures derived from two separate dissections. "Brain" indicates membranes prepared from adult (2-to 4-month-old) mouse hippocampi. (B) Fluorescent photo micrograph illustrating subcellular localization of RyR immunoreactivity (red) and phalloidin reactivity (green) in a 21-DIV mouse hippocampal neuron. Bar = 5 μM.   Figure S1). Expression in hippo campal neurons of siRNA against either RyR1 or RyR2 suppressed PCB95 stimulation of dendritic length and branching but had no effect on basal dendritic growth ( Figure 5).
In contrast, expression of control siRNA had no effect on either PCB95-induced or basal dendritic growth ( Figure 5).

Discussion
We previously reported that develop mental A1254 exposure promotes dendritic growth in cerebellar Purkinje cells and cortical pyramidal neurons of weanling rats and that nanomolar concentrations of PCB95 promote dendritic growth in cultured neocortical neurons (Yang et al. 2009). Here, we extend these observa tions to show that a) develop mental expo sure to the NDL PCB95 in the maternal diet phenocopies the effect of develop mental A1254 exposure on dendritic arborization in the developing hippo campus, b) PCB95 promotes dendritic growth in cultured hippo campal neurons at picomolar to nanomolar concentrations, and c) the dendrite promoting activity of PCB95 requires RyR activity. As a first test of the cause-effect relation ship between PCBenhanced RyR activity and PCBinduced dendritic growth, we compared dendritic growth in cultured hippo campal neurons exposed to PCB95 vs. PCB66. PCB95 is a triortho substituted congener that potently sensitizes the RyR channel to physiological and pharmacological activators (Wong and Pessah 1996) and preferentially stabilizes the RyR in its full open conforma tion (Samso et al. 2009). PCB66 has a single orthochlorine substitution, and despite similar physicochemical properties to PCB95, has negligible influence on RyR activity (Pessah et al. 2006). In cultured hippo campal neurons, PCB95 promotes dendritic growth, whereas PCB66 has no effect at the concentrations tested. As a second test, blocking RyR func tion by either pharmacological antagonism or siRNA knockdown prevented PCB95induced dendritic growth. Interestingly, RyR1 siRNA and RyR2 siRNA were equally effective in blocking the dendrite promoting activity of PCB95. Studies in HEK cells confirmed that each siRNA was specific for its target mRNA and did not crossreact with non target RyR mRNA. The native activities of both RyR iso forms are therefore required for the dendrite promoting activity of PCB95, and interference with either isoform is sufficient to prevent the influence of RyRactive PCBs, without altering basal dendritic growth or cell viabil ity. The biology underlying the dual require ment for RyR1 and RyR2 in PCBinduced dendritic growth remains to be determined. We see three possible explanations.
One possibility is that each isoform regu lates a complementary but distinct profile of downstream effectors necessary for dendritic growth. For example, both transcription and translationdependent pathways mediate (C) PCB-95, but not PCB-66, significantly enhanced dendritic arborization of TFP + neurons in hippocampal slice cultures, and this effect was blocked by FLA365. Data presented as mean ± SE (n = 35-50 neurons from three to four independent cultures per group).
A second possibility is that activation of spatially segregated RyR1 and RyR2 chan nels creates Ca 2+ microdomains within the soma and the dendritic processes and ter minals (Berridge 2006) whose coincident activation is necessary for enhancing activity dependent dendritic growth. As described in the companion paper (Wayman et al. 2012), PCB95 enhances spontaneous Ca 2+ oscilla tions within the soma and distal dendrites of the same neuron, and these effects are blocked by ryanodine. Collectively, these data indi cate that NDL PCBs mediate the gain of RyR function that promotes dendritic growth, and they suggest a role for RyR in normal activity dependent dendritic growth.
The human health relevance of these in vitro mechanistic studies is supported by our observations that develop mental PCB expo sure similarly promotes dendritic growth in the developing brains of weanling rats. Using an exposure paradigm relevant to human PCB exposures in terms of route of exposure, dose level, and dose duration (Yang et al. 2009), we observed that A1254 increases dendritic growth in pyramidal neurons of the CA1 hippo campus. A1254 is composed predom inantly of NDL PCB congeners with RyR activity (Kostyniak et al. 2005), consistent with our in vitro data establishing that PCBs promote dendritic growth via RyRdependent mechanisms. The proposal that NDL PCBs contribute to the dendrite promoting activ ity of A1254 is strengthened by the observa tion that develop mental exposure to PCB95 in the maternal diet similarly stimulates den dritic growth in vivo. Interestingly, over the dose ranges tested in this study, PCB95, but not A1254, exhibited an inverted doseresponse relationship. Although we cannot rule out the possibility that nonlinearity would also be observed with A1254 at doses > 6 mg/kg/day, the different dose-response relationships observed for A1254 vs. PCB95 may reflect the fact that A1254 is a mixture of DL and NDL PCBs with varying RyR potency (Kostyniak et al. 2005). Alternatively, upregulation of cytochrome P450 enzymes by DL PCBs in A1254 could result in different toxicokinetics of NDL PCBs in A1254 vs. PCB95-exposed animals (Gauger et al. 2007;Giera et al. 2011).
The nonlinear dose response of develop mental PCB95 exposure on dendritic growth in vivo is recapitulated in vitro. The loss of dendritepromoting activity in vitro at PCB95 concentrations > 2 μM is likely not due to decreased cell viability (Howard et al. 2003). A plausible explanation is that chronic RyR sensitization alters RyR expression and/or activity, in an inverted concentrationrelated manner, which is perhaps due to local pro duction of reactive oxygen species (ROS) by PCBs (Fonnum et al. 2006). Microsomal RyR complexes possess a small number of highly reactive cysteines that confer tight regulation of Ca 2+ channel activity in response to chang ing transmembrane redox potential (Feng et al. 2000;Liu et al. 1994). Thus the sensitiz ing and desensitizing influence of RyRactive PCBs on Ca 2+ signaling may be strongly influ enced by the level of ROS production (Pessah et al. 2002). A parallel mechanism has been demonstrated in which moderate increases in Ca 2+ promote dendritic growth, whereas large increases cause dendritic retraction (Lohmann and Wong 2005;Segal et al. 2000). Thus, when increasing the concentration of PCB95 from 200 nM to 2 μM, intracellular Ca 2+ con centrations may cross the threshold from con centrations that promote dendritic growth to those that cause dendritic retraction.
A third possibility is suggested by the report that transfection of cortical neurons with constitutively active CaMKIV (Ca 2+ / calmodulindependent protein kinase IV) promotes dendritic growth, whereas expres sion of constitutively active CaMKII (Ca 2+ / calmodulin dependent protein kinase II) inhibits dendritic growth (Redmond et al. 2002). Perhaps at higher concentrations that do not promote dendritic growth, PCBs are preferentially activating Ca 2+ dependent sig naling molecules that inhibit dendritic growth. Distinguishing between these possibilities is the focus of future studies.
These data linking a direct molecular effect of NDL PCBs (sensitized RyR activity) to dis ruption of a specific neuro developmental event (dendritic arborization) have significant impli cations for understanding how PCBs interfere with normal neurodevelopment in the human brain. Structural aberrations in the dendritic arbors of central neurons are thought to con tribute to clinical manifestations of diverse heritable and environmentally induced neuro developmental disorders in humans (Bourgeron 2009;Fukuda et al. 2005;Garey 2010;Penzes et al. 2011;Svitkina et al. 2010). Consistent with this, develop mental A1254 exposure altered dendritic arborization in weanling rats coincident with performance deficits in the Morris swim task (Yang et al. 2009) and exposure to PCB95 on gestation days 10-16 altered behavior in adult rats (Schantz et al. 1997). NDL PCB congeners with the highest activity towards RyRs, including PCB95, col lectively represent 40-50% of the total PCBs currently found in environmental and biotic samples, and their net effects are likely to be additive (Pessah et al. 2006). However, even low levels of PCB exposure might adversely influence neuronal connectivity in the develop ing brain of genetically susceptible individu als. Mutations in RYR (ryanodine receptor) genes have been linked to environmentally triggered disorders in humans including malig nant hyperthermia , cardiac Figure 5. RyR-specific siRNA blocks PCB-induced dendritic growth in vitro. (A) Representative photomicrographs of 9-DIV hippocampal neurons transfected with MAP2B-EGFP alone or in combination with RyR siRNA before 48-hr treatment with vehicle or PCB-95 (200 nM). Expression of RyR1-or RyR2-specific siRNA, but not scrambled siRNA, blocked PCB-95 effects on dendritic length (B) and branching (C). Data presented as mean ± SE (n = 30 neurons from three independent cultures per group). **p < 0.01 relative to vehicle control. ‡ p < 0.001 relative to untransfected (control) PCB-95-treated hippocampal neurons.  (Wehrens et al. 2005), and sudden death (Laitinen et al. 2004). Recent studies demonstrate that specific RYR mutations con fer sex-and gene-dosedependent susceptibil ity to pharmacological (halogenated anesthetic) and environmental (heat) stressors that trigger malignant hyperthermia and muscle damage in otherwise asymptomatic individuals (Barrientos et al. 2012;Yuen et al. 2012). Importantly, PCB95 is significantly more potent and effica cious in disrupting cation regulation of mutant R615CRYR1 compared with wild type RyR1 (Ta and Pessah 2007). Considered together, these observations identify PCBs, and in par ticular NDL PCBs with high RyR activity, as candidate environmental risk factors in neuro developmental disorders and provide important new clues about the possible role of RyRs in contributing to heritable and environmentally triggered neuro developmental deficits.