Although banned from production for decades, PCBs remain a significant risk to human health. A primary target of concern is the developing brain. Epidemiological studies link PCB exposures in utero or during infancy to increased risk of neuropsychiatric deficits in children. Nonclinical studies of legacy congeners found in PCB mixtures synthesized prior to the ban on PCB production suggest that non-dioxin-like (NDL) congeners are predominantly responsible for the developmental neurotoxicity associated with PCB exposures. Mechanistic studies suggest that NDL PCBs alter neurodevelopment via ryanodine receptor-dependent effects on dendritic arborization. Lightly chlorinated congeners, which were not present in the industrial mixtures synthesized prior to the ban on PCB production, have emerged as contemporary environmental contaminants, but there is a paucity of data regarding their potential developmental neurotoxicity. PCB 11, a prevalent contemporary congener, is found in the serum of children and their mothers, as well as in the serum of pregnant women at increased risk for having a child diagnosed with a neurodevelopmental disorder (NDD). Recent data demonstrates that PCB 11 modulates neuronal morphogenesis via mechanisms that are convergent with and divergent from those implicated in the developmental neurotoxicity of legacy NDL PCBs. This review summarizes these data and discusses their relevance to adverse neurodevelopmental outcomes in humans.

Although banned from production for decades, PCBs remain a significant risk to human health. A primary target of concern is the developing brain. Epidemiological studies link PCB exposures in utero or during infancy to increased risk of neuropsychiatric deficits in children. Nonclinical studies of legacy congeners found in PCB mixtures synthesized prior to the ban on PCB production suggest that non-dioxin-like (NDL) congeners are predominantly responsible for the developmental neurotoxicity associated with PCB exposures. Mechanistic studies suggest that NDL PCBs alter neurodevelopment via ryanodine receptor-dependent effects on dendritic arborization. Lightly chlorinated congeners, which were not present in the industrial mixtures synthesized prior to the ban on PCB production, have emerged as contemporary environmental contaminants, but there is a paucity of data regarding their potential developmental neurotoxicity. PCB 11, a prevalent contemporary congener, is found in the serum of children and their mothers, as well as in the serum of pregnant women at increased risk for having a child diagnosed with a neurodevelopmental disorder (NDD). Recent data demonstrates that PCB 11 modulates neuronal morphogenesis via mechanisms that are convergent with and divergent from those implicated in the developmental neurotoxicity of legacy NDL PCBs. This review summarizes these data and discusses their relevance to adverse neurodevelopmental outcomes in humans.

PCB 11 (3,3'-dichlorobiphenyl), a contemporary congener produced as a byproduct of current pigment production processes, has recently emerged as a prevalent worldwide pollutant. We recently demonstrated that exposure to PCB 11 increases dendritic arborization in vitro, but the mechanism(s) mediating this effect remain unknown. To address this data gap, primary cortical neuron-glia co-cultures derived from neonatal Sprague-Dawley rats were exposed for 48 h to either vehicle (0.1% DMSO) or PCB 11 at concentrations ranging from 1 fM to 1 nM in the absence or presence of pharmacologic antagonists of established molecular targets of higher chlorinated PCBs. Reporter cell lines were used to test activity of PCB 11 at the aryl hydrocarbon receptor (AhR) and thyroid hormone receptor (THR). PCB 11 lacked activity at the AhR and THR, and antagonism of these receptors had no effect on the dendrite-promoting activity of PCB 11. Pharmacologic antagonism of various calcium channels or treatment with antioxidants also did not alter PCB 11-induced dendritic arborization. In contrast, pharmacologic blockade or shRNA knockdown of cAMP response element-binding protein (CREB) significantly decreased dendritic growth in PCB 11-exposed cultures, suggesting PCB 11 promotes dendritic growth via CREB-mediated mechanisms. Since CREB signaling is crucial for normal neurodevelopment, and perturbations of CREB signaling have been associated with neurodevelopmental disorders, our findings suggest that this contemporary pollutant poses a threat to the developing brain, particularly in individuals with heritable mutations that promote CREB signaling.

PCB 11 is an emerging global pollutant that we recently showed promotes axonal and dendritic growth in primary rat neuronal cell cultures. Here, we address the influence of sex and species on neuronal responses to PCB 11. Neuronal morphology was quantified in sex-specific primary hippocampal and cortical neuron-glia co-cultures derived from neonatal C57BL/6J mice and Sprague Dawley rats exposed for 48 h to vehicle (0.1% DMSO) or PCB 11 at concentrations ranging from 1 fM to 1 nM. Total axonal length was quantified in tau-1 immunoreactive neurons at day in vitro (DIV) 2; dendritic arborization was assessed by Sholl analysis at DIV 9 in neurons transfected with MAP2B-FusRed. In mouse cultures, PCB 11 enhanced dendritic arborization in female, but not male, hippocampal neurons and male, but not female, cortical neurons. In rat cultures, PCB 11 promoted dendritic arborization in male and female hippocampal and cortical neurons. PCB 11 also increased axonal growth in mouse and rat neurons of both sexes and neuronal cell types. These data demonstrate that PCB 11 exerts sex-specific effects on neuronal morphogenesis that vary depending on species, neurite type, and neuronal cell type. These findings have significant implications for risk assessment of this emerging developmental neurotoxicant.

Early life exposures to environmental contaminants are implicated in the pathogenesis of many neurodevelopmental disorders (NDDs). These disorders often display sex biases, but whether environmental neurotoxicants act in a sex-dependent manner to modify neurodevelopment is largely unknown. Since altered dendritic morphology is associated with many NDDs, we tested the hypothesis that male and female primary mouse neurons are differentially susceptible to the dendrite-promoting activity of 2,2',3,5',6-pentachlorobiphenyl (PCB 95). Hippocampal and cortical neuron-glia co-cultures were exposed to vehicle (0.1% dimethylsulfoxide) or PCB 95 (100 fM-1 μM) from day in vitro 7-9. As determined by Sholl analysis, PCB 95-enhanced dendritic growth in female but not male hippocampal and cortical neurons. In contrast, both male and female neurons responded to bicuculline with increased dendritic complexity. Detailed morphometric analyses confirmed that PCB 95 effects on the number and length of primary and nonprimary dendrites varied depending on sex, brain region and PCB concentration, and that female neurons responded more consistently with increased dendritic growth and at lower concentrations of PCB 95 than their male counterparts. Exposure to PCB 95 did not alter cell viability or the ratio of neurons to glia in cultures of either sex. These results demonstrate that cultured female mouse hippocampal and cortical neurons are more sensitive than male neurons to the dendrite-promoting activity of PCB 95, and suggest that mechanisms underlying PCB 95-induced dendritic growth are sex-dependent. These data highlight the importance of sex in neuronal responses to environmental neurotoxicants.

Background

Primary neuronal cell cultures are useful for studying mechanisms that influence dendritic morphology during normal development and in response to various stressors. However, analyzing dendritic morphology is challenging, particularly in cultures with high cell density, and manual methods of selecting neurons and tracing dendritic arbors can introduce significant bias, and are labor-intensive. To overcome these challenges, semi-automated and automated methods are being developed, with most software solutions requiring computer-assisted dendrite tracing with subsequent quantification of various parameters of dendritic morphology, such as Sholl analysis. However fully automated approaches for classic Sholl analysis of dendritic complexity are not currently available.

New method

The previously described Omnisphero software, was extended by adding new functions to automatically assess dendritic mass, total length of the dendritic arbor and the number of primary dendrites, branch points, and terminal tips, and to perform Sholl analysis.

Results

The new functions for assessing dendritic morphology were validated using primary mouse hippocampal and rat cortical neurons transfected with a fluorescently tagged MAP2 cDNA construct. These functions allow users to select specific populations of neurons as a training set for subsequent automated selection of labeled neurons in high-density cultures.

Comparison with existing semi-automated methods

Compared to manual or semi-automated analyses of dendritic arborization, the new functions increase throughput while significantly decreasing researcher bias associated with neuron selection, tracing, and thresholding.

Conclusion

These results demonstrate the importance of using unbiased automated methods to mitigate experimenter-dependent bias in analyzing dendritic morphology.

Altered dendritic morphology is common in neurodevelopmental disorders (NDDs), many of which show sex biases in prevalence, onset and/or severity. However, whether dendritic morphology varies as a function of sex in juvenile mice or primary neuronal cell cultures is largely unknown even though both are widely used models for studying NDDs. To address this gap, we quantified dendritic morphology in CA1 pyramidal hippocampal and adjacent somatosensory pyramidal cortical neurons from male and female postnatal day (P)28 C57BL/6J mice. As determined by Sholl analysis of Golgi-stained brain sections, dendritic arbors of male hippocampal neurons are more complex than females. Conversely, dendritic morphology of female cortical neurons is more complex than males. In primary neuron-glia co-cultures from P0 mouse hippocampi, male neurons have more complex dendritic arbors than female neurons. Sex differences are less pronounced in cortical cultures. In vitro sex differences in dendritic morphology are driven in part by estrogen-dependent mechanisms, as evidenced by decreased dendritic complexity in male hippocampal neurons cultured in phenol red-free media or in the presence of an estrogen receptor antagonist. Evidence that sex influences dendritic morphogenesis in two models of neurodevelopment in a region-specific manner has significant mechanistic implications regarding sex biases in NDDs.

Background

Dichlorodiphenyltrichloroethane (DDT) was used extensively to control malaria, typhus, body lice, and bubonic plague worldwide, until countries began restricting its use in the 1970s. However, the use of DDT to control vector-borne diseases continues in developing countries. Prenatal DDT exposure is associated with elevated blood pressure in humans.

Objective

We hypothesized that perinatal DDT exposure causes hypertension in adult mice.

Methods

DDT was administered to C57BL/6J dams from gestational day 11.5 to postnatal day 5. Blood pressure (BP) and myocardial wall thickness were measured in male and female adult offspring. Adult mice were treated with an angiotensin converting enzyme (ACE) inhibitor, captopril, to evaluate sensitivity to amelioration of DDT-associated hypertension by ACE inhibition. We further assessed the influence of DDT exposure on the expression of mRNAs that regulate BP through renal ion transport.

Results

Adult mice perinatally exposed to DDT exhibited chronically increased systolic BP, increased myocardial wall thickness, and elevated expression of mRNAs of several renal ion transporters. Captopril completely reversed hypertension in mice perinatally exposed to DDT.

Conclusions

These data demonstrate that perinatal exposure to DDT causes hypertension and cardiac hypertrophy in adult offspring. A key mechanism underpinning this hypertension is an overactivated renin angiotensin system because ACE inhibition reverses the hypertension induced by perinatal DDT exposure. Citation: La Merrill M, Sethi S, Benard L, Moshier E, Haraldsson B, Buettner C. 2016. Perinatal DDT exposure induces hypertension and cardiac hypertrophy in adult mice. Environ Health Perspect 124:1722-1727; http://dx.doi.org/10.1289/EHP164.

Background

Primary neuronal cell cultures are useful for studying mechanisms that influence dendritic morphology during normal development and in response to various stressors. However, analyzing dendritic morphology is challenging, particularly in cultures with high cell density, and manual methods of selecting neurons and tracing dendritic arbors can introduce significant bias, and are labor-intensive. To overcome these challenges, semi-automated and automated methods are being developed, with most software solutions requiring computer-assisted dendrite tracing with subsequent quantification of various parameters of dendritic morphology, such as Sholl analysis. However fully automated approaches for classic Sholl analysis of dendritic complexity are not currently available.

New method

The previously described Omnisphero software, was extended by adding new functions to automatically assess dendritic mass, total length of the dendritic arbor and the number of primary dendrites, branch points, and terminal tips, and to perform Sholl analysis.

Results

The new functions for assessing dendritic morphology were validated using primary mouse hippocampal and rat cortical neurons transfected with a fluorescently tagged MAP2 cDNA construct. These functions allow users to select specific populations of neurons as a training set for subsequent automated selection of labeled neurons in high-density cultures.

Comparison with existing semi-automated methods

Compared to manual or semi-automated analyses of dendritic arborization, the new functions increase throughput while significantly decreasing researcher bias associated with neuron selection, tracing, and thresholding.

Conclusion

These results demonstrate the importance of using unbiased automated methods to mitigate experimenter-dependent bias in analyzing dendritic morphology.

Background

The Sholl technique is widely used to quantify dendritic morphology. Data from such studies, which typically sample multiple neurons per animal, are often analyzed using simple linear models. However, simple linear models fail to account for intra-class correlation that occurs with clustered data, which can lead to faulty inferences.

New method

Mixed effects models account for intra-class correlation that occurs with clustered data; thus, these models more accurately estimate the standard deviation of the parameter estimate, which produces more accurate p-values. While mixed models are not new, their use in neuroscience has lagged behind their use in other disciplines.

Results

A review of the published literature illustrates common mistakes in analyses of Sholl data. Analysis of Sholl data collected from Golgi-stained pyramidal neurons in the hippocampus of male and female mice using both simple linear and mixed effects models demonstrates that the p-values and standard deviations obtained using the simple linear models are biased downwards and lead to erroneous rejection of the null hypothesis in some analyses.

Comparison with existing methods

The mixed effects approach more accurately models the true variability in the data set, which leads to correct inference.

Conclusions

Mixed effects models avoid faulty inference in Sholl analysis of data sampled from multiple neurons per animal by accounting for intra-class correlation. Given the widespread practice in neuroscience of obtaining multiple measurements per subject, there is a critical need to apply mixed effects models more widely.