Douglas Weston

Lifetime Consequences of Combined Maternal Lead and Stress

Elevated lead (Pb) exposure and high stress both target low socio-economic status populations. Both also act on the hypothalamic-pituitary-adrenal (HPA) axis. Pb disrupts cognition through effects on the mesocorticolimbic dopamine pathway. Stress hormones act on this same pathway via the HPA axis. The fact that Pb and stress are likely interactive risk factors served as the rationale for a series of studies in our laboratory. These demonstrate that stress can modify Pb effects, that Pb can modify stress responsivity, and, notably, that Pb + stress effects can occur in the absence of an effect of either alone in rats. Furthermore, maternal only Pb exposure can permanently alter basal corticosterone levels, stress responsivity (i.e. permanent modification of HPA axis function) and brain catecholamines in offspring of both genders. Interactive effects of Pb + stress are not limited to early development: even Pb exposures initiated post-weaning alter basal corticosterone and stress responsivity. Outcomes differ in relation to gender, brain region, stressor and time of measurement, making Pb + stress interactions complex. Altered HPA axis function may serve as a mechanism for the behavioural and catecholaminergic neurotoxicity associated with Pb, as well as for the increased incidence of disease and dysfunctions associated with low socio-economic status. The permanent consequences of maternal only Pb exposure suggest that Pb screening programmes should include pregnant women at risk for elevated Pb exposure, and that stress should be considered as an additional risk factor. Pb + stress effects observed in the absence of either risk factor alone raise questions about the capacity of current hazard identification approaches to adequately identify human health risks posed by neurotoxicants.

Early postnatal exposure to ultrafine particulate matter air pollution: persistent ventriculomegaly, neurochemical disruption, and glial activation preferentially in male mice.

Background: Air pollution has been associated with adverse neurological and behavioral health effects in children and adults. Recent studies link air pollutant exposure to adverse neurodevelopmental outcomes, including increased risk for autism, cognitive decline, ischemic stroke, schizophrenia, and depression. Objectives: We sought to investigate the mechanism(s) by which exposure to ultrafine concentrated ambient particles (CAPs) adversely influences central nervous system (CNS) development. Methods: We exposed C57BL6/J mice to ultrafine (< 100 nm) CAPs using the Harvard University Concentrated Ambient Particle System or to filtered air on postnatal days (PNDs) 4–7 and 10–13, and the animals were euthanized either 24 hr or 40 days after cessation of exposure. Another group of males was exposed at PND270, and lateral ventricle area, glial activation, CNS cytokines, and monoamine and amino acid neurotransmitters were quantified. Results: We observed ventriculomegaly (i.e., lateral ventricle dilation) preferentially in male mice exposed to CAPs, and it persisted through young adulthood. In addition, CAPs-exposed males generally showed decreases in developmentally important CNS cytokines, whereas in CAPs-exposed females, we observed a neuroinflammatory response as indicated by increases in CNS cytokines. We also saw changes in CNS neurotransmitters and glial activation across multiple brain regions in a sex-dependent manner and increased hippocampal glutamate in CAPs-exposed males. Conclusions: We observed brain region– and sex-dependent alterations in cytokines and neurotransmitters in both male and female CAPs-exposed mice. Lateral ventricle dilation (i.e., ventriculomegaly) was observed only in CAPs-exposed male mice. Ventriculomegaly is a neuropathology that has been associated with poor neurodevelopmental outcome, autism, and schizophrenia. Our findings suggest alteration of developmentally important neurochemicals and lateral ventricle dilation may be mechanistically related to observations linking ambient air pollutant exposure and adverse neurological/neurodevelopmental outcomes in humans. Citation: Allen JL, Liu X, Pelkowski S, Palmer B, Conrad K, Oberdörster G, Weston D, Mayer-Pröschel M, Cory-Slechta DA. 2014. Early postnatal exposure to ultrafine particulate matter air pollution: persistent ventriculomegaly, neurochemical disruption, and glial activation preferentially in male mice. Environ Health Perspect 122:939–945; http://dx.doi.org/10.1289/ehp.1307984

Alterations in glucocorticoid negative feedback following maternal Pb, prenatal stress and the combination: A potential biological unifying mechanism for their corresponding disease profiles

Combined exposures to maternal lead (Pb) and prenatal stress (PS) can act synergistically to enhance behavioral and neurochemical toxicity in offspring. Maternal Pb itself causes permanent dysfunction of the bodys major stress system, the hypothalamic pituitary adrenal (HPA) axis. The current study sought to determine the potential involvement of altered negative glucocorticoid feedback as a mechanistic basis of the effects in rats of maternal Pb (0, 50 or 150 ppm in drinking water beginning 2 mo prior to breeding), prenatal stress (PS; restraint on gestational days 16-17) and combined maternal Pb+PS in 8 mo old male and female offspring. Corticosterone changes were measured over 24 h following an i.p. injection stress containing vehicle or 100 or 300 microg/kg (females) or 100 or 150 microg/kg (males) dexamethasone (DEX). Both Pb and PS prolonged the time course of corticosterone reduction following vehicle injection stress. Pb effects were non-monotonic, with a greater impact at 50 vs. 150 ppm, particularly in males, where further enhancement occurred with PS. In accord with these findings, the efficacy of DEX in suppressing corticosterone was reduced by Pb and Pb+PS in both genders, with Pb efficacy enhanced by PS in females, over the first 6 h post-administration. A marked prolongation of DEX effects was found in males. Thus, Pb, PS and Pb+PS, sometimes additively, produced hypercortisolism in both genders, followed by hypocortisolism in males, consistent with HPA axis dysfunction. These findings may provide a plausible unifying biological mechanism for the reported links between Pb exposure and stress-associated diseases and disorders mediated via the HPA axis, including obesity, hypertension, diabetes, anxiety, schizophrenia and depression. They also suggest broadening of Pb screening programs to pregnant women in high stress environments.

Influence of low level maternal Pb exposure and prenatal stress on offspring stress challenge responsivity

We previously demonstrated potentiated effects of maternal Pb exposure producing blood Pb(PbB) levels averaging 39microg/dl combined with prenatal restraint stress (PS) on stress challenge responsivity of female offspring as adults. The present study sought to determine if: (1) such interactions occurred at lower PbBs, (2) exhibited gender specificity, and (3) corticosterone and neurochemical changes contributed to behavioral outcomes. Rat dams were exposed to 0, 50 or 150ppm Pb acetate drinking water solutions from 2 mos prior to breeding through lactation (pup exposure ended at weaning; mean PbBs of dams at weaning were <1, 11 and 31microg/dl, respectively); a subset in each Pb group underwent prenatal restraint stress (PS) on gestational days 16-17. The effects of variable intermittent stress challenge (restraint, cold, novelty) on Fixed Interval (FI) schedule controlled behavior and corticosterone were examined in offspring when they were adults. Corticosterone changes were also measured in non-behaviorally tested (NFI) littermates. PS alone was associated with FI rate suppression in females and FI rate enhancement in males; Pb exposure blunted these effects in both genders, particularly following restraint stress. PS alone produced modest corticosterone elevation following restraint stress in adult females, but robust enhancements in males following all challenges. Pb exposure blunted these corticosterone changes in females, but further enhanced levels in males. Pb-associated changes showed linear concentration dependence in females, but non-linearity in males, with stronger or selective changes at 50ppm. Statistically, FI performance was associated with corticosterone changes in females, but with frontal cortical dopaminergic and serotonergic changes in males. Corticosterone changes differed markedly in FI vs. NFI groups in both genders, demonstrating a critical role for behavioral history and raising caution about extrapolating biochemical markers across such conditions. These findings demonstrate that maternal Pb interacts with prenatal stress to further modify both behavioral and corticosterone responses to stress challenge, thereby suggesting that studies of Pb in isolation from other disease risk factors will not reveal the extent of its adverse effects. These findings also underscore the critical need to extend screening programs for elevated Pb exposure, now restricted to young children, to pregnant, at risk, women.

Interactions of Lifetime Lead Exposure and Stress: Behavioral, Neurochemical and HPA Axis Effects

Lead (Pb) and stress co-occur as risk factors, share biological substrates and produce common adverse effects. We previously found that prenatal restraint stress (PS) or offspring stress (OS) could enhance maternal Pb-induced behavioral, brain neurotransmitter level and HPA axis changes. The current study examined how lifetime Pb exposure, consistent with human environmental exposure, interacts with stress. Dams were exposed to Pb beginning 2 mos prior to breeding (0, 50 or 150ppm in drinking water), PS on gestational days 16 and 17, or the combination. Offspring continued on the same Pb exposure as the dam. A subset of Pb+PS offspring also received 3 additional stress challenges (OS), yielding 9 exposure groups/gender: 0-NS, 0-PS, 0-OS, 50-NS, 50-PS, 50-OS, 150-NS, 150-PS and 150-OS. As with maternal Pb (Virgolini et al., 2008a), lifetime Pb and stress influenced Fixed Interval (FI) behavior primarily in females. Relative to 0-NS control, reductions in postreinforcement pause (PRP) times were seen only with combined Pb+PS (50-PS, 50-OS, 150-PS). Stress increased FI response rates when Pb alone was without effect (150-PS, 150-OS), but gradually mitigated rate increases produced by Pb alone (50-PS, 50-OS), effects that appear to be due primarily to PS, as they were of comparable magnitude in PS and OS groups. Individual subject data suggest that enhanced Pb and PS effects reflect increasing numbers of subjects shifting to the high end of the normal range of FI performance values, consistent with a dose-response type of Pb+stress additivity. Consistent with reports of cortico-striatal mediation of both interval timing (PRP) and FI rates, principal component analyses suggested potential mediation via altered frontal cortex norepinephrine, reduced nucleus accumbens dopaminergic control and enhanced striatal monoamine control. Altered FI performance, whether occurring through changes in response rate, PRP, or both, represent behavioral inefficiency and potentially sub-optimal or even dysfunctional resource/energy use.

Developmental Exposure to Concentrated Ambient Ultrafine Particulate Matter Air Pollution in Mice Results in Persistent and Sex-Dependent Behavioral Neurotoxicity and Glial Activation

The brain appears to be a target of air pollution. This study aimed to further ascertain behavioral and neurobiological mechanisms of our previously observed preference for immediate reward (Allen, J. L., Conrad, K., Oberdorster, G., Johnston, C. J., Sleezer, B., and Cory-Slechta, D. A. (2013). Developmental exposure to concentrated ambient particles and preference for immediate reward in mice. Environ. Health Perspect. 121, 32-38), a phenotype consistent with impulsivity, in mice developmentally exposed to inhaled ultrafine particles. It examined the impact of postnatal and/or adult concentrated ambient ultrafine particles (CAPS) or filtered air on another behavior thought to reflect impulsivity, Fixed interval (FI) schedule-controlled performance, and extended the assessment to learning/memory (novel object recognition (NOR)), and locomotor activity to assist in understanding behavioral mechanisms of action. In addition, levels of brain monoamines and amino acids, and markers of glial presence and activation (GFAP, IBA-1) were assessed in mesocorticolimbic brain regions mediating these cognitive functions. This design produced four treatment groups/sex of postnatal/adult exposure: Air/Air, Air/CAPS, CAPS/Air, and CAPS/CAPS. FI performance was adversely influenced by CAPS/Air in males, but by Air/CAPS in females, effects that appeared to reflect corresponding changes in brain mesocorticolimbic dopamine/glutamate systems that mediate FI performance. Both sexes showed impaired short-term memory on the NOR. Mechanistically, cortical and hippocampal changes in amino acids raised the potential for excitotoxicity, and persistent glial activation was seen in frontal cortex and corpus callosum of both sexes. Collectively, neurodevelopment and/or adulthood CAPS can produce enduring and sex-dependent neurotoxicity. Although mechanisms of these effects remain to be fully elucidated, findings suggest that neurodevelopment and/or adulthood air pollution exposure may represent a significant underexplored risk factor for central nervous system diseases/disorders and thus a significant public health threat even beyond current appreciation.

Variations in the Nature of Behavioral Experience Can Differentially Alter the Consequences of Developmental Exposures to Lead, Prenatal Stress, and the Combination

Behavioral experience (BE) can critically influence later behavior and brain function, but the central nervous system (CNS) consequences of most developmental neurotoxicants are examined in the absence of any such context. We previously demonstrated marked differences in neurotransmitter changes produced by developmental lead (Pb) exposure ± prenatal stress (PS) depending upon whether or not rats had been given BE (Cory-Slechta, D. A., Virgolini, M. B., Rossi-George, A., Weston, D., and Thiruchelvam, M. (2009). The current study examined the hypothesis that the nature of the BE itself would be a critical determinant of outcome in mice that had been continually exposed to 0 or 100 ppm Pb acetate in drinking water alone or in combination with prenatal restraint stress. Half of the offspring in each of the four resulting groups/gender were exposed to positively reinforced (food-rewarded Fixed Interval schedule-controlled behavior) or negatively reinforced (inescapable forced swim) BE. Brain monoamines and amino acids differed significantly in relation to BE, even in control animals, as did the trajectory of effects of Pb ± PS, particularly in frontal cortex, hippocampus (both genders), and midbrain (males). In males, Pb ± PS-related changes in neurotransmitters correlated with behavioral performance. These findings suggest that CNS consequences of developmental toxicants studied in the absence of a broader spectrum of BEs may not necessarily be predictive of human outcomes. Evaluating the role of specific BEs as a modulator of neurodevelopmental insults offers the opportunity to determine what specific BEs may ameliorate the associated impacts and can assist in establishing underlying neurobiological mechanisms.

Sex-dependent impacts of low-level lead exposure and prenatal stress on impulsive choice behavior and associated biochemical and neurochemical manifestations

A prior study demonstrated increased overall response rates on a fixed interval (FI) schedule of reward in female offspring that had been subjected to maternal lead (Pb) exposure, prenatal stress (PS) and offspring stress challenge relative to control, prenatal stress alone, lead alone and lead+prenatal stress alone (Virgolini et al., 2008). Response rates on FI schedules have been shown to directly relate to measures of self-control (impulsivity) in children and in infants (Darcheville et al., 1992, 1993). The current study sought to determine whether enhanced effects of Pb±PS would therefore be seen in a more direct measure of impulsive choice behavior, i.e., a delay discounting paradigm. Offspring of dams exposed to 0 or 50ppm Pb acetate from 2 to 3 months prior to breeding through lactation, with or without immobilization restraint stress (PS) on gestational days 16 and 17, were trained on a delay discounting paradigm that offered a choice between a large reward (three 45mg food pellets) after a long delay or a small reward (one 45mg food pellet) after a short delay, with the long delay value increased from 0s to 30s across sessions. Alterations in extinction of this performance, and its subsequent re-acquisition after reinforcement delivery was reinstated were also examined. Brains of littermates of behaviorally-trained offspring were utilized to examine corresponding changes in monoamines and in levels of brain derived neurotrophic factor (BDNF), the serotonin transporter (SERT) and the N-methyl-d-aspartate receptor (NMDAR) 2A in brain regions associated with impulsive choice behavior. Results showed that Pb±PS-induced changes in delay discounting occurred almost exclusively in males. In addition to increasing percent long delay responding at the indifference point (i.e., reduced impulsive choice behavior), Pb±PS slowed acquisition of delayed discounting performance, and increased numbers of both failures to and latencies to initiate trials. Overall, the profile of these alterations were more consistent with impaired learning/behavioral flexibility and/or with enhanced sensitivity to the downshift in reward opportunities imposed by the transition from delay discounting training conditions to delay discounting choice response contingencies. Consistent with these behavioral changes, Pb±PS treated males also showed reductions in brain serotonin function in all mesocorticolimbic regions, broad monoamine changes in nucleus accumbens, and reductions in both BDNF and NMDAR 2A levels and increases in SERT in frontal cortex, i.e., in regions and neurotransmitter systems known to mediate learning/behavioral flexibility, and which were of greater impact in males. The current findings do not fully support a generality of the enhancement of Pb effects by PS, as previously seen with FI performance in females (Virgolini et al., 2008), and suggest a dissociation of the behaviors controlled by FI and delay discounting paradigms, at least in response to Pb±PS in rats. Collectively, however, the findings remain consistent with sex-dependent differences in the impacts of both Pb and PS and with the need to understand both the role of contingencies of reinforcement and underlying neurobiological effects in these sex differences.

Consequences of developmental exposure to concentrated ambient ultrafine particle air pollution combined with the adult paraquat and maneb model of the Parkinson's disease phenotype in male mice

Current evidence suggests suceptibility of both the substantia nigra and striatum to exposure to components of air pollution. Further, air pollution has been associated with increased risk of PD diagnsosis in humans or PD-like pathology in animals. This study examined whether exposure of mice to concentrated ambient ultrafine particles (CAPS; <100nm diameter) during the first two weeks of life would alter susceptibility to induction of the Parkinsons disease phenyotype (PDP) in a pesticide-based paraquat and maneb (PQ+MB) model during adulthood utilizing i.p. injections of 10mg/kg PQ and 30mg/kg MB 2× per week for 6 weeks. Evidence of CAPS-induced enhancement of the PQ+MB PDP was limited primarily to delayed recovery of locomotor activity 24 post-injection of PQ+MB that could be related to alterations in striatal GABA inhibitory function. Absence of more extensive interactions might also reflect the finding that CAPS and PQ+MB appeared to differentially target the nigrostriatal dopamine and amino acid systems, with CAPS impacting striatum and PQ+MB impacting dopamine-glutamate function in midbrain; both CAPS and PQ+MB elevated glutamate levels in these specific regions, consistent with potential excitotoxicity. These findings demonstrate the ability of postnatal CAPS to produce locomotor dysfunction and dopaminergic and glutamateric changes, independent of PQ+MB, in brain regions involved in the PDP.

Experimental manipulations blunt time-induced changes in brain monoamine levels and completely reverse stress, but not Pb+/−stress-related modifications to these trajectories

This study sought to further understand how environmental conditions influence the outcomes of early developmental insults. It compared changes in monoamine levels in frontal cortex, nucleus accumbens and striatum of male and female Long-Evans rat offspring subjected to maternal Pb exposure (0, 50 or 150ppm in drinking water from 2 months pre-breeding until pup weaning)+/-prenatal (PS) (restraint on GD16-17) or PS+offspring stress (OS; three variable stress challenges to young adults) determined at 2 months of age and at 6 months of age in littermates subsequently exposed either to experimental manipulations (EM: daily handling and performance on an operant fixed interval (FI) schedule of food reward), or to no experience (NEM; time alone). Time alone (NEM conditions), even in normal (control) animals, modified the trajectory of neurochemical changes between 2 and 6 months across brain regions and monoamines. EM significantly modified the NEM trajectories, and except NE and striatal DA, which increased, blunted the changes in monoamine levels that occurred over time alone. Pb+/-stress modified the trajectory of monoamine changes in both EM and NEM conditions, but these predominated under NEM conditions. Stress-associated modifications, occurring mainly with NEM OS groups, were fully reversed by EM procedures, while reversals of Pb+/-stress-associated modifications occurred primarily in nucleus accumbens, a region critical to mediation of FI response rates. These results extend the known environmental conditions that modify developmental Pb+/-stress insults, which is critical to ultimately understanding whether early insults lead to adaptive or maladaptive behavior and to devising behavioral therapeutic strategies. That time alone and a set of EM conditions typically used as outcome measures in intervention studies can themselves invoke neurochemical changes, moreover, has significant implications for experimental design of such studies.