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.

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.

CNS effects of developmental Pb exposure are enhanced by combined maternal and offspring stress.

Lead (Pb) exposure and elevated stress are co-occurring risk factors. Both impact brain mesolimbic dopamine/glutamate systems involved in cognitive functions. We previously found that maternal stress can potentiate Pb-related adverse effects in offspring at blood Pb levels averaging approximately 40 microg/dl. The current study of combined Pb exposure and stress sought to extend those results to lower levels of Pb exposure, and to examine relationships among consequences in offspring for fixed interval (FI) schedule-controlled behavior, neurochemistry and corticosterone levels. Dams were exposed to maternal Pb beginning 2 months prior to breeding (0, 50 or 150 ppm in drinking water), maternal restraint stress on gestational days 16 and 17 (MS), or the combination. In addition, a subset of offspring from each resultant treatment group was also exposed intermittently to variable stressors as adults (MS+OS). Marked "Pb-stress"-related increases in response rates on a fixed interval schedule, a behavioral performance with demonstrated sensitivity to Pb, occurred preferentially in female offspring even at mean blood Pb levels of 11 microg/dl when 50 ppm Pb was combined with maternal and offspring stress. Greater sensitivity of females to frontal cortex catecholamine changes may contribute to the elevated FI response rates as mesocorticolimbic systems are critical to the mediation of this behavior. Basal and final corticosterone levels of offspring used to evaluate FI performance differed significantly from those of non-behaviorally tested (NFI) littermates, demonstrating that purported mechanisms of Pb, stress or Pb/stress effects determined in non-behaviorally trained animals cannot necessarily be generalized to animals with behavioral histories. Finally, the persistent and permanent consequences of Pb, stress and Pb+stress in offspring of both genders suggest that Pb screening programs 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 (i.e., potentiated effects) raise questions about the capacity of current hazard identification approaches to adequately identify human health risks posed by neurotoxicants.

Heritability, correlations and in silico mapping of locomotor behavior and neurochemistry in inbred strains of mice.

The midbrain dopamine system mediates normal and pathologic behaviors related to motor activity, attention, motivation/reward and cognition. These are complex, quantitative traits whose variation among individuals is modulated by genetic, epigenetic and environmental factors. Conventional genetic methods have identified several genes important to this system, but the majority of factors contributing to the variation remain unknown. To understand these genetic and environmental factors, we initiated a study measuring 21 behavioral and neurochemical traits in 15 common inbred mouse strains. We report trait data, heritabilities and genetic and non-genetic correlations between pheno-types. In general, the behavioral traits were more heritable than neurochemical traits, and both genetic and non-genetic correlations within these trait sets were high. Surprisingly, there were few significant correlations between the behavioral and the individual neurochemical traits. However, striatal serotonin and one measure of dopamine turnover (DOPAC/DA) were highly correlated with most behavioral measures. The variable accounting for the most variation in behavior was mouse strain and not a specific neurochemical measure, suggesting that additional genetic factors remain to be determined to account for these behavioral differences. We also report the prospective use of the in silico method of quantitative trait loci (QTL) analysis and demonstrate difficulties in the use of this method, which failed to detect significant QTLs for the majority of these traits. These data serve as a framework for further studies of correlations between different midbrain dopamine traits and as a guide for experimental cross designs to identify QTLs and genes that contribute to these traits.

Expression of behavioral sensitization to the cocaine-like fungicide triadimefon is blocked by pretreatment with AMPA, NMDA and DA D1 receptor antagonists.

Triadimefon (TDF) is a triazole fungicide that blocks the reuptake of dopamine (DA), much like cocaine. A recent study in our laboratory found that intermittent injections of TDF led to robust locomotor sensitization in response to challenge TDF after a 2-week withdrawal period. The current study sought to determine whether the expression of TDF behavioral sensitization could be prevented by the DA D1-like receptor antagonist SCH 23390 (SCH), the DA D2-like receptor antagonist remoxipride (Rem), the competitive NMDA antagonist CPP, or the AMPA antagonist NBQX. Adult male C57/BL6 mice were injected with vehicle or 75 mg/kg TDF twice a week for 7 weeks, with locomotor activity measured periodically across the 14 doses. After a 2-week withdrawal period, mice were pretreated with SCH (0.015 mg/kg), Rem (0.3 mg/kg), CPP (2.5 mg/kg) or NBQX (10.0 mg/kg) followed 30 min later by vehicle or 75 mg/kg TDF and tested for the expression of TDF sensitization. Intermittent administration of TDF led to the development and robust expression of behavioral sensitization in terms of vertical activity. Pretreatment with SCH, NBQX and CPP successfully blocked the expression of vertical sensitization to TDF, while Rem pretreatment did not. All four antagonists, however, attenuated the neurochemical changes normally associated with TDF sensitization as measured 8 h after the 2-week TDF challenge. This paper reveals that NMDA, AMPA and DA D1-like receptors are necessary for the behavioral expression of sensitization to the fungicide triadimefon.

Risk factors for dopaminergic neuron loss in human alpha-synuclein transgenic mice.

Genetic background, pesticide exposure, age, gender, diet and lifestyle are implicated risk factors in Parkinson's disease. We demonstrate dopamine neuron loss and other features of Parkinsonism based on the interaction of several of these human risk factors in transgenic mice expressing human alpha-synuclein. Mice expressing different forms of human alpha-synuclein had progressive declines in locomotor activity and abnormal responses to apomorphine that were modified by transgenic status. Stereological counts of tyrosine hydroxylase-positive neurons significantly declined with age only in the transgenic lines, consistent with a constant or decreasing risk, with the line expressing a double-mutant form of human alpha-synuclein more severely affected than the line expressing wild-type human alpha-synuclein. Treatment with Mn2+-ethylenebisdithiocarbamate and paraquat resulted in significantly greater effects in the double-mutant line than the other lines. Inclusions were not identified in the transgenic lines. Overexpression of human alpha-synuclein had adverse effects on substantia nigra pars compacta dopaminergic neurons that were modified by risk factors interacting in humans, including human alpha-synuclein mutations, ageing, and exposure to pesticides.

Development of behavioral sensitization to the cocaine-like fungicide triadimefon is prevented by AMPA, NMDa, DA D1 but not DA D2 receptor antagonists.

Triadimefon (TDF) is a triazole fungicide that blocks the reuptake of dopamine (DA) and leads to increased locomotor activity levels in mice and rats, effects similar to those of indirect DA agonists such as cocaine. We recently found in mice that intermittent TDF administration led to robust locomotor sensitization, a phenomenon reflecting neuronal plasticity, following challenge with the same TDF dose after a 2-week withdrawal period. The current study sought to determine whether antagonists to DA D1-like receptors (SCH 23390; SCH), DA D2-like receptors (remoxipride; Rem), ionotropic glutamate n-methyl-d-aspartate (NMDA) receptors (CPP), or ionotropic glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (NBQX) could prevent the development of TDF behavioral sensitization, therefore indicating their mechanistic involvement in TDF sensitization. Mice were treated with either vehicle, SCH (0.015 mg/kg), remoxipride (Rem, 0.3 mg/kg), CPP (2.5 mg/kg) or NBQX (10.0 mg/kg), followed 30 min later by vehicle or 75 mg/kg TDF (TDF), twice a week for 7 weeks, with locomotor activity measured post-dosing once a week. After a 2-week withdrawal period, mice were challenged with 75 mg/kg TDF or vehicle, to test for the presence of behavioral sensitization. Pretreatment with SCH, CPP, or NBQX, but not Rem, blocked the development of behavioral sensitization to TDF specifically for vertical activity. Antagonists that blocked TDF vertical sensitization also attenuated the increase in extracellular DA turnover (homovanillic acid [HVA]/DA) normally associated with this behavioral response. Therefore, DA D1, NMDA and AMPA receptors appear to be necessary for the development of behavioral sensitization to TDF. As such, TDF may be considered an environmental risk factor for behavioral dysfunctions linked to glutamatergic and dopaminergic systems.

Interactions of paraquat and triadimefon: behavioral and neurochemical effects.

Triadimefon (TDF), a triazole fungicide, and paraquat (PQ), a non-selective herbicide/dessicant, are both known to adversely impact brain dopaminergic function and are used in overlapping geographical areas of the US. Since "real world" situations indicate humans are exposed to a diverse mixture of chemicals, this study hypothesized that combined exposures to PQ+TDF could produce interactive effects by simultaneously attacking multiple target sites of dopamine systems. Thus, 10 mg/kg PQ (PQ10) and 25 or 50 mg/kg TDF (TDF25 and 50, respectively) were administered i.p. to male C57BL/6 mice, 2x per week for 12 weeks, either alone or in combination. Acutely, TDF50 increased horizontal and vertical activity with increased vertical activity still occurring 24h later, indicative of sustained behavioral sensitization. Acutely, PQ decreased horizontal but not vertical activity with a lack of residual effects at 24h. PQ prevented the increased levels of activity associated with TDF50. These interactions differed for horizontal and vertical activity, indicating their differential neurochemical mediation, and suggesting that they did not arise from simple additivity of PQ and TDF effects. Nor could the interactive effects be readily ascribed to corresponding neurochemical interactions, since all treatments generally increased levels of DA and metabolites acutely in striatum and were associated with general reductions in levels of DA and metabolites and turnover in striatum and frontal cortex 7 days after the final treatment. Thus, TDF and PQ both separately and through interactions may serve as environmental risk factors through different mechanisms for dopaminergically-mediated behavioral dysfunctions.

Potentiated and preferential effects of combined paraquat and maneb on nigrostriatal dopamine systems: environmental risk factors for Parkinson's disease?

The absence of any compelling basis for a heritable basis of idiopathic Parkinson's disease (PD) has focused attention on environmental exposures as causative agents. While the herbicide paraquat has repeatedly been implicated, its impact on dopamine systems following systemic exposures is equivocal. The restricted focus on paraquat also ignores the extensive geographical overlap of its use with other agrichemicals known to adversely impact dopamine systems, including ethylenebisdithiocarbamate fungicides such as maneb. The present study sought to determine whether combined exposures to paraquat and maneb would produce additive effects and support a multiple-hit environmental contribution to PD. C57BL/6 mice were exposed to either paraquat (5-10 mg/kg) or maneb (15-30 mg/kg) i.p. alone or in combination once a week for 4 weeks. Sustained decreases in motor activity immediately following injections were consistently observed only with combined exposures, with activity levels returning to control values 24 h later. Concurrently, levels of dopamine and metabolites and dopamine turnover were increased immediately post-injection only by combined exposures, and returned to control levels or below within 48 h. Reductions in tyrosine hydroxylase immunoreactivity, measured 3 days after the last injection, resulted only from combined exposure and were detected in dorsal striatum, but not in the nucleus accumbens. The fact that combined exposures resulted in potentiated effects that appear to target nigrostriatal dopamine systems suggests that these combinations may be important environmental risk factors for Parkinsonism. These findings also raise questions about the adequacy of current risk assessment guidelines for these chemicals which are based on effect levels derived from exposures to single agents.

The nigrostriatal dopaminergic system as a preferential target of repeated exposures to combined paraquat and maneb: implications for Parkinson's disease.

Experimental evidence supporting 1,1'-dimethyl-4,4'-bipyridinium [paraquat (PQ)] as a risk factor for Parkinson's disease (PD) is equivocal. Other agricultural chemicals, including dithiocarbamate fungicides such as manganese ethylenebisdithiocarbamate [maneb (MB)], are widely used in the same geographical regions as paraquat and also impact dopamine systems, suggesting that mixtures may be more relevant etiological models. This study therefore proposed that combined PQ and MB exposures would produce greater effects on dopamine (DA) systems than would either compound administered alone. Male C57BL/6 mice were treated twice a week for 6 weeks with intraperitoneal saline, 10 mg/kg paraquat, 30 mg/kg maneb, or their combination (PQ + MB). MB, but not PQ, reduced motor activity immediately after treatment, and this effect was potentiated by combined PQ + MB treatment. As treatments progressed, only the combined PQ + MB group evidenced a failure of motor activity levels to recover within 24 hr. Striatal DA and dihydroxyphenylacetic acid increased 1-3 d and decreased 7 d after injections. Only PQ + MB reduced tyrosine hydroxylase (TH) and DA transporter immunoreactivity and did so in dorsal striatum but not nucleus accumbens. Correspondingly, striatal TH protein levels were decreased only by combined PQ + MB 5 d after injection. Reactive gliosis occurred only in response to combined PQ + MB in dorsal-medial but not ventral striatum. TH immunoreactivity and cell counts were reduced only by PQ + MB and in the substantia nigra but not ventral tegmental area. These synergistic effects of combined PQ + MB, preferentially expressed in the nigrostriatal DA system, suggest that such mixtures could play a role in the etiology of PD.

Spectral analysis of AC and DC components of the pulse photoplethysmograph at rest and during induction of anaesthesia.

We examined spectral components of beat to beat variability in AC and DC signals of the reflectance photoplethysmograph at finger and earlobe sites in 20 resting volunteers and 20 patients during propofol, alfentanil, isoflurane, nitrous oxide anaesthesia. We observed that at rest, the majority of spectral power at both sites and in both signals was in the low 'thermoregulatory' frequency band (0.01-0.08 Hz). These fluctuations were greater in the finger than in the earlobe and in the AC signal compared to the DC. With anaesthesia, low as well as mid (0.08-0.15 Hz) frequency variability decreased at both sites and in both signals whereas high frequency 'ventilatory' power (0.15-0.45 Hz) was maintained. During anaesthesia we found no significant differences between the spectral components of the AC or DC signals or between the finger and the earlobe sites. At all frequencies, the fluctuations in the AC and DC signals were out of phase with each other.