Developmental Manganese Exposure Causes Lasting Attention Deficits Accompanied by Dysregulation of mTOR Signaling and Catecholaminergic Gene Expression in Brain Prefrontal Cortex.

Elevated manganese (Mn) exposure is associated with attentional deficits in children, and is an environmental risk factor for attention deficit hyperactivity disorder (ADHD). We have shown that developmental Mn exposure causes lasting attention and sensorimotor deficits in a rat model of early childhood Mn exposure, and that these deficits are associated with a hypofunctioning catecholaminergic system in the prefrontal cortex (PFC), though the mechanistic basis for these deficits is not well understood. To address this, male Long-Evans rats were exposed orally to Mn (50 mg/kg/d) over PND 1-21 and attentional function was assessed in adulthood using the 5-Choice Serial Reaction Time Task. Targeted catecholaminergic system and epigenetic gene expression, followed by unbiased differential DNA methylation and gene regulation expression transcriptomics in the PFC, were performed in young adult littermates. Results show that developmental Mn exposure causes lasting focused attention deficits that are associated with reduced gene expression of tyrosine hydroxylase, dopamine transporter, and DNA methyltransferase 3a. Further, developmental Mn exposure causes broader lasting methylation and gene expression dysregulation associated with epigenetic regulation, inflammation, cell development, and hypofunctioning catecholaminergic neuronal systems. Pathway enrichment analyses uncovered mTOR and Wnt signaling pathway genes as significant transcriptomic regulators of the Mn altered transcriptome, and Western blot of total, C1 and C2 phospho-mTOR confirmed mTOR pathway dysregulation. Our findings deepen our understanding of the mechanistic basis of how developmental Mn exposure leads to lasting catecholaminergic dysfunction and attention deficits, which may aid future therapeutic interventions of environmental exposure associated disorders. Significance Statement: Attention deficit hyperactivity disorder (ADHD) is associated with environmental risk factors, including exposure to neurotoxic agents. Here we used a rodent model of developmental manganese (Mn) exposure producing lasting attention deficits to show broad epigenetic and gene expression changes in the prefrontal cortex, and to identify disrupted mTOR and Wnt signaling pathways as a novel mechanism for how developmental Mn exposure may induce lasting attention and catecholaminergic system impairments. Importantly, our findings establish early development as a critical period of susceptibility to lasting deficits in attentional function caused by elevated environmental toxicant exposure. Given that environmental health threats disproportionately impact communities of color and low socioeconomic status, our findings can aid future studies to assess therapeutic interventions for vulnerable populations.

Dietary and genetic manipulations of folate metabolism differentially affect neocortical functions in mice.

Converging evidence suggests that folate-mediated one-carbon metabolism may modulate cognitive functioning throughout the lifespan, but few studies have directly tested this hypothesis. This study examined the separate and combined effects of dietary and genetic manipulations of folate metabolism on neocortical functions in mice, modeling a common genetic variant in the MTHFD1 gene in humans. Mutant (Mthfd1(gt/+)) and wildtype (WT) male mice were assigned to a folate sufficient or deficient diet at weaning and continued on these diets throughout testing on a series of visual attention tasks adapted from the 5-choice serial reaction time task. WT mice on a deficient diet exhibited impulsive responding immediately following a change in task parameters that increased demands on attention and impulse control, and on trials following an error. This pattern of findings indicates a heightened affective response to stress and/or an inability to regulate negative emotions. In contrast, Mthfd1(gt/+) mice (regardless of diet) exhibited attentional dysfunction and a blunted affective response to committing an error. The Mthfd1(gt/+) mice also showed significantly decreased expression levels for genes encoding choline dehydrogenase and the alpha 7 nicotinic cholinergic receptor. The effects of the MTHFD1 mutation were less pronounced when combined with a deficient diet, suggesting a compensatory mechanism to the combined genetic and dietary perturbation of folate metabolism. These data demonstrate that common alterations in folate metabolism can produce functionally distinct cognitive and affective changes, and highlight the importance of considering genotype when making dietary folate recommendations.

A mouse model of fragile X syndrome exhibits heightened arousal and/or emotion following errors or reversal of contingencies.

This study was designed to further assess cognitive and affective functioning in a mouse model of Fragile X syndrome (FXS), the Fmr1(tm1Cgr) or Fmr1 "knockout" (KO) mouse. Male KO mice and wild-type littermate controls were tested on learning set and reversal learning tasks. The KO mice were not impaired in associative learning, transfer of learning, or reversal learning, based on measures of learning rate. Analyses of videotapes of the reversal learning task revealed that both groups of mice exhibited higher levels of activity and wall-climbing during the initial sessions of the task than during the final sessions, a pattern also seen for trials following an error relative to those following a correct response. Notably, the increase in both behavioral measures seen early in the task was significantly more pronounced for the KO mice than for controls, as was the error-induced increase in activity level. This pattern of effects suggests that the KO mice reacted more strongly than controls to the reversal of contingencies and pronounced drop in reinforcement rate, and to errors in general. This pattern of effects is consistent with the heightened emotional reactivity frequently described for humans with FXS.

Attentional dysfunction, impulsivity, and resistance to change in a mouse model of fragile X syndrome.

On a series of attention tasks, male mice with a mutation targeted to the fragile X mental retardation 1 (Fmrl) gene (Fmrl knockout [KO] mice) committed a higher rate of premature responses than wild-type littermates, with the largest differences seen when task contingencies changed. This finding indicates impaired inhibitory control, particularly during times of stress or arousal. The KO mice also committed a higher rate of inaccurate responses than controls, particularly during the final third of each daily test session, indicating impaired sustained attention. In the selective attention task, the unpredictable presentation of potent olfactory distractors produced a generalized disruption in the performance of the KO mice, whereas for controls, the disruption produced by the distractors was temporally limited. Finally, the attentional disruption seen following an error was more pronounced for the KO mice than for controls, further implicating impaired regulation of arousal and/or negative affect. The present study provides the first evidence that the Fmrl KO mouse is impaired in inhibitory control, attention, and arousal regulation, hallmark areas of dysfunction in fragile X syndrome. The resistance to change also seen in these mice provides a behavioral index for studying the autistic features of this disorder.

Prenatal cocaine exposure alters sensitivity to the effects of idazoxan in a distraction task.

The present study was designed to test whether prenatal cocaine (COC) exposure alters sensitivity to the attentional effects of idazoxan (IDZ), an alpha-2 adrenergic antagonist that increases coeruleocortical NE activity. The task assessed subjects' ability to selectively attend to an unpredictable light cue and disregard olfactory distractors. IDZ increased commission errors specifically under conditions of distraction, an effect that was similar in the COC and control groups. In contrast, COC animals were significantly more sensitive than controls to the effects of IDZ on omission errors and nontrials. The pattern of effects suggests that the differential treatment response to IDZ on these latter measures resulted from an alteration in norepinephrine (NE)-modulated dopamine release in the COC animals, reflecting lasting changes in dopaminergic and/or noradrenergic systems as a result of the early cocaine exposure. Based on the behavioral measures that showed a differential response to IDZ in the COC animals, it seems likely that these changes may contribute to the alterations in sustained attention and arousal regulation that have been reported in both animals and humans exposed to cocaine in utero.

Spatial reversal learning in Aroclor 1254-exposed rats: sex-specific deficits in associative ability and inhibitory control.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that have been associated with cognitive deficits in children exposed in utero. Cognitive deficits due to PCB exposure have also been documented in animal models, but the underlying behavioral mechanisms responsible for those deficits remain to be elucidated. The current study examined the effects of gestational and lactational exposure to PCBs on spatial discrimination-reversal learning (spatial RL) in rats using standard two-lever operant testing chambers. Pregnant Long-Evans rats (10/dose) received either 0 or 6 mg/kg Aroclor 1254 (A1254) po in corn oil from gestational day 6 to postnatal day 21. One male and one female from each litter were tested on spatial RL beginning at 190-220 days of age. Animals were reinforced with a 45-mg food pellet for pressing the lever associated with the correct spatial location (either left or right). After reaching 85% correct performance for 2 consecutive days, the opposite spatial location was reinforced. Five of these position reversals were given. Male rats exposed to A1254 made significantly more total errors (121.6 +/- 12.5) on the first reversal than controls (90.7 +/- 5.8). In contrast, female rats exposed to A1254 exhibited deficits on the fourth and fifth reversals (23.6 +/- 4.2, 17.0 +/- 2.8 and 36.7 +/- 4.7, 26.8 +/- 2.5 for control and exposed animals, respectively). Response-pattern analyses in the A1254-exposed male and female rats revealed fundamental differences in the underlying behavioral mechanisms responsible for the deficits. A1254-exposed males exhibited an increased tendency to incorrectly respond to the previously correct stimulus (i.e., perseverate) following a reversal while A1254-exposed females exhibited impairments in their ability to make new associations with a reinforced spatial location (i.e., associative deficit). These data provide new insights into the underlying behavioral mechanisms that may be responsible for the spatial learning deficits observed in PCB-exposed rodents and monkeys.

Recovery of associative function following early amygdala lesions in rats.

Adult rats with amygdala lesions made at either Postnatal Day (PND) 10 or PND40 were tested on a series of reversal tasks that tap the ability to form stimulus-reward associations. PND40 rats were significantly impaired relative to both controls and PND10 rats on learning rate of the original discrimination and subsequent reversals. Analyses of discrete learning phases revealed that the impairment was specific to the postchance phase. The PND10 group was not impaired relative to controls on any measure. These results confirm prior findings that amygdala lesions sustained in adulthood impair the formation of stimulus-reward associations. They also demonstrate that substantial sparing or recovery of function is possible when the lesion is made during early development. Furthermore, the findings support the view that behavioral recovery may be more likely if the lesion is sustained near the time of peak synaptogenesis.

Early lead exposure produces lasting changes in sustained attention, response initiation, and reactivity to errors.

The present study tested the hypothesis that early lead (Pb) exposure causes lasting attentional dysfunction. Long-Evans dams were fed Pb-adulterated water during gestation and/or lactation; the offspring were tested as adults. The results of a visual discrimination task revealed no Pb effects on learning rate or information-processing speed. However, lasting effects of the early Pb exposure were seen in the subsequent vigilance tasks, particularly in the final task in which onset of the visual cue and cue duration varied randomly across trials. Exposure during both gestation and lactation impaired response initiation. In addition, animals exposed to Pb during lactation only or lactation+gestation committed significantly more omission errors than controls under two specific conditions: (1) trials in which a delay was imposed prior to cue presentation and (2) trials that followed an incorrect response. The pattern of treatment differences indicated that early Pb exposure produced lasting impairment of sustained attention and increased reactivity to errors. Both effects may contribute to the cognitive impairment, problematic classroom behaviors, and increased delinquency associated with early Pb exposure in children. These findings also demonstrate that the developmental timing of the exposure determines the pattern of effects. Thus, conclusions regarding whether or not a particular cognitive or affective function is impaired or spared by early Pb exposure must be limited to the specific timing and intensity of exposure.

Attention as a target of intoxication: insights and methods from studies of drug abuse.

A symposium was convened to discuss recent developments in the assessment of attention and the effects of drugs and toxic chemicals on attention at the 17th annual meeting of the Behavioral Toxicology Society on May 1, 1999, in Research Triangle Park, NC. Speakers addressed issues including the methodology of assessing cognitive function, the neurobiology of specific aspects of attention, the dual roles of attention as a target of intoxication and as a mediating variable in the development of addiction to psychoactive drugs, the changes in attention that accompany neuropsychological disorders of schizophrenia, senile dementia of the Alzheimer type and attention deficit hyperactivity disorder, and potential therapies for these disorders. This article provides an overview of the objectives of the symposium, followed by summaries of each of the talks given.

Prenatal cocaine exposure impairs selective attention: evidence from serial reversal and extradimensional shift tasks.

This study assessed the effects of prenatal cocaine exposure on cognitive functioning, using an intravenous (IV) rodent model that closely mimics the pharmacokinetics seen in humans after smoking or IV injection and that avoids maternal stress and undernutrition. Cocaine-exposed males were significantly impaired on a 3-choice, but not 2-choice, olfactory serial reversal learning task. Both male and female cocaine-exposed rats were significantly impaired on extradimensional shift tasks that required shifting from olfactory to spatial cues; however, they showed no impairment when required to shift from spatial to olfactory cues. In-depth analyses of discrete learning phases implicated deficient selective attention as the basis of impairment in both tasks. These data provide clear evidence that prenatal cocaine exposure produces long-lasting cognitive dysfunction, but they also underscore the specificity of the impairment.

Effects of chronic lead exposure on learning and reaction time in a visual discrimination task.

Long-Evans rats exposed chronically to lead (Pb) acetate (0, 75, or 300 ppm) were tested as adults on an automated, three-choice visual discrimination task as part of a larger study designed to elucidate the cognitive effects of developmental Pb exposure. Median adult BPb levels for the groups were <5, 20, and 36 microgram/dl. The pattern of results suggested a linear effect, with increasing lead dose producing progressively slower learning and an increased incidence of "impaired" individuals. This latter measure proved to be slightly more sensitive than the former, suggesting individual differences in susceptibility to Pb neurotoxicity. Additional analyses revealed that the impairing effect of Pb was seen in both the chance and post-chance learning phases, indicating that the deficit was not limited to (but could include) attentional function. Reaction time on incorrect trials was reduced in the 300-ppm group, whereas no Pb effect was seen for correct trials. The present findings suggest that chronic developmental Pb exposure produces an associative deficit as well as a tendency to respond rapidly, but does not affect information-processing speed.

Enduring effects of early lead exposure: evidence for a specific deficit in associative ability.

Long-Evans dams were exposed to Pb acetate in the drinking water during both gestation and lactation, or lactation only. This report presents the results of an automated, olfactory, serial reversal task administered to the adult offspring. Although overall learning rate was not significantly affected by Pb exposure, analyses of specific phases of the learning process revealed that all three exposed groups required significantly more trials than controls to reach criterion from the point at which perseverative responding to the previously correct cue ended. These in-depth analyses revealed that the reversal learning impairment of the Pb-exposed animals was not due to a deficit in inhibiting responses to the previously correct cue, the mechanism commonly assumed to underlie impaired reversal learning. Instead, the analyses revealed that two other independent Pb effects were responsible for the prolonged postperseverative learning period: a response bias and an impaired ability to associate cues and/or actions with affective consequences. The contribution of these two factors varied as a function of the timing and intensity of the Pb exposure. It is hypothesized that the Pb-induced associative deficit may reflect lasting damage to the amygdala and/or nucleus accumbens, which comprise a system thought to modulate the process by which environmental cues acquire affective significance.

Prenatal cocaine exposure increases sensitivity to the attentional effects of the dopamine D1 agonist SKF81297.

Sensitivity to the attentional effects of SKF81297, a selective full agonist at dopamine D(1) receptors, was assessed in adult rats exposed to cocaine prenatally (via intravenous injections) and controls. The task assessed the ability of the subjects to monitor an unpredictable light cue of either 300 or 700 msec duration and to maintain performance when presented with olfactory distractors. SKF81297 decreased nose pokes before cue presentation and increased latencies and response biases (the tendency to respond to the same port used on the previous trial), suggesting an effect of SKF81297 on the dopamine (DA) systems responsible for response initiation and selection. The cocaine-exposed (COC) and control animals did not differ in sensitivity to the effects of SKF81297 on these measures. In contrast, the COC animals were significantly more sensitive than were controls to the impairing effect of SKF81297 on omission errors, a measure of sustained attention. This pattern of results provides evidence that prenatal cocaine exposure produces lasting changes in the DA system(s) subserving sustained attention but does not alter the DA system(s) underlying response selection and initiation. These findings also provide support for the role of D(1) receptor activation in attentional functioning.

Effects of dietary mixtures of amino acids on fetal growth and maternal and fetal amino acid pools in experimental maternal phenylketonuria.

BACKGROUND: Branched-chain amino acids have been reported to improve fetal brain development in a rat model in which maternal phenylketonuria (PKU) is induced by the inclusion of an inhibitor of phenylalanine hydroxylase, DL-p-chlorophenylalanine, and L-phenylalanine in the diet. OBJECTIVE: We studied whether a dietary mixture of several large neutral amino acids (LNAAs) would improve fetal brain growth and normalize the fetal brain amino acid profile in a rat model of maternal PKU induced by DL-alpha-methylphenylalanine (AMPhe). DESIGN: Long-Evans rats were fed a basal diet or a similar diet containing 0.5% AMPhe + 3.0% L-phenylalanine (AMPhe + Phe diet) from day 11 until day 20 of gestation in experiments to test various mixtures of LNAAs. Maternal weight gains and food intakes to day 20, fetal body and brain weights at day 20, and fetal brain and fetal and maternal plasma amino acid concentrations at day 20 were measured. RESULTS: Concentrations of phenylalanine and tyrosine in fetal brain and in maternal and fetal plasma were higher and fetal brain weights were lower in rats fed the AMPhe + Phe diet than in rats fed the basal diet. However, fetal brain growth was higher and concentrations of phenylalanine and tyrosine in fetal brain and in maternal and fetal plasma were lower in rats fed the AMPhe + Phe diet plus LNAAs than in rats fed the diet containing AMPhe + Phe alone. CONCLUSION: LNAA supplementation of the diet improved fetal amino acid profiles and alleviated most, but not all, of the depression in fetal brain growth observed in this model of maternal PKU.

Efficacy of succimer chelation for reducing brain Pb levels in a rodent model.

Increasing evidence indicates that early low-level lead (Pb) exposure produces enduring cognitive impairment in children, underscoring the need to develop improved therapeutic intervention. Although chelating agents have been shown to effectively reduce body Pb levels, it is not yet known whether this treatment ameliorates Pb-induced cognitive dysfunction. Clinical research in this area is hampered by the need to rely on reductions in blood Pb levels as the index of treatment efficacy, despite the fact that brain Pb level is the exposure parameter of greatest relevance to neurocognitive outcomes. The present studies were designed to provide information that will aid future research in this area in both human and animal models. The objectives of these studies were (1) to evaluate the efficacy of different doses and durations of succimer (meso-2,3-dimercaptosuccinic acid; DMSA) chelation for reducing brain and blood Pb levels and (2) to determine the extent to which blood Pb can serve as a surrogate of brain Pb following chelation. Long-Evans hooded rats were exposed to Pb from birth until day 31 (Study 1) or day 40 (Study 2) of life, followed by oral treatment with a vehicle or one of two succimer regimens for a duration of either 7 or 21 days. Results indicated that 7 days of succimer treatment produced a 1.5- to 2.5-fold greater reduction of Pb in blood than in brain, relative to time-matched vehicle groups. Prolonged treatment (21) days did not further reduce blood Pb levels (relative to 7-day succimer treatment), but did produce further reductions in brain Pb level compared to time-matched vehicle groups. Thus, chelation-mediated reductions in brain Pb did not parallel reductions in blood Pb over the course of treatment. While the relevance of these data to humans may be confounded by anatomical and physiological differences between rodents and primates, as well as differences in the metabolism of succimer (DMSA), they suggest that clinical studies should exercise caution when using blood Pb as an index of the efficacy of chelation treatment for reducing brain Pb levels.

Analyses of response patterns clarify lead effects in olfactory reversal and extradimensional shift tasks: assessment of inhibitory control, associative ability, and memory.

Rats exposed to lead (Pb) chronically from conception were tested on (a) an olfactory serial reversal task and (b) an extradimensional shift (EDS) task. Pb exposure did not impair learning of the original olfactory discrimination but did impair learning of the 5 reversals and the EDS task. In the reversals, Pb exposure tended to shorten the initial period of persistent responding to the previously correct cue, but significantly prolonged the postperseverative learning phase (both the "chance" and "greater-than-chance" components). These effects are similar to those produced by lesions of the amygdala, a structure implicated in the process by which stimuli acquire incentive value. This similarity, coupled with the pattern of findings, suggests that Pb-induced impairment of reversal learning is due to a deficiency in learning the new contingencies of the task (an associative deficit), not inflexibility or deficient inhibitory control. These findings also illustrate the importance of analyzing the types of errors committed, rather than focusing solely on learning rate.

An in-depth analysis of lead effects in a delayed spatial alternation task: assessment of mnemonic effects, side bias, and proactive interference.

This study examined the effects of chronic postweaning lead (Pb) exposure in Long-Evans rats on a series of spatial alternation tasks. All tasks were administered in automated testing chambers, with a nosepoke as the critical response. While neither Pb-exposed group (median blood lead levels: 19 and 39 micrograms/dl, respectively) was impaired in learning the alternation rule, both groups performed more poorly than controls on the alternation task with variable intertrial delays (0, 10, 20, and 40 s). The deficit was constant across delays, arguing against memory dysfunction. Analyses of the responses on individual trials shed further light on the impaired and spared processes in the Pb-exposed rats. First, these analyses revealed stronger side biases in the higher exposure group. One interpretation is that these animals experienced impatience when the longer delays were included, making it more difficult for them to inhibit a prepotent response to a preferred side. In contrast, these trial-by-trial analyses revealed that several other factors-retention interval, semantic proactive interference, and temporal discriminability-exerted similar effects on performance in the control and lead-exposed animals. The use of logistic regression for these trial-by-trial analyses provided a means of simultaneously assessing the influence of several variables on performance, a significant advantage when there is confounding or interactions between variables.

Specific effects of idazoxan in a distraction task: evidence that endogenous norepinephrine plays a role in selective attention in rats.

Rats were injected with the alpha 2-adrenergic antagonist idazoxan (IDZ) prior to testing on vigilance and distraction tasks. In the vigilance task, rats responded with nose pokes to brief visual cues presented at variable intervals following trial onset. The distraction task was similar except that irrelevant odor cues (distractors) were presented in the interval prior to light onset on some trials. IDZ injection had no effect on performance in the vigilance task. In the distraction task, however, the higher IDZ dose (1.0 mg/kg) modulated the propensity to make a premature response when the distractors were presented. Notably, the direction of the effect varied with the rats' baseline level of distractibility. This pattern of effects suggests that endogenous norepinephrine (NE) influences distractibility and/or selective attention.

Malnutrition and the brain: changing concepts, changing concerns.

Our conceptions of how malnutrition endured early in life affects brain development have evolved considerably since the mid-1960s. At that time, it was feared that malnutrition endured during certain sensitive periods in early development would produce irreversible brain damage possibly resulting in mental retardation and an impairment in brain function. We now know that most of the alterations in the growth of various brain structures eventually recover (to some extent), although permanent alterations in the hippocampus and cerebellum remain. However, recent neuropharmacological research has revealed long-lasting, if not permanent, changes in brain neural receptor function resulting from an early episode of malnutrition. These more recent findings indicate that the kinds of behaviors and cognitive functions impaired by malnutrition may be more related to emotional responses to stressful events than to cognitive deficits per se, the age range of vulnerability to these long-term effects of malnutrition may be much greater than we had suspected and the minimal amount of malnutrition (hunger) necessary to produce these long-term alterations is unknown.

Enduring cognitive effects of early malnutrition: a theoretical reappraisal.

This article presents a reappraisal of the literature on the enduring cognitive effects of early malnutrition. In addition to summarizing the existing empirical literature, we present a theoretical framework for determining whether the processes likely to be most vulnerable to early malnutrition were adequately assessed. The two types of information used to make this determination are clinical and experimental behavioral data as well as reported neural changes. One point of clear consensus is that animals exposed to early malnutrition exhibit lasting changes in the realm of emotionality, motivation, and/or anxiety. Because these alterations profoundly affect all aspects of behavioral functioning, including cognition, it is suggested that future research focus on these changes, rather than control for them as many past studies have done. The functional integrity of specific cognitive processes is less clear. The only cognitive processes for which enduring cognitive changes were demonstrated in rehabilitated animals--outside of effects mediated by these affective changes--are cognitive flexibility and, possibly, susceptibility to proactive interference. However, the inference that these are the only processes affected does not appear to be warranted on the basis of the evidence that several cognitive processes likely to be affected have not been fully assessed. Examples include executive functions linked to the prefrontal cortex (for example, attention), transfer of learning, procedural learning and long-term memory. Future research focusing on these specific cognitive functions as well as on these unequivocal affective changes should allow a more definitive conclusion regarding the enduring functional consequences of early malnutrition.