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.

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 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.

A mnemonic role for vasopressin: the evidence for and against.

This review critically evaluates the animal and human research concerning vasopressin's putative mnemonic role. Weaknesses in the interpretations of the early animal experiments as well as the implications of the later inconsistent findings are discussed. It is concluded that both the initial enthusiasm and the subsequent skepticism concerning this hypothesized role were premature. This conclusion applies equally to the human research. A review of these studies reveals that almost all of the negative reports involved cognitively-impaired individuals. The relatively few studies that have been conducted concerning vasopressin's effects in unimpaired human subjects are consistent with the hypothesis that vasopressin does affect cognition, though both the mechanism of action and the specific cognitive processes which are altered have yet to be elucidated.

Weight loss on a low-fat diet: consequence of the imprecision of the control of food intake in humans.

This study examined the degree to which humans compensate for a reduction in dietary fat by increasing energy intake. Thirteen females were randomly assigned to either a low-fat diet (20-25% of calories as fat) or a control diet (35-40% fat) for 11 wk. After a 7-wk washout period, the conditions were reversed for another 11 wk. Energy intake on the low-fat diet gradually increased by 0.092 kJ/wk resulting in a total caloric compensation of 35% by the end of the 11-wk treatment period. This failure to compensate calorically on the low-fat diet resulted in a deficit of 1.22 kJ/d and a weight loss of 2.5 kg in 11 wk, twice the amount of weight lost on the control diet. These results demonstrate that body weight can be lost merely by reducing the fat content of the diet without the need to voluntarily restrict food intake.

Dietary fat and the regulation of energy intake in human subjects.

The role of dietary fat in the regulation of energy intake was assessed by manipulating a conventional diet and measuring spontaneous food consumption. Twenty-four women each consumed a sequence of three 2-wk dietary treatments in which 15-20%, 30-35%, or 45-50% of the energy was derived from fat. These diets consisted of foods that were similar in appearance and palatability but differed in the amount of high-fat ingredients used. Relative to their energy consumption on the medium-fat diet, the subjects spontaneously consumed an 11.3% deficit on the low-fat diet and a 15.4% surfeit on the high-fat diet (p less than 0.0001), resulting in significant changes in body weight (p less than 0.001). A small amount of caloric compensation did occur (p less than 0.02), which was greatest in the leanest subjects (p less than 0.03). These results suggest that habitual, unrestricted consumption of low-fat diets may be an effective approach to weight control.

Variation in energy intake during the menstrual cycle: implications for food-intake research.

The relationship between spontaneous energy consumption and menstrual cycle was evaluated in 23 subjects who participated in one of two independent studies. Ad libitum intakes of experimental diets were measured by food weighing and bomb calorimetry for 56 or 42 d. Comparisons were made between each woman's mean energy during the 10 d before and after the onset of menstruation. The significant decline (364 kJ, or 87 kcal) between these two 10-d intervals was smaller than but consistent with findings from previous studies of data from food journals. In a separate analysis with time-series techniques, two distinct periods of elevated intake were identified (during the midluteal and midfollicular phases) that were independent of illness and menstrual symptoms. This pattern of food intake is discussed with reference to normal hormonal fluctuations. These findings confirm that menstrual cycle is a potential confounding variable that should be controlled in research on human food intake.

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.

Body composition and energy intake: do overweight women overeat and underreport?

The relationship between energy consumption and body composition was evaluated in 63 women by use of energy-intake values that were precisely measured in a metabolic unit and corrected for deviations from energy balance. Energy requirement for the maintenance of body weight was not significantly correlated with adiposity expressed as percent body fat. However, energy requirement was positively associated with lean mass (p less than 0.0001) whereas fat mass added no predictive value to the same multivariate regression equation. Self-reported energy intake (before the experiments) was not correlated with lean mass and was underestimated by lean subjects at least as much as by obese subjects. Discrepant findings in the literature concerning relationships between obesity and energy intake may be explained by reporting error and by the relative lean mass of obese vs nonobese women but not by systematic underreporting unique to obese subjects.

Improvement of memory by a vasopressin fragment: importance of individual differences in mnemonic function.

Three studies examined the effect of AVP4-9 on memory in an appetitively motivated task (the radial maze) to ascertain the generality of the mnemonic effects seen with vasopressin analogues in avoidance paradigms. Both pre- and posttrial administration of this vasopressin fragment significantly alleviated the forgetting seen in the control condition. However, a significant relation was observed between the proficiency of the rats and the degree to which their performance was improved by the posttrial AVP4-9 treatment. The more proficient subjects did not benefit from this treatment despite the fact that their performance provided evidence of significant forgetting. This pattern of results suggests that physiological differences of animals varying in mnemonic ability may account for their disparate response to the peptide treatment. The present findings support vasopressin's putative mnemonic role but indicate that exogenous administration of vasopressinlike peptides may improve memory only in certain subgroups, and possibly only under particular testing conditions.

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.

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.

A vasopressin metabolite increases attentional selectivity.

Two behavioral paradigms were used to assess the effect of a vasopressin metabolite, AVP4-9, on selectivity of attention. The effects observed in a multiple-cue task indicated that AVP4-9 treatment increased the extent to which attention was controlled by the dominant cues in the environment. When these stimuli predicted reward, the peptide treatment facilitated learning, but when these cues were nonpredictive, the treatment hindered learning. In a redundant learning paradigm, administration of the lower dose of AVP4-9 (1 microgram/kg) prevented animals from learning about an added, equally predictive (i.e., redundant) set of cues, suggesting that this treatment caused selective attending to the originally presented stimuli, whose relationship with reward had already been learned. The results of these two studies provide converging evidence that AVP4-9 treatment increases the selectivity of attention, with preferential processing of dominant information. Parallels with the putative attentional effects of increased arousal are discussed.

Enhancement and impairment of memory retrieval by a vasopressin metabolite: an interaction with the accessibility of the memory.

A series of 3 experiments was conducted to determine the generalizability of the improved recall seen with pretest administration of vasopressin in aversively motivated tasks. The learning paradigm selected for this purpose assessed the transmission of a food preference between rats; retention intervals of 8, 10, and 14 days were used in the 3 experiments, respectively. Pretest subcutaneous injection of a vasopressin metabolite, AVP4-9, significantly improved recall under conditions in which memory was poor in vehicle-treated rats but significantly impaired memory at a time when good recall was evident in controls. These findings support the postulate that administration of vasopressin or its metabolites modulate memory retrieval processes and suggest that the nature of this effect depends on the degree of forgetting that has occurred.

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.

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.

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.

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.

Deficient cumulative learning: an animal model of retarded cognitive development.

Biological insults that produce profound mental retardation (MR) in humans have generally been found to produce little cognitive dysfunction in animal models. Based on the fact that impaired transfer of learning is one of the hallmark characteristics of mentally retarded humans, we proposed that this discrepancy may largely reflect the common use of a single learning task as the critical cognitive index rather than an assessment of cumulative learning. Consistent with this hypothesis, rats exposed to prenatal hyperphenylalaninemia (a model of maternal PKU) evidenced significant impairment when tested on a series of 10 problems designed to allow for positive transfer of learning. This same treatment, however, did not alter learning rate of the individual tasks that comprised this series when presented singly to experimentally naive animals. Deficient transfer of learning contributed significantly to the impairment observed in the maternal PKU group. These results support the hypothesis that the assessment of cumulative learning is an important component of animal models of impaired cognitive development.