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Over the last decade, several clinical reports have outlined cases of early-onset manganese (Mn)-induced dystonia-parkinsonism, resulting from loss of function mutations of the Mn transporter gene SLC39A14. Previously, we have performed characterization of the behavioral, neurochemical, and neuropathological changes in 60-day old (PN60) Slc39a14-knockout (KO) murine model of the human disease. Here, we extend our studies to aging Slc39a14-KO mice to assess the progression of the disease. Our results indicate that 365-day old (PN365) Slc39a14-KO mice present with markedly elevated blood and brain Mn levels, similar to those found in the PN60 mice and representative of the human cases of the disease. Furthermore, aging Slc39a14-KO mice consistently manifest a hypoactive and dystonic behavioral deficits, similar to the PN60 animals, suggesting that the behavioral changes are established early in life without further age-associated deterioration. Neurochemical, neuropathological, and functional assessment of the dopaminergic system of the basal ganglia revealed absence of neurodegenerative changes of dopamine (DA) neurons in the substantia nigra pars compacta (SNc), with no changes in DA or metabolite concentrations in the striatum of Slc39a14-KO mice relative to wildtype (WT). Similar to the PN60 animals, aging Slc39a14-KO mice expressed a marked inhibition of potassium-stimulated DA release in the striatum. Together our findings indicate that the pathophysiological changes observed in the basal ganglia of aging Slc39a14-KO animals are similar to those at PN60 and aging does not have a significant effect on these parameters.

Childhood lead (Pb(2+)) intoxication is a global public health problem best known for producing deficits in learning and poor school performance. Human and preclinical studies have suggested an association between childhood Pb(2+) intoxication and proclivity to substance abuse and delinquent behavior. While environmental factors have been implicated in opioid addiction, less is known about the role of exposure to environmental pollutants on the brain opioid system. Opioid receptors are involved in the biological effects of opioids and other drugs of abuse. In this study, we examine the effect of chronic developmental Pb(2+) exposure (1500 ppm in the diet) on μ-opioid receptor (MOR) levels in the rat brain using [(3)H]-D-Ala2-MePhe4-Gly-ol5 enkephalin ([(3)H]-DAMGO) quantitative receptor autoradiography at different developmental stages (juvenile, early-adolescent, late adolescent and adult) in male and female rats. Our results indicate that chronic developmental Pb(2+) exposure increases the levels of [(3)H]-DAMGO specific binding to MOR in juvenile and early adolescent Pb(2+)-exposed male and female rat brain with no changes in late-adolescent (PN50) and minor changes in Pb(2+)-exposed adult male rats (PN120). Specifically, at PN14, Pb(2+)-exposed males had an increase in MOR binding in the lateral posthalamic nuclei (LPTN), and Pb(2+)-exposed females had increased MOR binding in LPTN, medial thalamus, and hypothalamus. At PN28, Pb(2+)-exposed males had increased MOR levels in the striatum, stria medullaris of the thalamus, LPTN, medial thalamus, and basolateral amygdala, while Pb(2+)-exposed females showed an increase in nucleus accumbens core, LPTN, and medial thalamus. No changes were detected in any brain region of male and female rats at PN50, and at PN120 there was a decrease in MOR binding of Pb(2+)-exposed males in the medial thalamus. Our findings demonstrate age and gender specific effects of MOR levels in the rat brain as a result of chronic developmental Pb(2+) exposure. These results indicate that the major changes in brain MOR levels were during pre-adolescence and early adolescence, a developmental period in which there is higher engagement in reward and drug-seeking behaviors in humans. In summary, we show that chronic exposure to Pb(2+), an ubiquitous and well-known environmental contaminant and neurotoxicant, alters MOR levels in brain regions associated with addiction circuits in the adolescent period, these findings have important implications for opioid drug use and abuse.

Manganese (Mn) is essential for a variety of physiological processes, but at elevated levels, can be neurotoxic. While cognitive dysfunction has been recently appreciated to occur as a result of chronic Mn exposures, it is still unclear as to which cognitive domains are most susceptible to disruption by Mn exposure. We previously described early appearing Mn-induced changes in performance on a paired associate learning task in monkeys chronically exposed to Mn and suggested that performance of this task might be a sensitive tool for detecting cognitive dysfunction resulting from Mn exposure. As chronic Mn exposure has been suggested to be associated with attention, working memory and executive function deficits, the present study was conducted to assess the extent to which detrimental effects of chronic Mn exposure could be detected using tasks specifically designed to preferentially assess attention, working memory, and executive function. Six cynomolgus monkeys received Mn exposure over an approximate 12 month period and three served as control animals. All animals were trained to perform a self-ordered spatial search (SOSS) task and a five choice serial reaction time (5-CSRT) task. Deficits in performance of the SOSS task began to appear by the fourth month of Mn exposure but only became consistently significantly impaired beginning at the ninth month of Mn exposure. Performance on the 5-CSRT became significantly affected by the third month of Mn exposure. These data suggest that in addition to the paired associate learning task, cognitive processing speed (as measured by the 5-CSRT) may be a sensitive measure of Mn toxicity and that brain circuits involved in performance of the SOSS task may be somewhat less sensitive to disruption by chronic Mn exposure.

N-methyl-D-aspartate receptor (NMDAR) ontogeny and subunit expression are altered during developmental lead (Pb²+) exposure. However, it is unknown whether these changes occur at the synaptic or cellular level. Synaptic and extra-synaptic NMDARs have distinct cellular roles, thus, the effects of Pb²+ on NMDAR synaptic targeting may affect neuronal function. In this communication, we show that Pb²+ exposure during synaptogenesis in hippocampal neurons altered synaptic NMDAR composition, resulting in a decrease in NR2A-containing NMDARs at established synapses. Conversely, we observed increased targeting of the obligatory NR1 subunit of the NMDAR to the postsynaptic density (PSD) based on the increased colocalization with the postsynaptic protein PSD-95. This finding together with increased binding of the NR2B-subunit specific ligand [³H]-ifenprodil, suggests increased targeting of NR2B-NMDARs to dendritic spines as a result of Pb²+ exposure. During brain development, there is a shift of NR2B- to NR2A-containing NMDARs. Our findings suggest that Pb²+ exposure impairs or delays this developmental switch at the level of the synapse. Finally, we show that alter expression of NMDAR complexes in the dendritic spine is most likely due to NMDAR inhibition, as exposure to the NMDAR antagonist aminophosphonovaleric acid (APV) had similar effects as Pb²+ exposure. These data suggest that NMDAR inhibition by Pb²+ during synaptogensis alters NMDAR synapse development, which may have lasting consequences on downstream signaling.

Chronic manganese (Mn) exposure produces neurological deficits including a form of parkinsonism that is different from Parkinson's disease (PD). In chronic Mn exposure, dopamine neurons in the substantia nigra (SN) do not degenerate but they appear to be dysfunctional. Further, previous studies have suggested that the substantia nigra pars reticulata (SNr) is affected by Mn. In the present study, we investigated whether chronic Mn exposure induces microglia activation in the substantia nigra pars compacta (SNc) and SNr in Cynomolgus macaques. Animals were exposed to different weekly doses of Mn (3.3-5.0, 5.0-6.7, 8.3-10 mg Mn/kg body weight) and microglia were examined in the substantia nigra using LN3 immunohistochemistry. We observed that in control animals, LN3 labeled microglia were characterized by a resting phenotype. However, in Mn-treated animals, microglia increased in number and displayed reactive changes with increasing Mn exposure. This effect was more prominent in the SNr than in the SNc. In the SNr of animals administered the highest Mn dose, microglia activation was the most advanced and included dystrophic changes. Reactive microglia expressed increased iNOS, L-ferritin, and intracellular ferric iron which were particularly prominent in dystrophic compartments. Our observations indicate that moderate Mn exposure produces structural changes on microglia, which may have significant consequences on their function.

Chronic manganese (Mn) exposure produces a neurological syndrome with psychiatric, cognitive and parkinsonian features. Gene expression studies in the frontal cortex of Cynomolgus macaques exposed to different doses of Mn showed gene expression changes associated with cell cycle regulation, DNA repair, apoptosis, ubiquitin-proteasome system, protein folding, cholesterol homeostasis, axonal/vesicular transport and inflammation. Amyloid-beta (A-beta) precursor-like protein 1 (APLP1), a member of the amyloid precursor family, was the most highly up-regulated gene. Immunohistochemistry confirmed increased APLP1 expression and revealed the presence of A-beta diffuse plaques. Cortical neurons and white matter fibers from Mn-exposed animals exhibited accumulation of silver grains indicative of on-going degeneration. Cortical neurons also expressed nuclear hypertrophy, intracytoplasmic vacuoles, and apoptotis stigmata. The levels of p53 were increased in neurons and glial cells in Mn-exposed tissue. Analysis of another amyloidogenic protein, alpha-synuclein, also exhibited aggregation in the gray and white matter from Mn-exposed animals. In summary, chronic Mn exposure in non-human primates produces a cellular stress response leading to neurodegenerative changes, diffuse A-beta plaques and alpha-synuclein aggregation in the frontal cortex. These changes may help explain the cognitive and working memory deficits expressed by these animals.

Pavlovian fear conditioning is a model of emotional learning in which a neutral stimulus such as a tone is paired with an aversive stimulus such as a foot shock. Presentation of a tone with a foot shock in a context (test box) elicits a freezing response representative of stereotypic fear behavior. After conditioning has occurred, presentation of the context (test box) or tone in the absence of the unconditioned stimulus (shock) causes extinction of the fear response. Rats chronically exposed to environmentally relevant levels of lead (Pb(2+)) and controls were tested in a fear-conditioning (FC) paradigm at 50 days of age (PN50). Littermates to FC rats received an immediate shock (IS) when placed in the test box with no tone. Blood Pb(2+) levels in control and Pb(2+)-exposed animals were (mean+/-S.E.M.): 0.76+/-0.11 (n=15) and 25.8+/-1.28microg/dL (n=14). Freezing behavior was recorded during acquisition (day of training) or during 4 consecutive extinction days. Control and Pb(2+)-exposed FC rats exhibited the same level of freezing time on the acquisition day. No freezing behavior occurred in IS rats regardless of treatment. Presentation of context 24h later produced a freezing response on both control and Pb(2+)-exposed FC rats but not in IS rats. When tested in the extinction phase, Pb(2+)-exposed FC rats exhibited deficits in extinction compared to control FC rats. That is, when presented with context on 4 consecutive days after acquisition of the fear response, Pb(2+)-exposed FC rats exhibited a greater freezing response than control FC rats. These findings indicate that chronic Pb(2+) exposure produces a deficit in extinction learning and the animals remain more fearful than controls.

Oxidative stress has been implicated in the etiology of Parkinson's disease (PD) and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of PD. In this report we show that Nrf2, a transcription factor that regulates the expression of phase 2 and antioxidative enzymes, modulates MPTP neurotoxicity in rodents. Nrf2 knockout and wild-type mice were administered MPTP doses ranging from 20 to 60mg/kg. Seven days after MPTP administration dopamine transporter (DAT) levels were measured using [(125)I]-RTI-121 quantitative autoradiography as an index of dopamine terminal integrity in the striatum. The results indicate that MPTP administration resulted in a greater loss of DAT levels in the striatum of Nrf2 knockout mice than in wild-type at all MPTP doses tested. Activation of the Nrf2 pathway by oral administration of the Nrf2 inducer 3H-1,2-dithiole-3-thione (D3T) to wild-type mice produced partial protection against MPTP-induced neurotoxicity. The protective effect of D3T was not due to a change in MPTP metabolism since the level of the MPTP metabolite MPP+ was not significantly different in the D3T treated striatum relative to vehicle control. Administration of D3T to Nrf2 knockout mice did not protect against MPTP neurotoxicity suggesting that the Nrf2 pathway is necessary for the D3T-mediated attenuation of MPTP neurotoxicity. This study demonstrates the significance of activating intrinsic antioxidative mechanisms in an in vivo model of neurodegeneration. The in vivo activation of the Nrf2 pathway in the brain may be an important strategy to mitigate the effects of oxidative stress in neurodegenerative disorders and neurological disease.

What are the molecular bases for the neurotoxicity that occurs after developmental exposure to low levels of Pb2+, and are these effects persistent and detrimental in adults? Our inability to understand specific mechanisms behind Pb2+ neurotoxicity has long been one of many problem areas of this preventable childhood disease. The sensitivity of the developing brain to Pb2+-induced neurotoxicity is an outcome of the many unique characteristics that comprise the developing central nervous system. The developing brain can be exposed to significant concentrations of Pb2+ during vulnerable periods of development such as synapse formation, gene and protein expression, and other diverse molecular changes associated with these processes. Recently, changes in NMDA receptor subunits were identified in animals that showed cognitive deficits induced by exposure to Pb2+. This molecular association is important because it provides new evidence in the characterization of developmental Pb2+ neurotoxicity that supports physiological findings of impairments in synaptic plasticity and behavior. This review updates information from molecular studies that can be directly associated with impairments of behavior and synaptic plasticity, and outlines the functional consequences of molecular differences in Pb2+-exposed animals that illuminate potential mechanisms of Pb2+-induced neurotoxicity.