Pathophysiological studies of aging Slc39a14 knockout mice to assess the progression of manganese-induced dystonia-parkinsonism.

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.

Chronic early-life lead exposure sensitizes adolescent rats to cocaine: Role of the dopaminergic system.

Exposure to heavy metals has been associated with psychiatric disorders and recent studies suggest an association between childhood lead (Pb2+) intoxication and schizophrenia (SZ). In animal models, Pb2+ exposure recapitulates key neuropathological and dopaminergic system alterations present in SZ. Given the high comorbidity of mental disorders such as SZ and substance abuse, coupled with evidence showing that Pb2+ exposure affects addiction circuits, we hypothesized that early life Pb2+ exposure could sensitize neuronal systems relevant to SZ and substance abuse. To this goal, we examined the effects of chronic developmental Pb2+ exposure on the acute locomotor response to cocaine (0, 5, and 15 mg kg-1) and behavioral sensitization. We also examined the role of the dopaminergic system in the psychostimulant effects of cocaine, and measured D1-dopamine receptor (D1R) levels in the rat brain using [3H]-SCH23390 quantitative receptor autoradiography, as well as the ability of the D1R antagonist SCH23390 to block the cocaine effects on locomotor activation. These studies were performed in male and female rats at different developmental ages consisting of juveniles (postnatal, PN14), early-adolescent (PN28), late adolescent (PN50), and adults (PN120). Our results show that chronic developmental Pb2+ exposure increases the acute locomotor response to the higher dose of cocaine in Pb2+-exposed male adolescent (PN28 and PN50) rats, and to the lower dose of cocaine in adolescent female rats. No changes in the locomotor activity were detected in adult rats. Behavioral sensitization experiments showed a sustained sensitization in early adolescent Pb2+-exposed male but not female rats. The cocaine-induced effects on locomotor activity were abrogated by injection of a D1R antagonist suggesting the involvement of this dopamine receptor subtype. Furthermore, Pb2+-induced increases D1R levels in several brain regions were prominent in juveniles and early adolescence but not in late adolescence or in adults. In summary, early chronic developmental Pb2+ exposure results in age and sex-dependent effect on the locomotor response to cocaine, suggesting differential susceptibilities to the neurotoxic effects of Pb2+ exposure. Our data provides further support to the notion that Pb2+ exposure is an environmental risk factor for psychiatric disorders and substance abuse.

Imaging neuroinflammation with TSPO: A new perspective on the cellular sources and subcellular localization.

Translocator Protein 18 kDa (TSPO), previously named Peripheral Benzodiazepine Receptor, is a well-validated and widely used biomarker of neuroinflammation to assess diverse central nervous system (CNS) pathologies in preclinical and clinical studies. Many studies have shown that in animal models of human neurological and neurodegenerative disease and in the human condition, TSPO levels increase in the brain neuropil, and this increase is driven by infiltration of peripheral inflammatory cells and activation of glial cells. Therefore, a clear understanding of the dynamics of the cellular sources of the TSPO response is critically important in the interpretation of Positron Emission Tomography (PET) studies and for understanding the pathophysiology of CNS diseases. Within the normal brain compartment, there are tissues and cells such as the choroid plexus, ependymal cells of the lining of the ventricles, and vascular endothelial cells that also express TSPO at even higher levels than in glial cells. However, there is a paucity of knowledge if these cell types respond and increase TSPO in the diseased brain. These cells do provide a background signal that needs to be accounted for in TSPO-PET imaging studies. More recently, there are reports that TSPO may be expressed in neurons of the adult brain and TSPO expression may be increased by neuronal activity. Therefore, it is essential to study this topic with a great deal of detail, methodological rigor, and rule out alternative interpretations and imaging artifacts. High levels of TSPO are present in the outer mitochondrial membrane. Recent studies have provided evidence of its localization in other cellular compartments including the plasma membrane and perinuclear regions which may define functions that are different from that in mitochondria. A greater understanding of the TSPO subcellular localization in glial cells and infiltrating peripheral immune cells and associated function(s) may provide an additional layer of information to the understanding of TSPO neurobiology. This review is an effort to outline recent advances in understanding the cellular sources and subcellular localization of TSPO in brain cells and to examine remaining questions that require rigorous investigation.

Behavioral and neurochemical studies of inherited manganese-induced dystonia-parkinsonism in Slc39a14-knockout mice.

Inherited autosomal recessive mutations of the manganese (Mn) transporter gene SLC39A14 in humans, results in elevated blood and brain Mn concentrations and childhood-onset dystonia-parkinsonism. The pathophysiology of this disease is unknown, but the nigrostriatal dopaminergic system of the basal ganglia has been implicated. Here, we describe pathophysiological studies in Slc39a14-knockout (KO) mice as a preclinical model of dystonia-parkinsonism in SLC39A14 mutation carriers. Blood and brain metal concentrations in Slc39a14-KO mice exhibited a pattern similar to the human disease with highly elevated Mn concentrations. We observed an early-onset backward-walking behavior at postnatal day (PN) 21 which was also noted in PN60 Slc39a14-KO mice as well as dystonia-like movements. Locomotor activity and motor coordination were also impaired in Slc39a14-KO relative to wildtype (WT) mice. From a neurochemical perspective, striatal dopamine (DA) and metabolite concentrations and their ratio in Slc39a14-KO mice did not differ from WT. Striatal tyrosine hydroxylase (TH) immunohistochemistry did not change in Slc39a14-KO mice relative to WT. Unbiased stereological cell quantification of TH-positive and Nissl-stained estimated neuron number, neuron density, and soma volume in the substantia nigra pars compacta (SNc) was the same in Slc39a14-KO mice as in WT. However, we measured a marked inhibition (85-90%) of potassium-stimulated DA release in the striatum of Slc39a14-KO mice relative to WT. Our findings indicate that the dystonia-parkinsonism observed in this genetic animal model of the human disease is associated with a dysfunctional but structurally intact nigrostriatal dopaminergic system. The presynaptic deficit in DA release is unlikely to explain the totality of the behavioral phenotype and points to the involvement of other neuronal systems and brain regions in the pathophysiology of the disease.

Chronic developmental lead exposure increases µ-opiate receptor levels in the adolescent rat brain.

Childhood lead (Pb2+) 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 Pb2+ 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 Pb2+ exposure (1500 ppm in the diet) on µ-opioid receptor (MOR) levels in the rat brain using [3H]-d-Ala2-MePhe4-Gly-ol5 enkephalin ([3H]-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 Pb2+ exposure increases the levels of [3H]-DAMGO specific binding to MOR in juvenile and early adolescent Pb2+-exposed male and female rat brain with no changes in late-adolescent (PN50) and minor changes in Pb2+-exposed adult male rats (PN120). Specifically, at PN14, Pb2+-exposed males had an increase in MOR binding in the lateral posthalamic nuclei (LPTN), and Pb2+-exposed females had increased MOR binding in LPTN, medial thalamus, and hypothalamus. At PN28, Pb2+-exposed males had increased MOR levels in the striatum, stria medullaris of the thalamus, LPTN, medial thalamus, and basolateral amygdala, while Pb2+-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 Pb2+-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 Pb2+ 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 Pb2+, 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.

Awake delta and theta-rhythmic hippocampal network modes during intermittent locomotor behaviors in the rat.

Delta-frequency network activity is commonly associated with sleep or behavioral disengagement accompanied by a dearth of cortical spiking, but delta in awake behaving animals is not well understood. We show that hippocampal (HC) synchronization in the delta frequency band (1-4 Hz) is related to animals' locomotor behavior using detailed analyses of the HC local field potential (LFP) and simultaneous head- and body-tracking data. In contrast to running-speed modulation of the theta rhythm (6-10 Hz), delta was most prominent when animals were stationary or moving slowly, that is, when theta and fast gamma (65-120 Hz) were weak, and often developed rapidly when animals paused briefly between runs. We next combined time-frequency decomposition of the LFP with hierarchical clustering algorithms to categorize momentary estimations of the power spectral density (PSD) into putative modes of HC activity. Delta and theta power were strikingly orthogonal across spectral modes, as well as across bouts of precisely defined running and stationary behavior. Delta-band and theta-band coherences between HC recording sites were monotonically related to theta-delta ratios across modes; and whereas theta coherence between HC and medial prefrontal cortex (mPFC) increased during running, delta-band coherence between mPFC and HC increased during stationary bouts. Taken together, our findings suggest that delta-dominated network modes (and corresponding mPFC-HC couplings) represent functionally distinct circuit dynamics that are temporally and behaviorally interspersed among theta-dominated modes during navigation. As such, delta modes could play a fundamental role in coordinating encoding and retrieval mechanisms or decision-making processes at a timescale that segments event sequences within behavioral episodes. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

PET imaging of dopamine release in the frontal cortex of manganese-exposed non-human primates.

Humans and non-human primates exposed to excess levels of manganese (Mn) exhibit deficits in working memory and attention. Frontal cortex and fronto-striatal networks are implicated in working memory and these circuits rely on dopamine for optimal performance. Here, we aimed to determine if chronic Mn exposure alters in vivo dopamine release (DAR) in the frontal cortex of non-human primates. We used [11 C]-FLB457 positron emission tomography with amphetamine challenge to measure DAR in Cynomolgus macaques. Animals received [11 C]-FLB457 positron emission tomography scans with and without amphetamine challenge prior to Mn exposure (baseline), at different time points during the Mn exposure period, and after 10 months of Mn exposure cessation. Four of six Mn-exposed animals expressed significant impairment of frontal cortex in vivo DAR relative to baseline. One Mn animal had no change in DAR and another Mn animal expressed increased DAR relative to baseline. In the reversal studies, one Mn-exposed animal exhibited complete recovery of DAR while the second animal had partial recovery. In both animals, frontal cortex Mn concentrations normalized after 10 months of exposure cessation based on T1-weighted magnetic resonance imaging. D1-dopamine receptor (D1R) autoradiography in frontal cortex tissue indicates that Mn animals that experienced cessation of Mn exposure expressed D1R levels that were approximately 50% lower than Mn animals that did not experience cessation of Mn exposure or control animals. The present study provides evidence of Mn-induced alterations in frontal cortex DAR and D1R that may be associated with working memory and attention deficits observed in Mn-exposed subjects.

From the Cover: 7,8-Dihydroxyflavone Rescues Lead-Induced Impairment of Vesicular Release: A Novel Therapeutic Approach for Lead Intoxicated Children.

Childhood lead (Pb2+) intoxication is a public health problem of global proportion. Lead exposure during development produces multiple effects on the central nervous system including impaired synapse formation, altered synaptic plasticity, and learning deficits. In primary hippocampal neurons in culture and hippocampal slices, Pb2+ exposure inhibits vesicular release and reduces the number of fast-releasing sites, an effect associated with Pb2+ inhibition of NMDA receptor-mediated trans-synaptic Brain-Derived Neurotrophic Factor (BDNF) signaling. The objective of this study was to determine if activation of TrkB, the cognate receptor for BDNF, would rescue Pb2+-induced impairments of vesicular release. Rats were chronically exposed to Pb2+ prenatally and postnatally until 50 days of age. This chronic Pb2+ exposure paradigm enhanced paired-pulse facilitation of synaptic potentials in Schaffer collateral-CA1 synapses in the hippocampus, a phenomenon indicative of reduced vesicular release probability. Decreased vesicular release probability was confirmed by both mean-variance analysis and direct 2-photon imaging of vesicular release from hippocampal slices of rats exposed to Pb2+in vivo. We also found a Pb2+-induced impairment of calcium influx in Schaffer collateral-CA1 synaptic terminals. Intraperitoneal injections of Pb2+ rats with the TrkB receptor agonist 7,8-dihydroxyflavone (5 mg/kg) for 14-15 days starting at postnatal day 35, reversed all Pb2+-induced impairments of presynaptic transmitter release at Schaffer collateral-CA1 synapses. This study demonstrates for the first time that in vivo pharmacological activation of TrkB receptors by small molecules such as 7,8-dihydroxyflavone can reverse long-term effects of chronic Pb2+ exposure on presynaptic terminals, pointing to TrkB receptor activation as a promising therapeutic intervention in Pb2+-intoxicated children.

TSPO in a murine model of Sandhoff disease: presymptomatic marker of neurodegeneration and disease pathophysiology.

Translocator protein (18 kDa), formerly known as the peripheral benzodiazepine receptor (PBR), has been extensively used as a biomarker of active brain disease and neuroinflammation. TSPO expression increases dramatically in glial cells, particularly in microglia and astrocytes, as a result of brain injury, and this phenomenon is a component of the hallmark response of the brain to injury. In this study, we used a mouse model of Sandhoff disease (SD) to assess the longitudinal expression of TSPO as a function of disease progression and its relationship to behavioral and neuropathological endpoints. Focusing on the presymptomatic period of the disease, we used ex vivo [(3)H]DPA-713 quantitative autoradiography and in vivo [(125)I]IodoDPA-713 small animal SPECT imaging to show that brain TSPO levels markedly increase prior to physical and behavioral manifestation of disease. We further show that TSPO upregulation coincides with early neuronal GM2 ganglioside aggregation and is associated with ongoing neurodegeneration and activation of both microglia and astrocytes. In brain regions with increased TSPO levels, there is a differential pattern of glial cell activation with astrocytes being activated earlier than microglia during the progression of disease. Immunofluorescent confocal imaging confirmed that TSPO colocalizes with both microglia and astrocyte markers, but the glial source of the TSPO response differs by brain region and age in SD mice. Notably, TSPO colocalization with the astrocyte marker GFAP was greater than with the microglia marker, Mac-1. Taken together, our findings have significant implications for understanding TSPO glial cell biology and for detecting neurodegeneration prior to clinical expression of disease.

Presynaptic mechanisms of lead neurotoxicity: effects on vesicular release, vesicle clustering and mitochondria number.

Childhood lead (Pb2+) intoxication is a global public health problem and accounts for 0.6% of the global burden of disease associated with intellectual disabilities. Despite the recognition that childhood Pb2+ intoxication contributes significantly to intellectual disabilities, there is a fundamental lack of knowledge on presynaptic mechanisms by which Pb2+ disrupts synaptic function. In this study, using a well-characterized rodent model of developmental Pb2+ neurotoxicity, we show that Pb2+ exposure markedly inhibits presynaptic vesicular release in hippocampal Schaffer collateral-CA1 synapses in young adult rats. This effect was associated with ultrastructural changes which revealed a reduction in vesicle number in the readily releasable/docked vesicle pool, disperse vesicle clusters in the resting pool, and a reduced number of presynaptic terminals with multiple mitochondria with no change in presynaptic calcium influx. These studies provide fundamental knowledge on mechanisms by which Pb2+ produces profound inhibition of presynaptic vesicular release that contribute to deficits in synaptic plasticity and intellectual development.

Regional brain distribution of translocator protein using [(11)C]DPA-713 PET in individuals infected with HIV.

Imaging the brain distribution of translocator protein (TSPO), a putative biomarker for glial cell activation and neuroinflammation, may inform management of individuals infected with HIV by uncovering regional abnormalities related to neurocognitive deficits and enable non-invasive therapeutic monitoring. Using the second-generation TSPO-targeted radiotracer, [(11)C]DPA-713, we conducted a positron emission tomography (PET) study to compare the brains of 12 healthy human subjects to those of 23 individuals with HIV who were effectively treated with combination antiretroviral therapy (cART). Compared to PET data from age-matched healthy control subjects, [(11)C]DPA-713 PET of individuals infected with HIV demonstrated significantly higher volume-of-distribution (VT) ratios in white matter, cingulate cortex, and supramarginal gyrus, relative to overall gray matter VT, suggesting localized glial cell activation in susceptible regions. Regional TSPO abnormalities were evident within a sub-cohort of neuro-asymptomatic HIV subjects, and an increase in the VT ratio within frontal cortex was specifically linked to individuals affected with HIV-associated dementia. These findings were enabled by employing a gray matter normalization approach for PET data quantification, which improved test-retest reproducibility, intra-class correlation within the healthy control cohort, and sensitivity of uncovering abnormal regional findings.

Impairment of nigrostriatal dopamine neurotransmission by manganese is mediated by pre-synaptic mechanism(s): implications to manganese-induced parkinsonism.

The long-term consequences of chronic manganese (Mn) exposure on neurological health is a topic of great concern to occupationally-exposed workers and in populations exposed to moderate levels of Mn. We have performed a comprehensive assessment of Mn effects on dopamine (DA) synapse markers using positron emission tomography (PET) in the non-human primate brain. Young male Cynomolgus macaques were given weekly i.v. injections of 3.3-5.0 mg Mn/kg (n = 4), 5.0-6.7 mg Mn/kg (n = 5), or 8.3-10.0 mg Mn/kg (n = 3) for 7-59 weeks and received PET studies of various DA synapse markers before (baseline) and at one or two time points during the course of Mn exposure. We report that amphetamine-induced DA release measured by PET is markedly impaired in the striatum of Mn-exposed animals. The effect of Mn on DA release was present in the absence of changes in markers of dopamine terminal integrity determined in post-mortem brain tissue from the same animals. These findings provide compelling evidence that the effects of Mn on DA synapses in the striatum are mediated by inhibition of DA neurotransmission and are responsible for the motor deficits documented in these animals.

Developmental lead exposure impairs extinction of conditioned fear in young adult rats.

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.

Dysregulation of glutamate carboxypeptidase II in psychiatric disease.

Experimental evidence is beginning to converge on an important role for dysregulation of glutamate carboxypeptidase II (GCPII) in schizophrenia. The goal of this study was to determine GCPII levels in postmortem brain specimens of patients with schizophrenia, bipolar disorder or unipolar depression and age-matched control subjects. We used N-[N-(S)-1,3-dicarboxypropyl]carbamoyl]-S-3-[(125)I]iodo-l-tyrosine ([(125)I]DCIT), a high-affinity radioligand for GCPII, to probe for GCPII expression in prefrontal cortex (PFC) and mesial temporal lobe, two brain regions implicated in the pathophysiology of schizophrenia. We found that GCPII levels measured by [(125)I]DCIT quantitative autoradiography were significantly lower in the PFC and entorhinal cortex in patients with schizophrenia compared to age-matched controls. Patients with bipolar disorder also expressed significantly lower GCPII levels in PFC than controls. The decrease in [(125)I]DCIT binding in schizophrenia and bipolar disorder remained significant after adjusting for drug abuse. A significant difference in GCPII levels was also observed between schizophrenia relative to bipolar disorder and depressed subjects in the hippocampus-stratum lucidum and between schizophrenia and bipolar in the CA2 region of the hippocampus, with bipolar and depressed subjects expressing higher levels of GCPII than subjects with schizophrenia. These differences in hippocampal GCPII levels may implicate differences in the etiologies of these mental disorders. In summary, this study demonstrates a regional dysregulation of GCPII expression in the brain of patients with schizophrenia and other psychiatric disorders and supports a hypoglutamatergic state of the former illness. GCPII may represent a viable therapeutic target for intervention in psychiatric disease.

VMAT2 and dopamine neuron loss in a primate model of Parkinson's disease.

We used positron emission tomography (PET) to measure the earliest change in dopaminergic synapses and glial cell markers in a chronic, low-dose MPTP non-human primate model of Parkinson's disease (PD). In vivo levels of dopamine transporters (DAT), vesicular monoamine transporter-type 2 (VMAT2), amphetamine-induced dopamine release (AMPH-DAR), D2-dopamine receptors (D2R) and translocator protein 18 kDa (TSPO) were measured longitudinally in the striatum of MPTP-treated animals. We report an early (2 months) decrease (46%) of striatal VMAT2 in asymptomatic MPTP animals that preceded changes in DAT, D2R, and AMPH-DAR and was associated with increased TSPO levels indicative of a glial response. Subsequent PET studies showed progressive loss of all pre-synaptic dopamine markers in the striatum with expression of parkinsonism. However, glial cell activation did not track disease progression. These findings indicate that decreased VMAT2 is a key pathogenic event that precedes nigrostriatal dopamine neuron degeneration. The loss of VMAT2 may result from an association with alpha-synuclein aggregation induced by oxidative stress. Disruption of dopamine sequestration by reducing VMAT2 is an early pathogenic event in the dopamine neuron degeneration that occurs in the MPTP non-human primate model of PD. Genetic or environmental factors that decrease VMAT2 function may be important determinants of PD.

Evidence for cortical dysfunction and widespread manganese accumulation in the nonhuman primate brain following chronic manganese exposure: a 1H-MRS and MRI study.

Exposure to high levels of manganese (Mn) is known to produce a complex neurological syndrome with psychiatric disturbances, cognitive impairment, and parkinsonian features. However, the neurobiological basis of chronic low-level Mn exposure is not well defined. We now provide evidence that exposure to levels of Mn that results in blood Mn concentrations in the upper range of environmental and occupational exposures and in certain medical conditions produces widespread Mn accumulation in the nonhuman primate brain as visualized by T1-weighted magnetic resonance imaging. Analysis of regional brain Mn distribution using a "pallidal index equivalent" indicates that this approach is not sensitive to changing levels of brain Mn measured in postmortem tissue. Evaluation of longitudinal 1H-magnetic resonance spectroscopy data revealed a significant decrease (p = 0.028) in the N-acetylaspartate (NAA)/creatine (Cr) ratio in the parietal cortex and a near significant decrease (p = 0.055) in frontal white matter (WM) at the end of the Mn exposure period relative to baseline. Choline/Cr or myo-Inositol/Cr ratios did not change at any time during Mn exposure. This indicates that the changes in the NAA/Cr ratio in the parietal cortex are not due to changes in Cr but in NAA levels. In summary, these findings suggest that during chronic Mn exposure a significant amount of the metal accumulates not only in the basal ganglia but also in WM and in cortical structures where it is likely to produce toxic effects. This is supported by a significantly decreased, in the parietal cortex, NAA/Cr ratio suggestive of ongoing neuronal degeneration or dysfunction.

Nigrostriatal dopamine system dysfunction and subtle motor deficits in manganese-exposed non-human primates.

We tested the hypothesis that movement abnormalities induced by chronic manganese (Mn) exposure are mediated by dysfunction of the nigrostriatal dopamine system in the non-human primate striatum. Motor function and general activity of animals was monitored in parallel with chronic exposure to Mn and Positron Emission Tomography (PET) studies of in vivo dopamine release, dopamine transporters and dopamine receptors in the striatum. Analysis of metal concentrations in whole blood and brain was obtained and post-mortem analysis of brain tissue was used to confirm the in vivo PET findings. Chronic Mn exposure resulted in subtle motor function deficits that were associated with a marked decrease of in vivo dopamine release in the absence of a change in markers of dopamine (DA) terminal integrity or dopamine receptors in the striatum. These alterations in nigrostriatal DA system function were observed at blood Mn concentrations within the upper range of environmental, medical and occupational exposures in humans. These findings show that Mn-exposed non-human primates that exhibit subtle motor function deficits have an apparently intact but dysfunctional nigrostriatal DA system and provide a novel mechanism of Mn effects on the dopaminergic system.

Experience-dependent regulation of zif268 gene expression and spatial learning.

Environmental enrichment (EE) is known to enhance the cognitive ability of rodents. To translate EE to the human condition, it is important to understand the parameters of its efficacy. In this study, we examine if the cognitive enhancement associated with EE is permanent and whether a developmental window exists for its efficacy. Rats were housed in continuous isolation (ISO), continuous enrichment (EE), enrichment from postnatal day (PN) 21-50, and then isolation from PN50-79 (PM), or isolation from PN21-50 and then enriched from PN50-79 (CW). Spatial learning ability and basal expression of the immediate-early genes zif268 and Arc as well as the NR1 subunit of the NMDA receptor were assessed. Rats housed in an enriched environment at the time of testing (EE and CW) performed significantly better in the spatial learning task than rats housed in an isolated environment at the time of testing (ISO and PM). Enhanced performance in the spatial learning task was associated with a higher expression of zif268 only in the CA3/CA4 region of the hippocampus. Our study further defines parameters that make environmental enrichment effective in enhancing learning performance and the findings may be helpful in the translation of this intervention to the human condition.

Acute manganese administration alters dopamine transporter levels in the non-human primate striatum.

We used positron emission tomography (PET) to measure non-invasively the effect of acute systemic administration to manganese sulfate (MnSO4) on dopamine transporter (DAT) levels in the living non-human primate brain. Baboons received [11C]-WIN 35,428 PET scans to measure DAT levels before and after acute MnSO4 administration. In one animal, we observed a 46% increase in DAT binding potential (BP), a measure of DAT binding site availability, 1 week after Mn administration. DAT levels returned to baseline values at 4 months and remained constant at 10 months after treatment. A subsequent single MnSO4 injection to the same animal also resulted in a 57% increase in DAT-BP, 2 days after administration. In a second animal, a 76% increase in DAT-BP relative to baseline was observed at 3 days after Mn injection. In this animal, the DAT-BP returned to baseline levels after 1 month. Using in vitro receptor binding assays, we found that Mn inhibits [3H]-WIN 35,428 binding to rat striatal DAT with an inhibitory constant (Ki) of 2.0+/-0.3mM (n=4). Saturation isotherms and Scatchard analysis of [3H]-WIN 35,428 binding to rat striatal DAT showed a significant decrease (30%, p<0.001) in the maximal number of binding sites (Bmax) in the presence of 2mM MnSO4. No significant effect of Mn was found on binding affinity (Kd). We also found that Mn inhibits [3H]-dopamine uptake with an IC50 of 11.4+/-1.5mM (n=4). Kinetic studies and Lineweaver-Burk analysis showed a significant decrease (40%, p<0.001) in the maximal velocity of uptake (Vmax) with 5mM MnSO4. No significant effect of Mn was found on Michaelis-Menten constant (Km). These in vitro findings suggest that the increase in DAT levels in vivo following acute Mn administration may be a compensatory response to its inhibitory action on DAT. These findings provide helpful insights on potential mechanisms of Mn-induced neurotoxicity and indicate that the DAT in the striatum is a target for Mn in the brain.

Glutamate carboxypeptidase II levels in rodent brain using [125I]DCIT quantitative autoradiography.

The ability to visualize quantitatively glutamate carboxypeptidase II (GCPII) levels in vivo could advance our understanding of its function in health and disease. In the current study, we synthesized and evaluated a radiolabeled (iodine-125) analog of N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-S-3-iodo-L-tyrosine (DCIT), a potent antagonist of GCPII activity. We examined the regional distribution of [125I]DCIT binding in the rodent brain using quantitative autoradiography in order to confirm the validity of this radioligand as a marker of GCPII in the brain. The ultimate goal is to develop an imaging agent for assessing GCPII levels in the living brain. The specific binding of [125I]DCIT to rat brain followed a regional distribution consistent with previous studies describing regional brain GCPII gene expression and activity. We found a modest rostrocaudal gradient in which specific binding of [125I]DCIT to GCPII was lowest in cortical regions, with increasing levels of binding in midbrain structures and high levels of binding in hindbrain and brainstem. Autoradiography of [125I]DCIT in GCPII knockout and wild type mouse brain showed a gene-dose dependency confirming the selectivity of this radioligand for GCPII. We propose that [125I]DCIT is a selective radioligand that can be used to quantify brain GCPII levels in vitro using quantitative autoradiography.