Waterborne Manganese Exposure Alters Plasma, Brain, and Liver Metabolites Accompanied by Changes in Stereotypic Behaviors

Overexposure to waterborne manganese (Mn) is linked with cognitive impairment in children and neurochemical abnormalities in other experimental models. In order to characterize the threshold between Mn-exposure and altered neurochemistry, it is important to identify biomarkers that positively correspond with brain Mn-accumulation. The objective of this study was to identify Mn-induced alterations in plasma, liver, and brain metabolites using liquid/gas chromatography–time of flight–mass spectrometry metabolomic analyses; and to monitor corresponding Mn-induced behavior changes. Weanling Sprague–Dawley rats had access to deionized drinking water either Mn-free or containing 1 g Mn/L for 6 weeks. Behaviors were monitored during the sixth week for a continuous 24 h period while in a home cage environment using video surveillance. Mn-exposure significantly increased liver, plasma, and brain Mn concentrations compared to control, specifically targeting the globus pallidus (GP). Mn significantly altered 98 metabolites in the brain, liver, and plasma; notably shifting cholesterol and fatty acid metabolism in the brain (increased oleic and palmitic acid; 12.57 and 15.48 fold change (FC), respectively), and liver (increased oleic acid, 14.51 FC; decreased hydroxybutyric acid, - 14.29 FC). Additionally, Mn-altered plasma metabolites homogentisic acid, chenodeoxycholic acid, and aspartic acid correlated significantly with GP and striatal Mn. Total distance traveled was significantly increased and positively correlated with Mn-exposure, while nocturnal stereotypic and exploratory behaviors were reduced with Mn-exposure and performed largely during the light cycle compared to unexposed rats. These data provide putative biomarkers for Mn-neurotoxicity and suggest that Mn disrupts the circadian cycle in rats

Organic Cation Transporter 3 and the Dopamine Transporter Differentially Regulate Catecholamine Uptake in the Basolateral Amygdala and Nucleus Accumbens

Regional alterations in kinetics of catecholamine uptake are due in part to variations in clearance mechanisms. The rate of clearance is a critical determinant of the strength of catecholamine signaling. Catecholamine transmission in the nucleus accumbens core (NAcc) and basolateral amygdala (BLA) is of particular interest due to involvement of these regions in cognition and motivation. Previous work has shown that catecholamine clearance in the NAcc is largely mediated by the dopamine transporter (DAT), but clearance in the BLA is less DAT‐dependent. A growing body of literature suggests that organic cation transporter 3 (OCT3) also contributes to catecholamine clearance in both regions. Consistent with different clearance mechanisms between regions, catecholamine clearance is more rapid in the NAcc than in the BLA, though mechanisms underlying this have not been resolved. We compared the expression of DAT and OCT3 and their contributions to catecholamine clearance in the NAcc and BLA. We found DAT protein levels were ~ 4‐fold higher in the NAcc than in the BLA, while OCT3 protein expression was similar between the two regions. Immunofluorescent labeling of the two transporters in brain sections confirmed these findings. Ex vivo voltammetry demonstrated that the magnitude of catecholamine release was greater, and the clearance rate was faster in the NAcc than in the BLA. Additionally, catecholamine clearance in the BLA was more sensitive to the OCT3 inhibitor corticosterone, while clearance in the NAcc was more cocaine sensitive. These distinctions in catecholamine clearance may underlie differential effects of catecholamines on behavioral outputs mediated by these regions

Age-dependent effects of protein restriction on dopamine release

FUNDING AND DISCLOSURE This work was supported by the Biotechnology and Biological Sciences Research Council [grant # BB/M007391/1 to J.E.M.], the European Commission [grant # GA 631404 to J.E.M.], The Leverhulme Trust [grant # RPG-2017-417 to J.E.M.] and the Tromsø Research Foundation [grant # 19-SG-JMcC to J. E. M.). The authors declare no conflict of interest. ACKNOWLEDGEMENTS The authors would like to acknowledge the help and support from the staff of the Division of Biomedical Services, Preclinical Research Facility, University of Leicester, for technical support and the care of experimental animals.Peer reviewedPublisher PD

Extracellular norepinephrine, norepinephrine receptor and transporter protein and mRNA levels are differentially altered in the developing rat brain due to dietary iron deficiency and manganese exposure

Abstract: Manganese (Mn) is an essential trace element, but overexposure is characterized by Parkinson's like symptoms in extreme cases. Previous studies have shown that Mn accumulation is exacerbated by dietary iron deficiency (ID) and disturbances in norepinephrine (NE) have been reported. Because behaviors associated with Mn neurotoxicity are complex, the goal of this study was to examine the effects of Mn exposure and ID-associated Mn accumulation on NE uptake in synaptosomes, extracellular NE concentrations, and expression of NE transport and receptor proteins. Sprague-Dawley rats were assigned to four dietary groups: control (CN; 35 mg Fe/kg diet), iron-deficient (ID; 6 mg Fe/kg diet), CN with Mn exposure (via the drinking water; 1 g Mn/L) (CNMn), and ID with Mn (IDMn) . 3 H-NE uptake decreased significantly (R = -0.753, p=0.001) with increased Mn concentration in the locus coeruleus, while decreased Fe was associated with decreased uptake of 3 H-NE in the caudate putamen (R = 0.436, p = 0.033) and locus coeruleus (R = 0.86; p < 0.001). Extracellular concentrations of NE in the caudate putamen were significantly decreased in response to Mn exposure and ID (p < 0.001). A diverse response of Mn exposure and ID was observed on mRNA and protein expression of NE transporter (NET) and α 2 adrenergic receptor. For example, elevated brain Mn and decreased Fe caused an approximate 50% decrease in NET and α 2 adrenergic receptor protein expression in several brain regions, with reductions in mRNA expression also observed. These data suggest that Mn exposure results in a decrease in NE uptake and extracellular NE concentrations via altered expression of transport and receptor proteins

Manganese exposure inhibits the clearance of extracellular GABA and influenced taurine homeostasis in the striatum of developing rats.

Abstract: Manganese (Mn) accumulation in the brain has been shown to alter the neurochemistry of the basal ganglia. Mn-induced alterations in dopamine biology are fairly well understood, but recently more evidence has emerged characterizing the role of γ-aminobutyric acid (GABA) in this dysfunction. The purpose of this study was to determine if the previously observed Mninduced increase in extracellular GABA (GABA EC ) was due to altered GABA transporter (GAT) function, and whether Mn perturbs other amino acid neurotransmitters, namely taurine and glycine (known modulators of GABA). Extracellular GABA, taurine, and glycine concentrations were collected from the striatum of control (CN) or Mn-exposed Sprague-Dawley rats using in vivo microdialysis, and the GAT inhibitor nipecotic acid (NA) was used to probe GAT function. Tissue and extracellular Mn levels were significantly increased, and the Fe:Mn ratio was decreased 36-fold in the extracellular space due to Mn-exposure. NA led to a 2-fold increase in GABA EC of CNs, a response that was attenuated by Mn. Taurine responded inversely to GABA, and a novel 10-fold increase in taurine was observed after the removal of NA in CNs. Mn blunted this response and nearly abolished extracellular taurine throughout collection. Striatal taurine transporter (Slc6a6) mRNA levels were significantly increased with Mn-exposure, and Mn significantly increased 3 H-Taurine uptake after 3-min exposure in primary rat astrocytes. These data suggest that Mn increases GABA EC by inhibiting the function of GAT, and that perturbed taurine homeostasis potentially impacts neural function by jeopardizing the osmoregulatory and neuromodulatory functions of taurine in the brain. Article: INTRODUCTION An essential trace element and a cofactor for several enzymes (Hurley and Keen, 1987), manganese (Mn) is involved in immune function, regulation of metabolism, reproduction, digestion, bone growth, and blood clotting (see review by With the intriguing findings that striatal extracellular GABA (GABA EC ) concentrations are higher due to Mn-exposure , and uptake of 3 H-GABA is attenuated by Mn-exposure in striatal synaptosomes Taurine is an abundant non-essential amino acid in the brain formed from cysteine. Traditionally, brain taurine is thought to function as an osmoregulator in cells (cell volume regulation), but has also been implicated in neuromodulation, possibly functioning as a neurotransmitter. Data exist suggesting that taurine functions as an anxiolytic agent We chose to look at the taurine/GABA relationship in the striatum because it is a known region for Mn accumulation In addition to GABA and taurine, we felt it was prudent to examine the effect of Mn on another amino acid neurotransmitter, glycine. Glycine is an abundant inhibitor neurotransmitter, similar to GABA, and it is known that taurine is a glycine receptor agonist Within the brain, astrocytes are the primary cells that maintain the composition of the extracellular fluid MATERIALS AND METHODS Animals Male weanling (post-natal day 21) Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) (n = 8 for microdialysis study; n = 6 for PCR gene expression and metal analysis studies) were randomly divided into two dietary treatment groups used in previous studies Cell cultures Rat primary cortical astrocyte cultures were purchased from Invitrogen (Carlsbad, CA) and certified for purity with >95% staining positive for the astrocytic marker glial fibrillary acidic protein (GFAP). Cells were grown in Dulbecco's Modified Eagle Media (D-MEM) with 15% fetal bovine serum (FBS), and maintained in a humidified atmosphere of 95% air/5% CO 2 at 37 °C. Manganese treatments were delivered using 0, 100, or 300 μM Mn in the form of MnCl 2 . These dose concentrations are based on previous studies in non-human primates reporting clinical symptoms of Mn neurotoxicity at brain concentrations of 300 μM, while 100 μM concentrations appeared to be asymptomatic H-Taurine Uptake of tritiated taurine ( 3 H-Taurine) was measured as described by Stereotaxic surgery After 5 weeks of dietary treatment and 1 week prior to microdialysis experiments, rats were anesthetized with ketamine-HCl (80 mg/kg) and xylazine (12 mg/kg) and maintained on a heating pad at 37 °C. The heads of the rats were shaved and wiped with a 5% povidone-iodine solution to reduce risk of infection. Sterile instruments and gloves were used throughout the surgical procedure. The rats were secured in the stereotaxic frame and an incision was made perpendicular to the bregma. A guide cannula (CMA/12, CMA Microdialysis, Acton, MA) was implanted into the striatum using the following coordinates: 2.4 mm lateral to the midline, 7.5 mm anterior to the lambda. The cannula was lowered to a depth of 2.5 mm, positioning it in the medial area of the striatum Microdialysis During week six of the dietary protocol, a microdialysis probe (CMA/12 Elite, CMA Microdialysis, Acton, MA) was inserted into the guide cannula and the rat was perfused with artificial cerebral spinal fluid (aCSF) (155 mM Na + , 0.83 mM Mg 2+ , 2.9 mM K + , 132.76 mM Cl − , 1.1 mM Ca + , pH 7.4) for 1 h at a flow rate of 1 μL/min. After perfusion, the flow rate was adjusted to 0.5 μL/min and 30 min fractions were collected in microtubes for a total of four and a half hours (9 samples per rat) in a refrigerated fraction collector (CMA Microdialysis, Acton, MA). This protocol has been used successfully in previous studies with stable neurotransmitter recovery in the dialysate CE-LIF analysis A protocol by RNA isolation and cDNA synthesis Total RNA was isolated from astrocyte monolayers and the striatum of control and Mn-exposed rats for quantitative PCR analysis. Tissue samples were stored in 1 mL of RNAlater ® solution (Ambion Inc., Austin, TX) and kept at −80 °C until analysis. Astrocytes were cultured in 6-well plates, then treated for 24 h with media containing 0, 100, or 300 μM Mn. Astrocytes were harvested in 500 μL denaturation solution (Ambion Inc., Austin, TX). Tissue and cell culture RNA isolation were performed using the ToTALLY RNA™ system (Ambion Inc., Austin, TX), following manufacturer's instructions. RNA concentration and purity were determined by spectrophotometric analysis before carrying out cDNA synthesis. Synthesis of cDNA was performed using the High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA), following manufacturer's instructions. Quantitative PCR Quantitative real-time PCR analysis was utilized to determine differential mRNA expression between control and Mn-treated tissue or cell samples of the solute carrier family taurine transporter Slc6a6 (Applied Biosystems, Foster City, CA; Rn00567962_m1, Chr. 4 -125875817-125945795). Triplicate aliquots of cDNA were analyzed on 96-well plates using TaqMan ® Gene Expression assays (Applied Biosystems, Foster City, CA). Values of cDNA expression were normalized relative to the expression of β-actin (Rn00667869_m1, Chr. 12 -12047070-12050040) analyzed from the same sample on the same plate and reported as percent of control. Metal analyses Mn, Fe, and copper (Cu) concentrations were measured with graphite furnace atomic absorption spectrometry (Varian AA240, Varian, Inc., USA). Brain tissue from the striatum was digested in ultra-pure nitric acid (1:10, w/v dilution) for 48-72 h in a sand bath (60 °C). A 50 μL aliquot of digested tissue was brought to 1 mL total volume with 2% nitric acid for analysis. The extracellular striatal samples obtained via microdialysis were not diluted due to the small volume (20 μL) and the likelihood that this biological compartment has a low concentration of metals. Bovine liver (NBS Standard Reference Material, USDC, Washington, DC) (10 μg Mn/g; 184 μg Fe/g; 80 μg Cu/g) was digested in ultra-pure nitric acid and used as an internal standard for analysis (final concentration 5 μg Mn/L; 92 μg Fe/L; 10 μg Cu/L). Statistical analysis Data were analyzed using SPSS v14 for Windows (Microsoft, Redmond, WA). Metal, baseline microdialysis, and 3 H-Taurine uptake data were analyzed using paired-samples t-tests to examine the difference between Mn-treated samples and controls. Independent sample t-tests were used to examine time-point percent change differences in the microdialysis data, time-point 3 H-Taurine uptake changes, and significance between Mn-exposed versus control mRNA expression of Scl6a6. A p-value of <0.05 was considered significant. RESULTS Manganese and iron concentrations Mn-exposure resulted in significant alterations in compartmental metal concentrations. As expected, tissue Mn levels were significantly higher in Mn-exposed rats versus control (p = 0.001) ( Extracellular concentrations of taurine, GABA, and glycine Extracellular amino acid concentrations are differentially altered by Mn-exposure. Baseline levels of taurine and glycine were more abundant than GABA in the extracellular space, though Mn does not have a statistically significant effect on their levels compared to control No significant difference in baseline taurine levels was found between control and Mn-exposed animals Gly EC levels were similar in control and Mn-exposed groups, and no significant percent changes were observed between time-points within either control or Mn groups Limits of detection of the CE-LIF method employed for each neurotransmitter were found by serial dilution of derivatized standards until no discernable analyte peak could be obtained. Accordingly, limits of detection for GABA, glycine, and taurine were 6.9 ± 1.7 nM, 24 ± 5 nM, and 42 ± 21 nM, respectively, with linear dynamic ranges of 3.6 decades, 3.1 decades, and 3.3 decades, respectively. H-Taurine uptake Mn-exposure results in increased 3 H-Taurine uptake in astrocytes. After observing the unique effects of Mn-exposure on Tau EC in the striatum of rats in vivo, we decided to examine the effect of Mn-exposure on 3 H-Taurine uptake in primary rat astrocytes in vitro. Primary astrocytes exposed to Mn revealed a slight (30%) decrease in taurine uptake after 1 min, followed by a significant (219%) increase after 3 min (p = 0.034) Fig. 2: 3 H-Taurine uptake in primary astrocytes. Primary astrocytes, seeded 2 × 10 −5 in 6-well plates (n = 6) then grown to confluence, were cultured with either Mn-treated (300 μM MnCl 2 ) or control media. After 24 h cultures were exposed to 3 H-Taurine for 1, 3, or 6 min and analyzed for 3 H-Taurine retention. The inset represents percent change in uptake due to Mn-exposure expressed as percent control ± SEM. A significant (p = 0.034) increase in 3 HTaurine uptake was observed after 3 min of exposure in the Mn-treated astrocytes versus control. *p < 0.05 via independent samples t-test between Mn and control treatment groups at each time-point. Gene expression of taurine transporter Mn-exposure increased taurine transporter gene expression in the rat brain, but not cultured astrocytes. Quantitative RT-PCR analysis was conducted on primary astrocytes and striatal brain tissue to determine whether or not taurine transporter (Slc6a6) gene expression reflected the observed Mn-induced alterations in Tau EC and 3 H-uptake. Chronic Mn-exposure caused a significant (p = 0.045) increase in striatal Slc6a6 mRNA levels compared to control DISCUSSION The purpose of this study was to examine the effect of Mn on GAT-mediated GABA uptake. Knowing that glycine and taurine are important amino acid neurotransmitters that are known to modulate GABA neurochemistry Fig. 4: Working model for Mn-induced GABA and taurine alterations. The dynamic shifts in neurotransmitter concentrations observed in response to nipecotic acid (NA) (panels A, B, and C) are mitigated by Mn (panels D, E, and F). We hypothesize this lack of response in Mn-exposed rats is driven by decreased GABA transporter (GAT-1) function. (A) The control panel displays GABA EC and Tau EC under normal conditions, representing baseline microdialysis measurements. All percent change (% change) comparisons in subsequent panels are based on the % change from baseline levels, represented in the control panel. Under normal conditions GABA EC binds to GABA A receptors (GABA A -R) allowing chloride ion (Cl 2− ) movement for inhibitory hyperpolarization of post-synaptic neurons, while pre-synaptic binding to GABA B receptors (GABA B -R) regulates GABA releas