Certain metals that are necessary for regulating biological function at trace levels hold the potential tobecome neurotoxic when in excess. Specifically, chronic exposure to high levels of manganese leads tomanganism, a neurological disorder that exhibits both motor and learning deficits similar to Parkinson’sdisease. Since Parkinson’s disease symptomatology is primarily attributed to dopamine neurodegener-ation in the striatum, dopamine system dysfunction has been implicated in the onset of manganism. Inthis study, dopamine system function in the dorsal striatum was evaluated in C57Bl/6 mice, 1, 7, and 21days following repeated injections of manganese(II) chloride (50 mg/kg, subcutaneous) intermittently for7 days. Tissue content analysis confirmed the presence of persistent accumulation of manganese in thestriatum up to 21 days after cessation of treatment. In vitro fast scan cyclic voltammetry examined theeffect of sub-acute manganese on electrically stimulated dopamine release and uptake in the striatum.While no difference was observed in uptake rates following manganese treatment, dopamine release was
attenuated on days 7 and 21, compared to control levels. Basal levels of extracellular dopamine deter-mined by the zero net flux microdialysis method were significantly lower in manganese-treated mice at 7days post-treatment. On the other hand, potassium stimulated increases in extracellular dopamine wereattenuated at all three time points. Together, these findings indicate that repeated manganese exposurehas long-term effects on the regulation of exocytotic dopamine release in the striatum, which may be
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. Introduction
Manganese (Mn) is a key component for cellular function (Namnd Kim, 2008), immunological response, adenosine triphosphateATP) regulation, digestion, and reproduction (Burton and Guilarte,009). Mn is also required for metalloenzymes such as superoxideismutase and glutamine synthetase (Aschner and Aschner, 2005).ormal levels of Mn intake by dietary consumption are around–5 mg per day and regulating homeostatic levels of Mn is crucialor sustained functioning of the body in several aspects (Aschnernd Aschner, 2005). Mn deficiency has been linked to fertility prob-ems, birth defects, stunted growth, and impaired formation ofones (Aschner and Aschner, 2005). On the other end of the spec-rum, overexposure to Mn can lead to accumulation in the brainnd subsequent neurotoxicity. High exposure can occur throughccupations in welding (Bowler et al., 2007), mining (Montes et al.,008; Rodriguez-Agudelo et al., 2006), and battery assembly (Badert al., 1999), amongst others. Elevated Mn levels can also result
rom medical conditions such as liver (Hauser et al., 1994) or renalda Silva et al., 2007; Ohtake et al., 2005) failure or iron deficiencyFitsanakis et al., 2008; Kim et al., 2005), both of which inhibit the
clearance of Mn from the body. Manganism, the resultant neurolog-ical disorder caused by overexposure to Mn, leads to a progressionof symptoms that mimic those of Parkinson’s disease. In the earlystages of manganism, general symptoms such as headaches, hyper-somnia, psychosis, irritability, anorexia, and apathy (Mergler andBaldwin, 1997) begin to appear. As the disease progresses, work-ing memory impairment, reduced attention concentration (Josephset al., 2005; Klos et al., 2006; Mergler et al., 1994), and speechdeficits (Mergler and Baldwin, 1997) manifest. The final symptomsto emerge are primarily motor deficits, such as muscular rigidity,“intention tremors” (Pal et al., 1999), dystonia, bradykinesia, andgait changes (Burton and Guilarte, 2009).
Clinical magnetic resonance imaging has demonstrated thatpreferential accumulation of Mn occurs in the dopamine (DA)rich regions of the basal ganglia (Dietz et al., 2001). Although themethod by which Mn passes through the blood brain barrier (BBB)is not fully understood, calcium channels (Crossgrove and Yokel,2005), transferrin (Aschner and Gannon, 1994), the divalent metaltransporter 1 (DMT-1; Au et al., 2008), and N-methyl-d-aspartate(NMDA; Itoh et al., 2008) have been suggested to regulate BBBtransport of Mn.
Despite the similarities between the symptoms of manganismand Parkinson’s disease and the evidence implicating Mn overex-posure in the onset of Parkinson’s disease (Racette et al., 2001),these two neurological disorders appear to act on different path-
(20 �L) was injected onto a pyrocoated graphite atomizer tube withan integrated L’vov platform within a GFAAS system (Perkin Elmer,Uberlingen, Germany) equipped with an electrothermal atomizerand an autosampler. The detection of each metal was achieved
Table 1Analysis of trace metals, DA, and the DA metabolite DOPAC in dorsal striatum tissuehomogenized with HNO3 or HClO4. Within the HNO3 group, homogenization is com-pared to overnight digestion of tissue as a preparation method. All data reported asaverage (n = 5–6) of wet tissue weight (ng/mg) ± SEM. DA and DOPAC levels cannotbe detected by overnight digestion and are therefore reported as ‘not determined’(ND).
ays and regions in the brain. Within the basal ganglia, excess Mnccumulation associated with manganism occurs primarily in thelobus pallidus, caudate–putamen (CPu), and nucleus accumbensNAc) (Calne et al., 1994; Pal et al., 1999; Perl and Olanow, 2007). Inontrast, Parkinson’s disease etiology is primarily associated with
greater than 80% loss of nigrostriatal DA neurons in the CPu,hich originate in the substantia nigra pars compacta, as well as the
ppearance of Lewy bodies (Hornykiewicz, 1998). This significantegeneration of DA neurons in the substantia nigra pars compacta isot observed with the onset of manganism (Olanow, 2004; Olanowt al., 1996; Perl and Olanow, 2007). Furthermore, manganismatients show no difference in radiolabeled fluorodopa uptake (aseasured by positron emission tomography, PET), while Parkin-
on’s patients show reduced uptake (Kim, 2006; Pal et al., 1999).aired with the clinical differences seen in symptoms between thewo disorders, such as the “resting tremors” of Parkinson’s patientsersus the “intention tremors” of manganism’s, this informationuggests that manganism is distinct from Parkinson’s (Pal et al.,999). As a result, there is a need for further studies to better under-tand how Mn specifically acts upon the DA system, without relyingn the findings of Parkinson’s studies as an indirect means to under-tand the alterations occurring in the DA system upon Mn exposure.
Previous studies performed on non-human primates and ratsave indicated several impairments of the DA system as a resultf Mn administration (Guilarte et al., 2006, 2008; McDougallt al., 2008; Newland, 1999; Serra et al., 2000; Vidal et al., 2005).ecreases in amphetamine-induced DA release have been observed
n non-human primates (via PET), emphasizing that excessive accu-ulation of Mn impairs DA release (Guilarte et al., 2006, 2008).owever, reports of alterations in other presynaptic componentsf DA dynamics have been inconsistent. For example, some studieshow an increase in DA transporter expression/binding upon excessn exposure (Chen et al., 2006), while others show a decrease
r no change (Guilarte et al., 2006; Kim et al., 2002; McDougallt al., 2008). Additionally, the influence of Mn toxicity on the DA2 autoreceptor shows variable effects, ranging from decreased
o increased expression to no effect at all (Guilarte et al., 2006;essler et al., 2003; McDougall et al., 2011). The inconclusive evi-ence obtained thus far begs for a more in depth analysis of theffect of Mn exposure on the presynaptic DA system.
In this study, we used several complementary analytical tech-iques to probe the effect of a sub-acute manganese(II) chlorideMnCl2) exposure on DA dynamics in C57Bl/6 mice over a period ofhree weeks. The integration of three different techniques allowsor a novel methodological approach to correlate Mn tissue levelsith the functionality of the DA system. Tissue content analy-
is quantified intracellular Mn and DA levels present in the brainfter treatment was halted. Using in vivo microdialysis, the con-equences of Mn treatment on striatal extracellular DA levels, asell as levels of its metabolites, 3,4-dihydroxyphenylacetic acid
DOPAC) and homovanillic acid (HVA), were assessed. Additionally,ast scan cyclic voltammetry (FSCV) was used to characterize DAynamics, specifically DA clearance, electrically evoked DA release,nd functionality of presynaptic DA D2 autoreceptors. Althoughur results demonstrate no serious deleterious effects from theub-acute administration of MnCl2 on DA tissue content levels, theicrodialysis and voltammetry results highlight subtle but signif-
cant alterations on the DA release mechanism.
. Materials and methods
.1. Animals
C57Bl/6 mice were obtained from Jackson Laboratories and bredn-house for all experiments. Mice were group housed (3–4 animals
e Methods 202 (2011) 182– 191 183
per cage) at Wayne State University’s animal care facilities. A 12 hlight/dark cycle was used with food and water available ad libitum.Tissue content and microdialysis studies were performed on malesonly, weighing 23–25 g. Voltammetry experiments were conductedon both male and female mice weighing 23–33 g. All protocols andanimal care followed guidelines set by the National Institutes ofHealth Office of Animal Care and Use and were approved by theWayne State University Institutional Animal Care and Use Com-mittee.
2.2. Manganese treatment
Mice were injected subcutaneously with manganese(II) chlo-ride tetrahydrate (MnCl2·4H2O) in a sub-acute treatment based ona published protocol shown to significantly increase Mn concen-trations in the basal ganglia (Dodd et al., 2005). Briefly, animalswere randomly assigned to two treatment groups and injected witheither 0.1 mL of Mn (50 mg/kg, in saline) or saline (0.9% NaCl). Thistreatment was repeated every third day for a week, for a total of 3treatments on days 1, 4, and 7. After the last injection, neurochemi-cal analyses by three different techniques were conducted at threetime points, 1, 7, and 21 days later, to assess both the short and longterm effects of Mn exposure.
2.3. Tissue content analysis for neurotransmitter and metaldetection
Brain tissue samples were analyzed for metal accumulation, DA,and its metabolite DOPAC. Briefly, mice were sacrificed by cervi-cal dislocation and brains were removed, dissected, and weighed.To determine which acid provided optimal recovery for both DAand Mn from the CPu, samples were homogenized in 400 �L ofeither 0.1 M HNO3 or 0.1 M HClO4. Overall, HNO3 provided the bestrecovery (Table 1) and it was used to prepare all subsequent tissuesamples. For all experiments, tissue homogenates were centrifugedfor 15 min at 12,000 × g at 4 ◦C in 400 �L of 0.1 M HNO3. Metal lev-els for Mn, Cu, and Fe were assayed using graphite furnace atomicabsorption spectrometery (GFAAS). DA and DOPAC were detectedin the supernatant using high performance liquid chromatography(HPLC) coupled to an electrochemical detector.
All tissue samples were analyzed for Mn, Cu, or Fe using GFAASas previously described (Erikson et al., 1997; Jaganathan andAggarwal, 1993; Liu et al., 2000). After tissue samples were homog-enized and centrifuged, they were diluted with 0.2% HNO3 and5 �g/mL Mg(NO3)2 was added as a matrix modifier. Each sample
* P < 0.001 compared to homogenized sample in HNO3 group (two-tailed Stu-dent’s t-test).
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sing an element specific hollow cathode lamp. The lamp wasperated at 20 mA, with a wavelength of 279.5 nm (Mn), 324.8 nmCu), or 248.3 nm (Fe). The slit width was 0.2 nm for both Mn ande and 0.7 nm for Cu. The GFAAS parameters were optimized tobtain the highest absorbance value and maximum pyrolysis tem-erature possible without loss of analyte. Metal peak areas were
ntegrated using Perkin Elmer WinLab32 software and quantifiedgainst known standards.
To assay tissue samples for the neurotransmitter DA and itsetabolite DOPAC, tissue samples were homogenized and cen-
rifuged as described above. DA and DOPAC from each sampleupernatant (20 �L) were separated and detected in a single chro-atogram using a Shimadzu LC-20AD HPLC (Shimadzu, Columbia,D) with electrochemical detection (ESA 5011 cell; +220 mV rela-
ive to a Pd reference electrode). The isocratic separation of analytesas achieved using a Luna C18 (100 mm × 3 mm) column with 3 �marticles (Phenomenex, Torrance, CA) with the same mobile phasesed for microdialysis sample separation (see Section 2.5) at a flowate of 0.55 mL/min (Szapacs et al., 2004). Neurotransmitter peakreas were integrated using Shimadzu LC Solutions software anduantified against known standards.
.4. Microdialysis surgery
Male C57Bl/6 mice (5–8 weeks old) were anesthetized withvertin (20 mL/kg) by intraperitoneal injection (Papaioannou andox, 1993). Once anesthetized, eyes were protected with sterilephthalmic ointment and the skin over the skull was shaved andterilized with Betadine and alcohol. A small incision was madebove the skull, which was cleaned and dehydrated with 10% H2O2.ice were placed on a stereotaxic frame for implantation of a
uide cannula (CMA/Microdialysis, Chelmsford, MA) targeted to theorsal striatum (coordinates in mm: A +0.8, L −1.3, V −2.5 fromregma), based on the Paxinos mouse atlas (Paxinos and Franklin,001). The guide cannula was secured in place using fast dryingental cement (Teets, Diamond Springs, CA). Following dialysisxperiments, mice were euthanized by carbon dioxide asphyxia-ion and brains were removed for histological confirmation of probelacement.
.5. Microdialysis
Approximately 6 h after surgery, the dummy cannulaas removed and the microdialysis probe (CMA/7, 2 mm
ength, 240 �m diameter, Cuprophane, 6 kDa MW cutoff;MA/Microdialysis, Chelmsford, MA) was inserted and per-
used overnight with artificial cerebrospinal fluid (aCSF; in mM:45 NaCl, 3.5 KCl, 2.0 Na2HPO4, 1.2 MgCl2, 1.0 CaCl2, pH 7.4) at aow rate of 1.1 �L/min. After the dialysis probe was inserted, theialysate outlet was inserted into a microcentrifuge tube on icend an overnight sample (collection period ∼12 h) was collected.he next morning, at least three metabolite baseline samples wereollected at 20-min intervals to measure extracellular DOPAC andVA levels. Next, at least 3 baseline samples were collected toeasure extracellular concentrations of DA. After the collection of
aseline samples, the saline- and Mn-treated mice were dividednto two groups for two different types of experiments: (1) zeroet flux analysis or (2) stimulation of DA release with 120 mM KClCSF.
The quantitative microdialysis method of zero net flux waserformed to determine basal extracellular DA levels in salinend Mn-treated mice. A programmable gradient infusion pump
CMA/402) was used to perfuse 5, 10, and 20 nM DA (prepared inCSF) through the microdialysis probe to the striatum for 90 minach (Cosford et al., 1996; Justice, 1993; Lonnroth et al., 1987;athews et al., 2004). Typically, when using this method, the
e Methods 202 (2011) 182– 191
amount of analyte being perfused into the microdialysis probe isnot measured, as it is assumed that DAin equals the targeted con-centration chosen for perfusion. Prior to the in vivo experiments,an in vitro calibration was performed to more accurately determinethe actual DAin values (Mathews et al., 2004). Dialysate DA concen-trations were determined following perfusion of 5, 10, and 20 nMDA via the same set up, only in the absence of the animal. SinceDA is easily oxidized to its ortho-quinone, the DA stock standardsolution (1 mM) was prepared in 0.1 M perchloric acid, 10 �g/mLascorbic acid, and 10% methanol, and aliquots (250 �L) were storedat −80 ◦C (Acworth and Cunningham, 1999). Zero net flux solutionswere made fresh daily by successive dilution of the stock standardsolution in aCSF containing 200 �M ascorbic acid.
Microdialysis samples (20 �L) were manually injected onto aLuna 100 mm × 3 mm, C18, 2.6 �m column (Phenomenex, Torrance,CA) for the separation of DA and its metabolites (DOPAC andHVA), followed by electrochemical detection using an ESA 5041 cell(ESA Coulochem III, Chelmsford, MA) with an applied potential of+220 mV relative to a palladium (Pd) reference electrode (Szapacset al., 2004). A guard cell (ESA 5020) was placed in line before theinjection loop and set at a potential of +350 mV. The isocratic sepa-ration was achieved using a mobile phase that consisted of 75 mMof NaH2PO4, 3.0 mM 1-octanesulfonic acid, 0.125 mM EDTA, 9.0%acetonitrile, and 0.2–0.5% triethylamine (pH ∼ 3.0). A flow rate of0.4 mL/min was used to detect the neurotransmitters in a singlechromatogram within 20 min. DA and its metabolite peak areaswere integrated using Shimadzu LC Solutions software and quan-tified against known standards.
In a second cohort of mice, isotonic aCSF containing 120 mM KCl(in mM: 120 KCl, 30.5 NaCl, 2.0 Na2HPO4, 1.2 MgCl2, 1.0 mM CaCl2,pH 7.4) was perfused through the probe for 20 min following base-line sample collections (Mathews et al., 2004; Trillat et al., 1997).Immediately after the 20 min of high K+ stimulation, the perfusatewas switched back to standard aCSF. This influx of K+ depolarizesneurons, inducing exocytosis and subsequently increasing extra-cellular DA levels. The effect of 120 mM K+ on extracellular DAlevels was determined in both saline- and Mn-treated mice. Thesesamples were analyzed by HPLC and electrochemical detection asdescribed for zero net flux experiments above. Additionally, thearea under the curve (AUC) for high K+ DA release was calculatedfrom 80 to 140 min (Fig. 3, inset) for all animal groups.
2.6. Fast scan cyclic voltammetry
For in vitro fast scan cyclic voltammetry (FSCV), mice were sac-rificed by decapitation following CO2 asphyxiation. The brain wasimmediately removed and placed in pre-oxygenated (95% O2/5%CO2) high sucrose aCSF (in mM: 180 sucrose, 30 NaCl, 26 NaHCO3,10 d-glucose, 4.5 KCl, 1.0 MgCl2·6H2O, 1.2 NaH2PO4, pH 7.4) on icefor 5–10 min (Lack et al., 2007). Coronal 400 �m thick slices thatencompassed the striatal brain region (which includes the CPu andNAc) were prepared using a vibrating tissue slicer (Vibratome®, St.Louis, MO). Slices were kept in a continuously oxygenated reservoirof aCSF (in mM: 126 NaCl, 25 NaHCO3, 11 glucose, 2.5 KCl, 2.4 CaCl,1.2 NaH2PO4, 1.2 MgCl2, 0.4 ascorbate, pH 7.4) for at least 1 h beforeuse. At the time of the experiment, a brain slice was transferred toa custom-made submersion chamber (Custom Scientific, Denver,CO) that was continuously perfused with aCSF and kept at 32 ◦C.
For all experiments, a two-electrode system was employed inwhich a Ag/AgCl reference electrode was paired with a carbon-fiber working microelectrode, inserted ∼75 �m deep into the slice.Carbon-fiber microelectrodes were made by first aspirating car-
bon fiber (diameter = 7 �m, Goodfellow, Oakdale, PA) through aglass capillary (A-M Systems, Carlsborg, WA). The capillary wassubsequently heated using a micropipette puller (Narishige, Tokyo,Japan) to form two electrodes, each with a tight seal at the glass-
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M. Khalid et al. / Journal of Neuro
ber junction. The exposed length of the carbon fiber was cut topproximately 50–200 �m. Each electrode was then filled with50 mM KCl and a lead wire (Squires Electronics, Cornelius, OR) toomplete the electrical connection. Using a ChemClamp potentio-tat (Dagan Corporation, Minneapolis, MN), potential was appliedt the carbon-fiber microelectrode in the shape of a triangularaveform, initially held at −0.4 V. That potential was ramped up to
1.2 V, then back down to −0.4 V at a scan rate of 400 V/s. This wave-orm was repeated every 100 ms. To evoke DA release in the brainlice, a bipolar stimulating tungsten electrode was placed approx-mately 100–200 �m away from the carbon-fiber microelectrode,ontrolled by TH software (ESA Inc., Chelmsford, MA). An electricaltimulation was applied to the slice by a single pulse (monophasic,50 �A, 60 Hz, 4 ms width) every 5 min.
DA release and uptake measurements were made after slicesere allowed to equilibrate in the submersion chamber for at
east 30 min. For receptor characterization, dose–response curvesere generated using pre-drug values for each animal as their
wn controls. DA dynamics were analyzed only after subtractingut the background current. Electrode calibration was performedost-experiment using a flow injection system. The electrode wasalibrated with a 3 �M DA solution in aCSF to obtain a calibrationactor, which was later used to convert the current to a concentra-ion. A Michaelis–Menten based set of equations were used to fithe current versus time profile of each release event to quantify the
aximum concentration of DA released per pulse ([DA]p), as well ashe rate of uptake by the DA transporter (�M/s; Vmax; Heien et al.,003; John and Jones, 2007; Jones et al., 1995; Wightman et al.,988).
Data obtained from FSCV experiments can be presented in one ofhe three ways, as depicted by representative plots in Fig. 5. The topraces in Fig. 5A are current versus time plots. The rise phase of theeak is predominately indicative of electrically evoked DA releaseamplitude), which is converted from current (y-axis) to a con-entration (�M) using the aforementioned calibration factor. Theescending phase of the peak (decay of peak returning to baseline)epresents DA uptake rates (�M/s). The insets show the respectiveyclic voltammograms, which plot the measured current (y-axis)gainst the potential (x-axis) applied to the electrode. The voltam-ograms are used to confirm the detection of DA by the location of
he oxidation peak of DA at +0.6 V and reduction peak of dopamine-rtho-quinone at −0.2 V. Below the two-dimensional plots is anll inclusive three-dimensional color plot which combines timex-axis), applied voltage to the working electrode (y-axis), and gen-rated current (z-axis; color scale). The characteristic oxidationurrent of DA is revealed as a bright green mark (an increase in cur-ent that corresponds to the amount of dopamine being oxidized)t approximately +0.6 V on the y-axis.
.7. Chemicals
Components of the mobile phase and neurotransmitter stan-ards were either of HPLC grade or the highest quality obtainedrom Sigma–Aldrich (St. Louis, MO) and Fisher Scientific (Pitts-urgh, PA). Concentrated HNO3, HClO4, boric acid, triethylamine,nd citric acid were purchased from EMD (Gibbstown, NJ). All stan-ards for calibration curves used in GFAAS and HPLC analysis wererepared fresh daily.
.8. Statistical analysis
Data were analyzed using GraphPad Prism software (GraphPad,
a Jolla, CA). All values are expressed as means ± standard error ofean (SEM) with a difference of P < 0.05 considered statistically
ignificant. Comparisons of two means were analyzed by Student’s-tests. When comparisons of three or more means were made, a
e Methods 202 (2011) 182– 191 185
one way analysis of variance (ANOVA) was performed. Two-wayANOVA with Bonferroni post-test was used when testing for inter-action of multiple variables.
3. Results
3.1. Tissue content protocol and analysis
The levels of striatal Mn, Fe, and Cu were analyzed by GFAASfirst to assess the extent to which the acid used for tissue treatmentinfluences metal recovery. Levels of DA and DOPAC were concur-rently determined from acid-digested tissue by HPLC. The striatumwas similarily homogenized in two different acids, 0.1 M HNO3 or0.1 M HClO4 (Table 1). Striatal Mn levels were unaffected by thechoice of acid used for homogenization (P = 0.1203), as were levelsof DA (P = 0.4809) and DOPAC (P = 0.0542). Striatal Cu (P < 0.0001)and Fe (P < 0.0001) levels were significantly reduced when the tis-sue was treated with HClO4 as compared to HNO3. For this reason,HNO3 was selected for all subsequent tissue content experiments.
Typically, metal levels in brain tissue are measured follow-ing sample digestion with a strong acid at 60 ◦C. However, highheat can degrade biological molecules, such as neurotransmitters,necessitating the use of cold acid solutions (Liu et al., 2000). Todetermine if lower temperatures and acid content would be suit-able for the recovery of trace metals, the striatum was dividedin half and the sides were either homogenized in 0.1 M HNO3 atroom temperature or digested overnight at 60 ◦C in pure HNO3. Nodifference was observed in the Mn levels extracted from homoge-nized tissues (0.92 ± 0.03 ng/mg wet weight; n = 6) compared to theovernight digested samples (0.96 ± 0.04 ng/mg wet weight; n = 6;P = 0.38; Table 1). Since similar Mn levels, as well as comparablelevels of other trace metals (Table 1), were recovered from both tis-sue preparations, the cold acid homogenizing assay was chosen forall measurements so that parallel neurotransmitter analyses couldalso be performed.
Employing the optimized tissue content protocol discussedabove, Mn levels in the striatum were measured using GFAAS atthree time points (days 1, 7, and 21) after repeated Mn treatment(Fig. 1). Analysis by one-way ANOVA indicated a significant differ-ence in Mn tissue content in Mn-treated mice compared to salinecontrols (F3,33 = 112.9, P < 0.001, n = 7; Fig. 1A). On day 1 a 260%increase in Mn levels was observed in mice treated with Mn com-pared to saline controls (P < 0.001); this increase is similar to thatreported by Dodd et al. (2005) at the same time point. Mn accumu-lation persisted for up to 21 days after treatment, revealing a 150%(P < 0.001) and 30% (P < 0.05) increase on days 7 and 21, respectively.The same tissue homogenate was used to concurrently detect DAtissue levels, which primarily reflects the content of intraneuronalstores. No difference in intracellular striatal DA levels was observedbetween Mn and saline treated mice at any of the time points ofanalysis (F3,36 = 0.60, P = 0.62, n = 7–8; Fig. 1B).
3.2. Microdialysis
In vivo microdialysis was used to determine whether theaccumulation of Mn observed in striatal tissue influences theconcentration of DA in the extracellular space of freely movinganimals. Striatal extracellular DA levels (analyzed without correc-tion for probe recovery) showed a significant difference betweentreatment groups (F3,15 = 14.13, P = 0.0003; Fig. 2). Extracellular DAconcentrations averaged from three baseline samples were notdifferent between saline-treated mice (3.1 ± 0.1 nM; n = 10) and
Mn-treated mice when DA was measured 1 (3.3 ± 0.1 nM; n = 6) or21 (3.1 ± 0.3 nM; n = 8) days after Mn treatment. However, striatalDA levels were significantly decreased 7 days after Mn treatment(2.1 ± 0.1 nM; n = 8; P < 0.01).
186 M. Khalid et al. / Journal of Neuroscience Methods 202 (2011) 182– 191
Fig. 1. Striatal tissue (A) Mn and (B) DA levels in saline- and Mn-treated mice.DR*
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Fig. 3. Extracellular DA levels determined by zero net flux microdialysis (n = 5). (A)Striatal DA levels (Cext), corrected for in vivo probe recovery, are represented by thex-intercepts of the plotted linear regression lines. (B) Summary of extracellular DA
ashed line indicates the normalized values for saline-treated mice (set as 100%).esults are means (n = 7–8) expressed as percentage of saline-treated mice. *P < 0.05,**P < 0.001 compared to control group (one-way ANOVA).
Since a decrease in extracellular DA levels was observed only athe day 7 time point, we set out to determine if this DA decrease isctually persistent across all time points but is perhaps masked byther presynaptic events. Others have established that the methodf zero net flux can expose alterations in extracellular DA levelshat are masked by transporter-mediated changes in in vivo probeecovery (Menacherry et al., 1992; Parsons et al., 1991). Therefore,ero net flux was utilized to evaluate DA levels at the days 1 and1 time points, in addition to confirming the changes observed inxtracellular levels at day 7. Basal DA levels in saline-treated mice,etermined with zero net flux analysis, were similar at all timeoints and therefore the data were collapsed into one represen-ative group labeled saline controls. The average extracellular DAevels (corrected for probe recovery) for this combined saline con-rol group were 9.0 ± 1.0 nM (n = 5). Using zero net flux, a significant
ain effect of Mn treatment on extracellular DA in the striatum wasevealed (F3,19 = 6.31, P = 0.005; Fig. 3). In mice treated with Mn,
triatal DA levels were 7.0 ± 1.0 nM (n = 5) and 9.0 ± 1.0 nM (n = 5)
and 21 days after Mn treatment, respectively, which are not dif-erent from those observed in the saline control group. However,imilar to the results using conventional microdialysis, Mn-treated
ig. 2. Baseline dialysis levels of DA and its metabolites DOPAC and HVA in thetriatum of saline-treated versus Mn-treated mice. Results are reported as meann = 4–10) concentrations (nM) ± SEM. *P < 0.05, **P < 0.01 compared to respectiveontrols (one-way ANOVA).
levels corrected for in vivo probe recovery in saline- and Mn-treated mice. **P < 0.01compared to control group (one-way ANOVA).
mice showed a significant reduction in extracellular DA levels to2.6 ± 0.3 nM (n = 5; P < 0.05) 7 days after cessation of sub-acutetreatment.
In zero net flux experiments, the extraction fraction (Ed),determined from the slope of the regression lines, gives an approx-imation of the probe recovery (Lonnroth et al., 1987). The averageEd for saline controls (0.49 ± 0.04 nM) was found to be significantlydifferent (F3,18 = 7.87, P = 0.0022) from the extraction fraction at day1 (0.29 ± 0.03 nM; P < 0.01) and day 21 (0.29 ± 0.01 nM; P < 0.01) inMn-treated mice; however, day 7 values (0.37 ± 0.04 nM) showedno difference between the two groups.
Together, conventional and quantitative microdialysis resultsindicate that extracellular DA concentrations in the dorsal CPu aremore susceptible to the effects of Mn accumulation at the interme-diate time point of analysis (day 7) post-Mn exposure. In contrast,the relative recovery rate of DA was altered on days 1 and 21(but not on day 7), suggesting Mn treatment has different time-dependent biological effects on DA neurotransmission.
Extracellular levels of the DA metabolites DOPAC and HVA werealso evaluated to determine if Mn overexposure results in alter-ations in DA metabolism. One-way ANOVA demonstrated thatDOPAC levels were significantly different due to Mn treatment(F3,19 = 8.24, P = 0.002). On day 7, extracellular DOPAC in saline-treated mice was 800 ± 100 nM (n = 5), but only 300 ± 100 nM (n = 5)in Mn-treated mice. Similarly, striatal HVA levels showed a sig-nificant decrease after treatment (F3,18 = 7.00, P = 0.004). Dialysatesamples analyzed from saline- and Mn-treated mice at the day7 time point had mean HVA levels of 700 ± 80 nM (n = 6) and400 ± 40 nM (n = 4), respectively. No differences in DOPAC or HVAlevels were seen at any other time points. Thus, changes in extra-cellular levels of DA metabolites were only observed 7 days afterMn treatment, which is consistent with our baseline dialysate DAanalyses.
To investigate whether Mn treatment affects stimulated DArelease, extracellular DA levels were assessed by in vivo microdial-ysis following reverse dialysis with high-K+ aCSF. Twenty minutes
science Methods 202 (2011) 182– 191 187
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Fig. 4. Extracellular DA levels determined by in vivo microdialysis. Time versus DAconcentration plots of high K+ stimulated DA release. Black bar indicates 20 mintime fraction in which 120 mM KCl aCSF was applied. Results are means (n = 6–10)
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erfusion of high-K+ aCSF (120 mM KCl) maximally elevated extra-ellular DA levels (46 ± 5 nM) to ∼15-fold above the baseline levels3.1 ± 0.1 nM; n = 10) observed in saline-treated mice. Conversely,
n treatment attenuated stimulated DA release at all time points.A levels in Mn-treated mice were only increased to approximately2 ± 2 (n = 6), 24 ± 4 (n = 8), and 29 ± 5 (n = 8) nM at 1, 7, and 21 daysfter Mn treatment, respectively (Fig. 4). Two-way ANOVA showed
significant main effect of treatment (F1,126 = 13.19, P < 0.001)nd time (F8,126 = 83.57, P < 0.001) as well as a significant interac-ion (F8,126 = 13.02, P < 0.001), indicating Mn treatment significantlyltered high-K+ evoked increases in extracellular DA at all timeoints.
.3. Fast scan cyclic voltammetry
Due to the slow temporal resolution of microdialysis (20 minample intervals), this technique mainly detects net changes inhe extracellular levels of DA and its metabolites. To assess variousspects of neuronal DA dynamics on a sub-second time scale, FSCVas utilized. FSCV enables the investigation of DA system function-
lity by quantifying DA release and uptake. Electrically evoked DAelease was measured in the CPu of saline and Mn-treated mice 1,, and 21 days after treatment. Additionally, the rate at which theA transporter recycles extracellular DA back into the presynaptic
euron was compared between the control group and Mn-treatedroup at all three time points using a calculated Vmax value. SinceA release and uptake were comparable across all treatment timeoints for the saline-treated mice, these data were collapsed into
ig. 5. Electrically evoked DA release and uptake from the dorsal CPu as measured by
nd Mn-treated mice on day 21. Central figures show color plots integrating time, applief electrical stimulation (5 s). Current versus time traces (converted to concentration vhowing characteristic DA current versus voltage voltammograms. Summary of (B) electrn-treated mice. Data are mean (n = 8–14) ± SEM expressed as [DA]p (�M) or Vmax (�M/
of DA concentration (nM) ± SEM. Inset compares area under curve upon high K+
stimulation in saline- (control) versus Mn-treated mice. ***P < 0.001 compared tocontrol group (two-way ANOVA).
a single control group. This control group (n = 14) had an averageDA release of approximately 1.8 ± 0.1 �M and an average uptake
rate of 4.0 ± 0.1 �M/s. Analysis with one-way ANOVA determinedelectrically evoked DA release was significantly different in Mn-treated mice compared to controls (F3,131 = 4.136, P < 0.01; Fig. 5B).An approximate 33% attenuation in electrically evoked DA release
FSCV. (A) Representative concentration versus time and false color plot of saline-d potential, and current as false color 3D plots. Red triangle represents time point
ersus time using post-experiment electrode calibration) shown above, with insetically evoked DA release and (C) DA uptake rates in the CPu of saline- (control) ands). *P < 0.05 compared to control group (one-way ANOVA).
188 M. Khalid et al. / Journal of Neuroscienc
Fig. 6. Quinpirole dose–response curves in the CPu. Dose–response curves gener-ated by plotting the log concentration of quinpirole (M) versus the maximal releaseof DA per pulse, as a percent of baseline response (defined as 100%). Dose–responsect
wnnni
attMMft2sa
lar DA levels. Since the tissue content of DA is mainly reflective of
urve of quinpirole on stimulated release in saline-treated controls versus Mn-reated mice shown for (A) day 1, (B) day 7, and (C) day 21 (n = 3–10).
as observed 7 days after cessation of treatment (1.4 ± 0.1 �M; = 8); this decrease was sustained for up to 21 days (1.4 ± 0.1 �M; = 12). In contrast, the rate of DA uptake in the dorsal CPu wasot altered in Mn-treated mice at any time point (average approx-
mately 4.1 ± 0.2 �M/s, F3,105 = 0.53, P = 0.60; Fig. 5C).To test if alterations in the functionality of DA D2 autoreceptors
re the cause of the differences in DA levels detected following Mnreatment, the effect of the D2 receptor agonist quinpirole on elec-rically evoked DA release was measured in slices from saline- and
n-treated mice (Kennedy et al., 1992; Maina and Mathews, 2010;ottola et al., 2002). Dose–response curves were generated by per-
using increasing concentrations of quinpirole (0.001–10 �M) overhe brain slice at 30-min intervals (Fig. 6; Maina and Mathews,
010). For all treatment groups, quinpirole decreased electricallytimulated DA release (F7,66 = 119.5, P < 0.0001). The concentrationt which the pre-drug value of DA release was reduced by 50%
e Methods 202 (2011) 182– 191
from the maximum response (IC50) was determined for each of thetreatment groups. The IC50 for saline-treated mice was 71 ± 12 nM(n = 10) and 60 ± 12 nM (averaged across the 3 time points; n = 12)for Mn-treated mice, indicating that our protocol for Mn treatmentdoes not affect the functionality of release-regulating presynapticautoreceptors in the CPu (F3,29 = 0.13, P = 0.94).
4. Discussion
Herein, we demonstrated that a sub-acute MnCl2 exposure ele-vates striatal tissue Mn levels for at least 21 days after cessation oftreatment compared to saline-treated mice. Despite being unableto detect changes in intraneuronal DA levels, both in vivo microdial-ysis and in vitro FSCV reveal Mn-induced baseline and stimulatedrelease changes in extracellular DA, as well as differences in extra-cellular levels of the DA metabolites DOPAC and HVA. Contrary toliterature reports (Chen et al., 2006; Kessler et al., 2003; Kim et al.,2002; McDougall et al., 2008, 2011), these alterations in releasewere not coupled with any compensatory changes in DA uptake orD2 autoreceptor functionality.
Multiple administration methods (e.g. intraperitoneal, subcu-taneous, inhalation, and oral), Mn salts (e.g. MnCl2, MnSO4, andMn(OAc)3), and doses (e.g. 25–100 mg/kg, >300 mg/kg, single- ormultiple-dose regimes) have been used to study the subsequentneurological effects of Mn exposure (Aschner et al., 2005; Burtonand Guilarte, 2009; Guilarte, 2010). The primary consideration inchoosing an exposure protocol is whether or not the exogenousapplication of Mn results in a significant accumulation at the regionof interest. In a review by Burton and Guilarte (2009), it is sug-gested that cumulative doses any lower than 300 mg/kg do notsignificantly alter DA levels in the striatum of non-human primates.However in rodents, Dodd et al. (2005) have shown that an inter-mittent, subcutaneous dose of 50 mg/kg MnCl2 causes significantaccumulation of Mn in the basal ganglia. Therefore, to better under-stand the impact of a sub-acute Mn exposure on DA dynamics, weadopted the protocol developed by Dodd et al. (2005).
The development of a parallel detection method of Mn and DAin the striatum enabled a direct correlation between Mn accumula-tion and DA levels in the same tissue sample. This parallel analysissubsequently revealed that intraneuronal DA levels are not affectedeven though persistent accumulation of Mn is observed in the stria-tum for up to 21 days after subcutaneous MnCl2 exposure (Fig. 1).This increase in Mn is in accordance with what was observed byDodd et al. (2005), who also reported a significant effect on locomo-tor activity but conducted no analysis of the DA system. Our tissuecontent results indicate that there are no alterations in intracellularDA concentrations. However, there may still be underlying changesin DA dynamics that are masked by the inability of tissue contentanalysis to distinguish between intra- and extracellular DA levels.
In vivo microdialysis was used to determine whether sub-acuteMn treatment selectively alters extracellular DA, DOPAC, or HVAlevels in the striatum at three discrete time points after Mn expo-sure. Extraneuronal DA, DOPAC, and HVA levels, without correctionfor probe recovery, showed no differences immediately (24 h) or 21days post-Mn treatment but were significantly reduced 7 days aftersub-acute exposure compared to control mice (Fig. 2). These resultssuggest that alterations in presynaptic DA dynamics are delayed,despite the immediate increase in striatal Mn levels observed by tis-sue content. Furthermore, our results suggest that a sub-acute Mnexposure does not deplete intracellular DA, as total tissue DA levelsin the striatum were unchanged despite reductions in extracellu-
intraneuronal stores, this treatment protocol does not appear to beneurotoxic. However, the dialysate results from this study suggestthat the presynaptic DA system is vulnerable to the effects of exces-
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M. Khalid et al. / Journal of Neuro
ive Mn accumulation. Compared to previous studies that havexamined the acute or long-term consequence of Mn on presynap-ic dopamine dynamics (McDougall et al., 2008; Serra et al., 2000;idal et al., 2005), our study investigated more intermediate timeoints following Mn exposure. Acute treatment was shown to alterxtracellular DA levels (Serra et al., 2000; Vidal et al., 2005), whileo difference was observed in extracellular DA levels in either thePu or NAc 70 days following prolonged (20 consecutive days) post-atal Mn-treatment (McDougall et al., 2008). Although our studyid not detect any immediate changes in extracellular DA levels as
n the Serra and Vidal studies, which may be the result of differ-nces in Mn concentration or duration of Mn infusion, our resultsuggest that sub-acute Mn exposure in adults has the ability tonfluence presynaptic DA dynamics. However, these impairmentsre apparent for only a restricted period of time.
To investigate whether Mn exposure results in other compen-atory changes at presynaptic DA terminals, high K+ was perfusedvia reverse dialysis) into the striatum to measure DA efflux. Ouresults show that Mn-treated mice at all time points had an atten-ated response to high K+ infusion compared to controls. Theseesults are consistent with the reduction in K+ evoked DA releasebserved by Vidal et al. (2005) upon an intrastriatal infusion of Mn.urthermore, long-term consequences of Mn-treatment have beeneen in the form of a blunted striatal response to cocaine induced DAfflux (McDougall et al., 2008). Additionally, Guilarte et al. (2006,008) have shown a decrease in amphetamine-induced DA releasepon Mn accumulation in non-human primates. Taken together,hese results suggest that Mn profoundly influences the ability ofxogenous compounds to evoke presynaptic DA release. In furtherupport of this conclusion, when electrically evoked DA release wasonitored using FSCV, a blunted DA response was observed in Mn-
reated mice that paralleled the alterations observed with in vivoicrodialysis. Although the neurochemical techniques (microdial-
sis versus FSCV) and sample preparation methods (intact brainersus slice preparation) used to probe DA dynamics are differ-nt, together they suggest that sub-acute Mn exposure can induceevere impairments in presynaptic striatal DA efflux. One thing toote is that FSCV only shows impairments 7 and 21 days after Mn-reatment whereas alterations in microdialysis results are seen atll time points. This difference may be attributed to the stimu-ation parameters used for each of these techniques. Specifically,he microdialysis high K+ stimulation of 20 min is six orders of
agnitude greater in duration than the 4 ms electrical-stimulationpplied to the slice when using FSCV. As a result, a stimulationonger in duration may be able to overcome any release-regulatingmpairments that are elucidated by shorter stimulation methods.
To better understand whether subtle but biologically significantlterations in extracellular DA occur at any other time points in Mn-reated mice, the microdialysis technique of zero net flux was used.ero net flux microdialysis permits the estimation of basal extra-ellular DA levels by infusing in various concentrations of DA touantify the extracellular DA concentration (Cext). Cext is defined ashe point where there is no net diffusion of DA between the extra-ellular space and the microdialysis membrane (the x-intercept)Parsons and Justice, 1993, 1994). This method also provides anpproximation of in vivo extraction fraction (Ed) from the slope ofhe zero net flux regression line (Parsons et al., 1991, 1996). Theresent results obtained by zero net flux in the CPu agree with thoseithout correction for extraction fraction, as a significant decrease
n extracellular DA concentration was observed only 7 days aftern-treatment in both cases. Notably, zero net flux results showed a
ignificant difference in Ed, but this difference was observed at 1 and
1 days after Mn treatment (Fig. 3). Since previous studies by Justicend co-workers have shown that pharmacologic inhibition of DAransporter-mediated uptake alters Ed (Cosford et al., 1996; Justice,993; Parsons and Justice, 1992, 1993, 1994; Parsons et al., 1991,
e Methods 202 (2011) 182– 191 189
1996), this finding suggests Mn treatment may affect DA transport.However, zero net flux studies in monoamine transporter knockoutmice (heterozygous for DA and complete knockout for serotonintransporter) show no difference in striatal Ed (Jones et al., 1998;Mathews et al., 2004). Thus, there is considerable debate on howEd values correlate with transporter function (Bungay et al., 2003;Mathews et al., 2004; Tang et al., 2003). A more direct methodto measure the DA uptake function of the DA transporter is touse FSCV, which records the release and uptake of DA followingelectrical stimulation in brain slices with high temporal (100 ms)resolution (John and Jones, 2007; Robinson et al., 2003; Wightmanet al., 1988). This method has been used to show that DA is typicallycleared from the extrasynaptic space in less than 1 s in non-treatedanimals, highlighting the necessity of a higher temporal resolu-tion technique to detect uptake changes (John and Jones, 2007;Robinson et al., 2003; Wightman et al., 1988). In contrast to ourzero net flux results, no difference in DA uptake rates between con-trols and Mn-treated mice was observed by FSCV. Specifically, ouranalysis of DA Vmax, a quantification of the rate at which the DAtransporter reuptakes DA into the presynaptic neuron, showed nodifference compared to saline-treated mice (Fig. 5). Previous stud-ies have implicated the DA transporter for Mn transport into DAneurons (Anderson et al., 2007; Chen et al., 2006; Erikson et al.,2005; Guilarte et al., 2008; Kim et al., 2002; McDougall et al., 2008).However, there is uncertainty as to whether Mn directly interactswith the DA transporter (Anderson et al., 2007; Chen et al., 2006;Erikson et al., 2005; Guilarte et al., 2008; Ingersoll et al., 1999) or,once inside DA neurons, disrupts either presynaptic proteins or DAfunction (Guilarte et al., 2006, 2008). Our FSCV results argue thatMn does not directly alter DA uptake, as no difference in uptakerates was observed. However, a significant concomitant reductionin stimulated DA release was measured by both microdialysis andvoltammetry. Taken together, these results suggest that intracellu-lar accumulation of Mn has the ability to either directly or indirectlyinfluence presynaptic DA release dynamics for at least 21 days aftersub-acute Mn treatment.
There is considerable evidence suggesting that Mn can alter DAD2 receptor binding and protein expression, but the lack of consis-tent findings across Mn treatments and animal models has madeit difficult to ascertain the exact interaction between excess Mnand DA D2 receptors (Guilarte et al., 2006; Kessler et al., 2003;McDougall et al., 2011). Since our study shows that a sub-acuteMn exposure reduces DA release dynamics with both microdialysisand voltammetry, we sought to determine if the impaired stimu-lated DA release was modulated by presynaptic release-regulatingDA D2 autoreceptors. An advantage to using FSCV to probe DA D2autoreceptor functionality is that brain slices exclude contributionsfrom the DA D2 impulse-regulating receptors found on cell bodies,allowing for a selective evaluation of nerve terminal D2 autorecep-tors that control exocytotic DA release (Jones et al., 1999; Maina andMathews, 2010). The results from the present study show no dif-ference in the half maximal inhibitory concentration (IC50) valuesfor the DA D2 agonist quinpirole, suggesting that a sub-acute Mnexposure does not affect DA D2 autoreceptor functionality in theCPu. Similarly, but using a different protocol, non-human primatestreated with Mn for at least 27 weeks showed no alterations inD2 autoreceptor levels in the caudate or putamen (Guilarte, 2010;Guilarte et al., 2008), and a pre-weaning Mn exposure (21 dayslong) in rats also showed no differences in striatal D2 protein levels3 and 86 days after treatment (Kern and Smith, 2011; Kern et al.,2010). However, there are other studies showing either an increaseor decrease in DA D2 receptors after Mn treatment (Kessler et al.,
2003; McDougall et al., 2011), and it is postulated that these varia-tions in effects are due to the wide range of Mn treatment protocolsand/or animal models that are used to evaluate potential Mn–DAinteractions. The results from this study with a sub-acute Mn expo-
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ure protocol suggest that there is no direct effect of Mn on DA2 autoreceptor functionality in the CPu and furthermore, that the
mpaired DA release in these Mn-treated mice is not a result of DA2 receptor dysfunction.
Intraneuronal Mn accumulation in DA neurons has numerousntracellular targets that could disrupt presynaptic DA release. Forxample, one target protein that receives considerable attention is-synuclein (Peneder et al., 2011; Prabhakaran et al., 2011) which
s a fundamental component of Lewy bodies (a hallmark of Parkin-on’s disease). The exact role of �-synuclein in neurotransmitteregulation has remained elusive, but there is increasing evidencehat it is involved in modulating neurotransmitter release (Larsent al., 2006; Maroteaux et al., 1988; Nemani et al., 2010). Mice thatverexpress �-synuclein have impaired DA release, which does notppear to be a result of altered DA synthesis, vesicular activity, or DAransporter-mediated reuptake (Larsen et al., 2006). Interestingly,hese DA system changes observed by Larsen et al. parallel our find-ngs in which a reduction in DA release is observed with no changen uptake. Since Parkinson’s disease and manganism are both linkedo DA system dysfunction, manifested as a loss of motor control, its plausible that �-synuclein is a common mediator in both dis-ases (Guilarte et al., 2008; Peneder et al., 2011; Prabhakaran et al.,011). However, future experiments would need to be performedo affirm interactions between Mn and �-synuclein.
Although it is enticing to associate Mn with alterations in �-ynuclein expression because of its similarities to Parkinson’s dis-ase (Guilarte et al., 2008; Peneder et al., 2011), it is possible that thettenuated DA release is a result of dysfunction in other neuromod-lators that would be susceptible to excess Mn accumulation andubsequently cause dysfunctions in DA transmission. For example,n the presence of excess Mn, it is postulated that DA can be fur-her oxidized (Burton and Guilarte, 2009; Donaldson et al., 1981;raumann et al., 2002; Lloyd, 1995), leading to an elevation in themount of free radicals or hydrogen peroxide in the brain (Burtonnd Guilarte, 2009; Donaldson et al., 1981; Lloyd, 1995; Shen andryhurst, 1998). Hydrogen peroxide itself is a modulator of synap-
ic DA release (Chen et al., 2001). If excess Mn causes an imbalanceincrease) in hydrogen peroxide levels, then it is plausible thatither this by itself or in combination with �-synuclein could leado the reductions we observed in presynaptic DA transmission.
. Conclusion
In summary, this work used several neurochemical techniques,hich together, indicate that a sub-acute Mn exposure impairs
timulated DA release, with the most pronounced alterationsppearing 7 days after treatment. This is the first time that DA sys-em dynamics have been monitored after Mn exposure at 3 discreteime points (early, intermediate, and late) in mice. Specifically, ouresults suggest that the DA system appears to be most susceptibleo presynaptic alterations 7 days after Mn treatment. Specificallyt this time point, in vivo microdialysis revealed a unique concomi-ant attenuation in striatal extracellular DA (both with and withoutrobe correction), HVA, and DOPAC levels. The decreases in extra-ellular DA levels do not appear to be a result of altered DA uptaker DA D2 autoreceptor function. Most importantly, this effect onelease is finite, as extracellular DA levels appear to rebound to con-rol levels at a later time point of analysis (21 days). Taken together,hese results suggest that Mn accumulation within the striatuman severely impair DA release dynamics, without showing signsf neurotoxicity.
cknowledgements
The authors gratefully acknowledge Dr. Tamara Hendricksonnd Dr. Kelly E. Bosse for insightful discussion during the prepa-
e Methods 202 (2011) 182– 191
ration of this manuscript, as well as the technical support of Dr.Kelly E. Bosse for assistance with the tissue content experiments.Funding provided by Wayne State University start up funds.
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