Celia A. Dodd

Basal Ganglia Accumulation and Motor Assessment Following Manganese Chloride Exposure in the C57BL/6 Mouse

Equivocal clinical evidence for involvement of manganese in development of Parkinson’s disease necessitates experimental studies on this issue. The aged, 1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine–treated C57BL/6 mouse is one of the most common models for Parkinson’s disease. However, there is little information on brain bioaccumulation of manganese, and little or no information on clinical/behavioral manifestations of manganese neurotoxicity, in this strain. Male C57BL/6 retired breeder mice were given a single subcutaneous injection of either 0, 50, or 100 mg/kg of MnCl2 (single-dose regimen) or three injections of either of these doses over 7 days (multiple-dose regimen). Behavioral assessment was performed 24 h after final injection, followed by sacrifice, and body weight was recorded each day. There was a 105% increase in striatal manganese concentration 1 day after a single 100 mg/kg injection, and 421% and 647% increases, respectively, 1 day after multiple doses of 50 or 100 mg/kg of MnCl2. One day after a single injection, there were respective 30.9% and 38.9% decreases in horizontal movement (grid crossing) for the 50 and 100 mg/kg doses and a 43.2% decrease for the multiple dose of 100 mg/kg. There was no significant main effect of dose level on rearing, swimming, grip strength, or grip fatigue. Unlike previous work with the C57BL/6 strain using smaller intraperitoneal doses, this study established dosing regimens that produced significant increases in basal ganglia manganese concentration reminiscent of brain increases in the CD-1 mouse following subcutaneous doses close to our lowest. A decrease in locomotor behavior, significant but not severe in this study, has been reported following manganese exposure in other mouse strains. These data, particularly the significant increase in basal ganglia manganese concentration, provide guidance for designing studies of the potential role of manganese in Parkinson’s disease using the most common animal model for the disorder.

Short-term atrazine exposure causes behavioral deficits and disrupts monoaminergic systems in male C57BL/6 mice.

Excessive exposure to the widely used herbicide atrazine (ATR) affects several organ systems, including the brain, where neurochemical alterations reflective of dopamine (DA) circuitry perturbation have been reported. The present study aimed to investigate effects of short-term oral exposure to a dose-range (0, 5, 25, 125, or 250 mg/kg) of ATR on behavioral, neurochemical, and molecular indices of toxicity in adult male C57BL/6 mice. The experimental paradigm included open field, pole and grip tests (day 4), novel object recognition (NOR) and forced swim test (FST; day 9), followed by tissue collection 4h post dosing on day 10. After 4 days of exposure, ATR decreased locomotor activity (≥125 mg/kg). On day 9, ATR-exposed mice exhibited dose-dependent decreased performance in the NOR test (≥25 mg/kg) and spent more time swimming and less time immobile during the FST (≥125 mg/kg). Neurochemically, short-term ATR exposure increased striatal DA and DA turnover (its metabolite homovanillic acid [HVA] and the HVA/DA ratio; ≥125 mg/kg). In addition, ATR exposure increased the levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the striatum (≥125 mg/kg) and it also increased DA turnover (≥125 mg/kg), 5-HIAA (125 mg/kg), and norepinephrine (≥125 mg/kg) levels in the prefrontal cortex. In the hippocampus, the only effect of ATR was to increase the norepinephrine metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG; 250 mg/kg). At the molecular level, the expression of key striatal (protein) or nigral (mRNA) markers associated with nigrostriatal DA function, such as tyrosine hydroxylase, DA transporter, vesicular monoamine transporter 2, and DA receptors, was not affected by ATR. These results indicate that short-term ATR exposure targets multiple monoamine pathways at the neurochemical level, including in the striatum, and induces behavioral abnormalities suggestive of impaired motor and cognitive functions and increased anxiety. Impaired performance in the NOR behavioral test was the most sensitive endpoint affected by ATR; this should be taken into consideration for future low-dose ATR studies and for the assessment of risk associated with overexposure to this herbicide.

Immunohistochemical Changes in the Mouse Striatum Induced by the Pyrethroid Insecticide Permethrin

Epidemiological studies have linked insecticide exposure and Parkinsons disease. In addition, some insecticides produce damage or physiological disruption within the dopaminergic nigrostriatal pathway of non-humans. This study employed immunohistochemical analysis in striatum of the C57BL/6 mouse to clarify tissue changes suggested by previous pharmacological studies of the pyrethroid insecticide permethrin. Dopamine transporter, tyrosine hydroxylase, and glial fibrillary acidic protein immunoreactivities were examined in caudate-putamen to distinguish changes in amount of dopamine transporter immunoreactive protein from degeneration or other damage to dopaminergic neuropil. Weight-matched pairs of pesticide-treated and vehicle-control mice were dosed and sacrificed on the same days. Permethrin at 0.8, 1.5 and 3.0 mg/kg were the low doses and at 200 mg/kg the high dose. Brains from matched pairs of mice were processed on the same slides using the avidin-biotin technique. Four fields were morphometrically located in each of the serial sections of caudateputamen, digitally photographed, and immunopositive image pixels were counted and compared between members of matched pairs of permethrin-treated and vehicle-control mice. For low doses, only 3.0 mg/kg produced a significant decrease in dopamine transporter immunostaining. The high dose of permethrin did not produce a significant change in dopamine transporter or tyrosine hydroxylase immunostaining, but resulted in a significant increase in glial fibrillary acidic protein immunostaining. These data suggest that a low dose of permethrin can reduce the amount of dopamine transporter immunoreactive protein in the caudate-putamen. They also suggest that previously reported reductions in dopamine uptake of striatal synaptosomes of high-dose mice may be due to nondegenerative tissue damage within this region as opposed to reductions of dopamine transporter protein or death of nigrostriatal terminals. These data provide further evidence that insecticides can affect the primary neurodegenerative substrate of Parkinsons disease.

Role of glial cells in manganese neurotoxicity

The objectives of this focused review are to (i) provide a systematic overview of recent advances pertaining to the role of glia, namely microglia and astrocytes, in the neuropathology associated with excessive exposure to manganese (Mn), (ii) highlight possible mechanisms and factors involved in Mn‐modulated, glia‐derived neuroinflammation, and (iii) discuss the implications of excessive neuroinflammation on neuronal injury within the context of Mn overexposure. As this is not meant to be a comprehensive review on the topic of Mn neurotoxicity, the reader may wish to refer to several broader and more comprehensive reviews. After a brief introduction to Mn neurotoxicity, we first discuss the role of glial cells in neurodegeneration. Next, we review existing in vitro and in vivo studies that implicate Mn as a modulator of glial activation and ensuing neuroinflammation. This is followed by an examination of recognized and potential mechanisms that are involved in the modulation of glial inflammatory output by Mn; here the common pathways activated by Mn in glial and neuronal cells, including outcomes of such activation, are also addressed. We finish with a discussion of the implications of Mn‐modulated glial activation for neuronal survival and with a list of data gaps in the topic that need to be filled in the future. Copyright

Manganese Potentiates LPS-Induced Heme-Oxygenase 1 in Microglia but not Dopaminergic Cells: Role in Controlling Microglial Hydrogen Peroxide and Inflammatory Cytokine Output

Excessive manganese (Mn) exposure increases output of glial-derived inflammatory products, which may indirectly contribute to the neurotoxic effects of this essential metal. In microglia, Mn increases hydrogen peroxide (H(2)O(2)) release and potentiates lipopolysaccharide (LPS)-induced cytokines (TNF-α, IL-6) and nitric oxide (NO). Inducible heme-oxygenase (HO-1) plays a role in the regulation of inflammation and its expression is upregulated in response to oxidative stressors, including metals and LPS. Because Mn can oxidatively affect neurons both directly and indirectly, we investigated the effect of Mn exposure on the induction of HO-1 in resting and LPS-activated microglia (N9) and dopaminergic neurons (N27). In microglia, 24h exposure to Mn (up to 250 μM) had minimal effects on its own, but it markedly potentiated LPS (100 ng/ml)-induced HO-1 protein and mRNA. Inhibition of microglial HO-1 activity with two different inhibitors indicated that HO-1 is a positive regulator of the Mn-potentiated cytokine output and a negative regulator of the Mn-induced H(2)O(2) output. Mn enhancement of LPS-induced HO-1 does not appear to be dependent on H(2)O(2) or NO, as Mn+LPS-induced H(2)O(2) release was not greater than the increase induced by Mn alone and inhibition of iNOS did not change Mn potentiation of HO-1. However, because Mn exposure potentiated the LPS-induced nuclear expression of small Maf proteins, this may be one mechanism Mn uses to affect the expression of HO-1 in activated microglia. Finally, the potentiating effects of Mn on HO-1 appear to be glia-specific for Mn, LPS, or Mn+LPS did not induce HO-1 in N27 neuronal cells.

Pyrethroid and organophosphate insecticide exposure in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease: an immunohistochemical analysis of tyrosine hydroxylase and glial fibrillary acidic protein in dorsolateral striatum.

The pyrethroid insecticide permethrin and the organophosphate insecticide chlorpyrifos can experimentally produce Parkinson’s disease (PD)-associated changes in the dopaminergic nigrostriatal pathway, short of frank degeneration, although at doses considerably higher than from a likely environmental exposure. The ability of permethrin (200 mg/kg), chlorpyrifos (50 mg/kg), or combined permethrin + chlorpyrifos to facilitate nigrostriatal damage in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (30 mg/kg) C57BL/6 mouse model of PD was investigated in three separate experiments. Tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP) immunohistochemistry assessed nigrostriatal degeneration or nigrostriatal damage more subtle than frank degeneration. Four fields in the dorsolateral caudate-putamen were examined at two rostrocaudal locations. The dopaminergic neurotoxin MPTP decreased striatal TH immunopositive neuropil and increased GFAP immunopositive neuropil. Neither permethrin nor chlorpyrifos, alone or in combination, altered the effects of MPTP upon TH or GFAP immunostaining. Permethrin alone increased striatal GFAP immunopositive neuropil but not when combined with chlorpyrifos treatment. Therefore, combined administration of the two insecticides appeared to protect against an increase in a neuropathological indicator of striatal damage seen with permethrin treatment alone. Differences compared with analysis of entire striatum emphasize the value of varying the topographic focus used to assess nigrostriatal degeneration in studies of insecticides in PD.

Differentiation state-dependent effects of in vitro exposure to atrazine or its metabolite diaminochlorotriazine in a dopaminergic cell line

AIMS This study sought to determine the impact of in vitro exposure to the herbicide atrazine (ATR) or its major mammalian metabolite diaminochlorotriazine (DACT) on dopaminergic cell differentiation. MAIN METHODS N27 dopaminergic cells were exposed for 24 or 48 h to ATR or DACT (12-300 μM) and their effects on cell viability, ATP levels, ADP:ATP ratio and differentiation markers, such as soma size and neurite outgrowth, were assessed. KEY FINDINGS Overall, intracellular ATP levels and soma size (decreased by ATR at ≥12 μM; 48 h) were the two parameters most sensitive to ATR exposure in undifferentiated and differentiating dopaminergic cells, respectively. At the morphological level, ATR, but not DACT, increased the percentage of morphologically abnormal undifferentiated N27 cells. On the other hand, exposure to DACT (300 μM; 48 h), but not ATR, increased the ADP:ATP ratio regardless of the differentiation state and it moderately disrupted thin neurite outgrowth. Only the highest concentration of ATR or DACT (300 μM) was cytotoxic after a longer exposure (48 h) and undifferentiated N27 cells were the least sensitive to the cytotoxic effects of ATR or DACT. SIGNIFICANCE Our results suggest that the energy perturbation and morphological disruption of dopaminergic neuronal differentiation induced by ATR and, to a lesser extent, DACT, may be associated with reported neurological deficits caused by developmental ATR exposure in rodents.

Consequences of manganese administration for striatal dopamine and motor behavior in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-exposed C57BL/6 mice

Environmental compounds may be important contributors to Parkinson’s disease etiology. Epidemiological and experimental evidence for the facilitation of parkinsonism by manganese is equivocal. This work addressed methodological concerns in the few studies of manganese modulation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity in C57BL/6 mice. Male, retired breeder mice received 0 or 100 mg/kg of manganese chloride (MnCl2; subcutaneously on days 1, 4 and 7) and 0 or 20 mg/kg of MPTP (intraperitoneally on day 8) and survived up to day 15 or 22. On the day of sacrificing, horizontal (grid crossing) and vertical (rearing) open field movement, swimming, grip strength and grip fatigue were examined. Striata were analyzed for dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) using high-performance liquid chromatography. MPTP produced a main effect decrease in striatal dopamine (48.8%) and DOPAC (38.1%), but there was no main effect of MnCl2 or MnCl2 × MPTP interaction. However, modulatory interactions were observed between the effects of MnCl2 and MPTP for grid crossing, rearing and grip strength. Interestingly, these interactions reduced the severity of behavioral deficits attributable to either of these compounds alone. For rearing and grip strength, the MnCl2 × MPTP interaction was dependent upon survival time. The mechanistic nature of the MnCl2 × MPTP interaction upon these behaviors, in the absence of such an interaction for striatal dopamine and DOPAC, remains to be clarified.

Behavioral and monoamine perturbations in adult male mice with chronic inflammation induced by repeated peripheral lipopolysaccharide administration

Considering the limited information on the ability of chronic peripheral inflammation to induce behavioral alterations, including on their persistence after inflammatory stimuli termination and on associated neurochemical perturbations, this study assessed the effects of chronic (0.25 mg/kg; i.p.; twice weekly) lipopolysaccharide (LPS) treatment on selected behavioral, neurochemical and molecular measures at different time points in adult male C57BL/6 mice. Behaviorally, LPS-treated mice were hypoactive after 6 weeks, whereas significant hyperactivity was observed after 12 weeks of LPS and 11 weeks after 13 week LPS treatment termination. Similar biphasic responses, i.e., early decrease followed by a delayed increase were observed in the open field test center time, suggestive of, respectively, increased and decreased anxiety. In a forced swim test, mice exhibited increased immobility (depressive behavior) at all times they were tested. Chronic LPS also produced persistent increase in splenic serotonin (5-HT) and time-dependent, brain region-specific alterations in striatal and prefrontocortical dopamine and 5-HT homeostasis. Microglia, but not astrocytes, were activated by LPS early and late, but their activation did not persist after LPS treatment termination. Above findings demonstrate that chronic peripheral inflammation initially causes hypoactivity and increased anxiety, followed by persistent hyperactivity and decreased anxiety. Notably, chronic LPS-induced depressive behavior appears early, persists long after LPS termination, and is associated with increased splenic 5-HT. Collectively, our data highlight the need for a greater focus on the peripheral/central monoamine alterations and lasting behavioral deficits induced by chronic peripheral inflammation as there are many pathological conditions where inflammation of a chronic nature is a hallmark feature.