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

Abstract

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.

Manganese, a metal that is ubiquitous in soil and air, and present in many foods, is essential for the normal function of cells and tissues (Aschner, Vrana, and Zheng 1999; Normandin, Panisset, and Zayed 2002; Takeda 2003; Aschner, Erikson, and Dorman 2005). In industrial settings manganese is used as an alloy in the production of steel, is a component of welding flux, and the manganese-based compound methyl cyclopentadienyl manganese tricarbonyl (MMT) is used as a gasoline additive in several countries including Canada (Kaiser 2003). Industrial overexposure to manganese has long been recognized to produce a neurological disorder, termed manganism, the clinical signs of which are often difficult to distinguish from those of Parkinson’s disease (Calne et al. 1994; Pal, Samii, and Calne 1999; Lee 2000; Normandin, Panisset, and Zayed 2002). Although neuropathological studies of manganese-induced neurotoxicity are fairly small compared with neuropathological studies of Parkinson’s disease, evidence suggests that the globus pallidus is the focus of manganese overexposure (Canavan, Cobb, and Drinker 1934; Yamada et al. 1986; Olanow et al. 1996; Kim et al. 1999; Spadoni et al. 2000; Baek et al. 2003; Kim 2004). Thus, the similarity of symptoms between manganism and Parkinson’s disease is not surprising because both appear to target elements of the integrated circuitry of the basal ganglia: the globus pallidus and the dopaminergic nigrostriatal pathway, respectively (Olanow and Tatton 1999; Normandin, Panisset, and Zayed 2002; Samii, Nutt, and Ransom 2004). These foci of neurotoxicity of the basal ganglia are synaptically integrated in such a way that suggests the possibility of a synergistic influence of manganese neurotoxicity upon development or onset of idiopathic Parkinson’s disease (Bolam et al. 2000; Crossman 2000). In fact, it has been reported that welders, who frequently use manganese-containing flux, are prone to early-onset Parkinson’s disease and develop the disorder an average of 17 years earlier than controls (Racette et al. 2001). In addition, general occupational exposure to manganese for more than 20 (Gorell et al. 1997, 1999) or 30 (Zayed et al. 1990) years was associated with increased risk of developing Parkinson’s disease. However, other occupational studies (Semchuk, Love, and Lee 1993; Park et al. 2004, 2005), as well as studies of other risk factors such as well water consumption and rural living (Vieregge et al. 1995; Seidler et al. 1996), do not support a link between manganese exposure and Parkinson’s disease. Therefore, epidemiological evidence for the putative manganese–Parkinson’s disease link is equivocal.

The equivocal clinical evidence for the involvement of manganese in the development or time of onset of Parkinson’s disease, in addition to the expanding market for MMT-containing gasoline, has sparked a need for controlled experimental studies on manganese neurotoxicity and its putative interaction with experimental parkinsonism. The limited number of studies directly addressing the effects of manganese compounds upon chemically induced parkinsonism also fail to paint a clear picture of the nature of this interaction since they run a gamut from potentiation (Takahashi, Rogerio, and Zanin 1989; Witholt, Gwiazda, and Smith 2000), to lack of potentiation (Baek et al. 2003), to protection (Rojas and Rios 1995; Sziraki, Rauhala, and Chiueh 1995; Sziraki et al. 1998). An intuitively appropriate subject for such studies is the aged, 1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine (MPTP)-treated C57BL/6 mouse because it is one of the most common and sensitive animal models for Parkinson’s disease (Heikkila et al. 1984; Di Monte and Langston 1995; Royland and Langston 1998; Schmidt and Ferger 2001). In this strain there is little information on the brain bioaccumulation of manganese and, to our knowledge, little or no information on the clinical/behavioral manifestations of manganese neurotoxicity. Natt et al. (2002) has examined the distribution of manganese in the visual system of the C57BL/6 mouse using magnetic resonance imaging (MRI) but this was only done following injection of manganese chloride (MnCl₂) into the eye to illustrate the utility of manganese in neuronal tract tracing. In a study in which manganese failed to potentiate MPTP neurotoxicity in this strain, Baek et al. (2003) reported that MnCl₂ treatment alone, at a 2 mg/kg daily dose over 3 weeks, failed to change the concentration of manganese in the basal ganglia. However, in a different study by Baek et al. (2004), this dose was shown to increase the gene and protein expression of the astrocyte-associated protein S100β in striatum. No behavioral measures were performed in any of these studies.

It is possible that the aforementioned failure to demonstrate neurotoxic synergism between manganese and MPTP in the C57BL/6 mouse resulted from the absence of significant manganese accumulation in the basal ganglia, which as noted above, is the major regional focus of pathology that both manganism and Parkinson’s disease share. Therefore, as a precursor to further exploration of the effect of manganese on experimental parkinsonism, this study attempted to determine a dose of MnCl₂ that would produce significant accumulation of manganese within the basal ganglia of the C57BL/6 mouse. Such accumulation would not only indicate that manganese reached the target region for manganese- and MPTP-induced neurotoxicity, but would also suggest an appropriate time at which MPTP could be administered, in future studies, while basal ganglia manganese concentration is still elevated. The minimum MnCl₂ dose, and dosing route, used in this study were modeled after a report by Gianutsos et al. (1985) that demonstrated significant increases in brain manganese in the CD-1 mouse. The soluble chloride form of manganese was chosen because it was used by Baek et al. (2003, 2004) in their studies of the C57BL/6 mouse and Gianutsos et al. (1985) reported that the chloride form produced greater brain manganese concentration, in the CD-1 strain, than the insoluble oxide form or organic MMT. Higher manganese concentration in rodent striatum, following administration of soluble versus insoluble forms, has also been reported for intratracheal (Roels et al. 1997) as well as inhalation (Dorman et al. 2001; Dorman et al. 2004b; Normandin et al. 2004) exposure. Several measures of motor behavior were employed in the present study to address the acute clinical impact of the doses and dosing schedules used.

MATERIALS AND METHODS

Animals and Treatments

Male C57BL/6 retired breeder mice, 7 to 9 months old at the time of the experiment, were used. They were obtained from Harlan Sprague-Dawley, Dublin, VA, USA. Forty-eight mice were randomly assigned to six treatment groups in a 3 (dose) × 2 (survival time) factorial design. The three dose levels of MnCl₂ tetrahydrate (Sigma-Aldrich, St. Louis, MO, USA) were 0, 50, or 100 mg/kg. The two survival times, following initial injection, were 1 day or 7 days. Mice surviving 1 day received only a single injection of one of the three dose levels. In this single-day dose regimen, behavioral assessment was performed 24 h after the injection and was immediately followed by sacrifice and basal ganglia tissue collection. Mice surviving 7 days received three identical injections of one of the three dose levels on days 1 (initial injection), 4, and 7. In this multiple-day dose regimen, behavioral assessment was performed 24 h after the last injection and was immediately followed by sacrifice and basal ganglia tissue collection. MnCl₂ was delivered subcutaneously, at the scruff of the neck, as either a 1% (for 50 mg/kg dosing) or a 2% (for 100 mg/kg dosing) solution in distilled water. Mice in the 0 mg/kg groups were considered vehicle controls and received distilled water alone at an injection volume comparable to that received by the respective MnCl₂-treated groups. The 50 and 100 mg/kg doses respectively represent 15.6% and 31.3% of the subcutaneous LD₅₀ for MnCl₂ tetrahydrate in mouse as reported on the Sigma-Aldrich Material Safety Data Sheet. The average injection volume was 0.15 ml. Body weight was determined immediately prior to every injection and on all other survival days of the experiment. Animal housing, care and use protocols were in compliance with National Institute of Health guidelines. All procedures were approved by the Virginia Tech Institutional Animal Care and Use Committee.

Assessment of Motor Behavior

Five dependent variables were employed in the assessment of motor behavior. Two of the variables, respectively, assessed horizontal movement and vertical movement in the open field. Swimming behavior was also measured to assess coordinated, horizontal movement in an environment where there was a different energy requirement and muscle load compared with the terrestrial open field. Muscle strength was evaluated by examining grip force and muscle fatigue was assessed by the change in grip force over trials. All behavioral tests were chosen so that no pretest training or habituation to the testing apparatus was necessary. All behavioral tests were carried out in a room that was separate from that in which mice were housed or temporarily stored.

Open Field Movement

Movement in the open field was assessed in the horizontal and vertical planes using an empty, glass tank (46.5 cm long × 26 cm wide × 30 cm high). The floor was marked out into a grid of six equal 15.5 cm × 13 cm rectangles and the walls were covered with white opaque paper, on the outside, to limit visual distractions. The tank was mounted atop a wooden stand that allowed an unobstructed view of the tank floor through use of a mirror, mounted at a 45° angle, 24 cm below the tank. After a mouse was placed in the apparatus the experimenter sat on the floor, approximately 1 m from the apparatus, and observed movement for a period of 3 min. Movement in the horizontal plane was assessed by counting the number of grid crossings defined as complete movement of the forelimbs and hind limbs from one grid block to another. Movement in the vertical plane was assessed by counting the number of times the mouse reared up on its hind limbs, by lifting both forelimbs off the floor, followed by the exploratory mannerisms of head rotation and sniffing. A double counter was used to record the respective occurrence of the crossing and rearing behaviors. The inside of the apparatus was cleaned with a disinfectant spray and wiped dry before each mouse was tested.

Swimming

This procedure measured the time necessary to swim from one end of a narrow, water-filled trough to a visible, raised platform at the other end. The trough was a 91.5 cm long × 18.5 cm wide × 17 cm high rectangular basin of opaque plastic filled with 27°C tap water to within 4.5 cm of the top edge. The water was changed at the beginning of each day that trials were run. The platform was comprised of a 7.5 cm long × 3.3 cm wide × 1.5 cm thick piece of wood with a wire mesh grid attached to the top that extended an extra centimeter on all sides. The platform was attached to a weighted base by a wooden dowel. This configuration anchored the platform to the floor of the trough such that the top of the platform sat 14 cm above the floor and 1.5 cm above the water. The wire mesh edges of the platform sat 4.5 cm away from the end-wall and sides of the trough. Swim duration was recorded on four consecutive trials. For each trial the mouse was held by the base of the tail and gently lowered into the water next to the end-wall opposite to the platform. Timing of swim duration began when the mouse was released and ended when the mouse gripped the raised platform with at least one forepaw. The maximum time limit for a trial was 4 min. At the end of a trial the mouse was placed on a dry towel for about 30 s and then the next trial was initiated.

Grip Strength and Fatigue

The grip strength apparatus consisted of a 12 cm × 12 cm wire mesh grid (0.5 cm × 0.5 cm squares) attached to the end, and oriented perpendicular to the long axis, of the armature of a tensile load cell (strain gauge) (Instron Corporation, Canton MA). The load cell output was fed to a Measurements Group Conditoner/Amplifier (Model 2310) and was calibrated such that a 100 g load, suspended from the grid along the gripping axis, produced a signal of 0.5 volts. Analog voltage signals were digitized every 50 ms using a National Instruments (Austin, TX, Model PCI-MIO-16E-4) computer interface card. Labview (National Instruments, Austin, TX) software was used to store the voltage measurements that were displayed as force as a function of time. Grip force was measured on four consecutive trials. The typical trial duration was 5 to 8 s with about 20 s between trials. For a given trial, after the mouse gripped the grid, the experimenter gently and steadily pulled the mouse away from the load cell by the base of the tail. The trial was terminated when the mouse released the wire mesh grid. The analysis of grip force employed the force value immediately prior to release of the mesh grid, which was easily recognizable on a grip force versus time plot. Trial 1 was used for the statistical analysis of grip force. The slope of a best-fit line of trials 1 through 4 was used for the analysis of grip fatigue.

Tissue Collection and Manganese Measurement

Following behavioral testing mice were sacrificed by rapid cervical dislocation. Brains were immediately removed and placed on a chilled glass petri dish that sat atop a bed of crushed ice. The dish and brain were kept moist with physiological saline. Following removal of the olfactory bulbs a midsagittal section was made. The septum pellucidum was then punctured and the striatum was pinched from the lateral wall of the lateral ventricle using a microforceps. This procedure has been used previously by Karen et al. (2001), Klein et al. (2001), and Gillette and Bloomquist (2003). The striata from both sides of the brain were placed together in a preweighed 1.5-ml microcentrifuge tube. The combined sample was weighed and then frozen at −70°C for later analysis.

Striatal manganese content was measured by inductively coupled plasma–atomic emission spectroscopy (ICP) analysis, in the Virginia Tech Soil Testing Laboratory, using a SpectroFlame Modula Tablebletop ICP with autosampler (Spectro Analytical Instruments, Inc., Fitchburg, MA). The amount of manganese was determined by a calibration curve derived from serial dilutions of a single element standard (CPI, Santa Rosa, CA). Multi-element quality control standards (VHG Labs, Manchester, NH) were used to verify the calibration standards as well as the accuracy and precision of the system. Blanks to check for carryover were run after every 10 samples in addition to quality control standards. Calibration and quality control standards, as well as blanks, were prepared using the digestion solution matrix. The striata were digested in a solution of 20% perchloric acid–80% nitric acid and then diluted with 0.5% nitric acid to make a 5 ml sample volume. A Rf power of 1200 W was used in conjunction with a sample uptake of 1.2 ml/min, an argon gas pressure of 100 psi, and a nebulizer pressure of 36 psi. A 4-min wash with digestion solution was performed before and after each sample was run. Smart Analyzer Software for ICP-Spectrophotometer (Spectro A.I. GmbH, Kleve, Germany) was used for spectral line scanning and to quantify the element content of samples, standards and blanks. The detection limit was 0.0002 ppm in solution and final concentrations were expressed as micrograms of manganese per gram of brain tissue.

Statistical Analysis

An independent two-way analysis of variance (ANOVA), using SAS (SAS Institute, Cary, NC, USA), was performed for each of the dependent variables measured in the experiment. Plots of standardized residuals across every factor in the model were inspected to assess model adequacy (Dean and Voss 1999). For brain manganese concentration, the plot of standardized residuals across predicted values revealed a multiplicative error structure. Brain manganese concentrations were therefore log-transformed for analysis and means were back-transformed (along with their 95% confidence intervals) for presentation. All analyses were corrected for the effect of treatment block and, except for body weight, examined the fixed effects of dose level, dose regimen, and the interaction of dose level and dose regimen. If a significant main effect of dose alone was observed, the following post hoc pairwise comparisons of independent variables were performed: 0 versus 50 mg/kg, 0 versus 100 mg/kg, and 50 versus 100 mg/kg. Because only two dose regimens were used (single-day and multiple-day), no post hoc testing was necessary for significant main effects of dose regimen. If significant main effects of both dose level and dose regimen were found, or there was an interaction between the two variables, then the three pairwise dose level comparisons listed above were performed for each of the dose regimens (six tests total). This post hoc testing plan was devised a priori with the intent of maximizing the power of the analysis for each dependent variable. The pairwise comparisons were done using Tukey’s test, which keeps the overall alpha at .05. Therefore, significant differences revealed by these tests are simply reported as p < .05. ANOVA of body weight was only performed for the multiple day dose regimen and examined the fixed effects of dose, day, and the interaction of dose and day. No post hoc testing was necessary given the results of this analysis.

RESULTS

ANOVA revealed significant main effects of both dose level (df = 2,39; p < .0001) and dose regimen (df = 1,39; p < .0001) upon striatal manganese concentration, as well as a significant interaction between dose level and dose regimen (df = 2,39; p = .0001). Figure 1 presents mean striatal manganese concentration, as a function of dose level, for both the single-day dose regimen and the multiple-day dose regimen groups. As illustrated, 1 day after a single injection, only the 100 mg/kg dose produced a significant increase in striatal manganese relative to vehicle-control mice. This increase was 105% (df = 39; p < .05). For mice receiving three injections over a 7-day survival period, both the 50 and 100 mg/kg doses produced a significant increase in striatal manganese relative to vehicle control mice. These increases were 421% (df = 39; p < .05) and 647% (df = 39; p < .05), respectively. There was no significant difference between the 50 mg/kg and 100 mg/kg groups, for either the single-day or multiple-day dose regimens, although there was a numeric trend toward increased manganese concentration with the higher dose for both regimens.

For mice that received the multiple-day dose regimen, ANOVA of mean body weight for each dose level, on days 1 through 7, revealed no significant main effects across dose levels or across experimental days nor was there a significant interaction between these variables. The weight for day 1 was a predosing weight because it was measured immediately prior to the first injection of the experiment. The weight for day 8, the day of sacrifice, was eliminated from the ANOVA because it was measured several hours earlier than the other days in order to permit completion of behavioral testing prior to sacrifice. This earlier weighing time was associated with a consistent numerical reduction from day 7, in all three groups, including the vehicle control. However, even in the group receiving the highest dose level (100 mg/kg), mean weight on day 8 was only 2.75% lower than the predosing weight measured on day 1.

ANOVA indicated significant main effects of dose level (df = 2,39; p < .0001) and dose regimen (df = 1,39; p < .0001) upon horizontal movement in the open field (grid crossing) but no significant interaction between the two independent variables. Figure 2 illustrates grid-crossing frequency, as a function of dose level, for both the single-day dose regimen and the multiple-day dose regimen groups. As can be seen, 1 day after a single injection, both the 50 mg/kg dose and the 100 mg/kg dose produced significant decreases in grid crossing frequency relative to vehicle-control mice. These decreases were 30.9% (df = 39; p < .05) and 38.9% (df = 39; p < .05), respectively. For mice receiving three injections over a 7-day survival period only the 100 mg/kg dose produced a significant decrease in grid-crossing frequency relative to vehicle control mice. This decrease was 43.2% (df = 39; p < .05). There was no significant difference between the 50 mg/kg and 100 mg/kg groups for either the single-day or multiple-day dose regimens. Although not tested as part of the preplanned pairwise statistical comparisons, a notably higher grid-crossing frequency for vehicle controls of the single-day, compared to the multiple-day, dose regimen can be observed upon visual inspection of Figure 2. The difference between these means is approximately four times the magnitude of the standard deviation of either mean and the main effect of dose regimen should be considered in light of this difference (see Discussion).

For vertical movement in the open field there was only a significant main effect of dose regimen (df = 1,39; p < .029) with no significant interactions among the independent variables. Mean rearing frequency (per 3 min) was 26.7% less under the multiple dose regimen (mean = 15.6, SE = 1.8) compared with the single dose regimen (mean = 21.3, SE = 1.7). No significant effects of experimental treatments, nor interactions among the independent variables, were revealed by ANOVA for either swimming, grip force, or grip fatigue. Although the maximum time limit for a swim trial was 4 min, this limit was reached in only one swim trial for each of two different mice. The results of the parametric ANOVA were the same when run with or without these extreme values.

DISCUSSION

In general, the results of this experiment have established dosing regimens of MnCl₂ that can produce substantial increases in the concentration of manganese within the basal ganglia of the C57BL/6 mouse as well as significant, but not severe, changes in motor behavior. The basal ganglia is the neural focus of both manganism and Parkinson’s disease and the C57BL/6 strain is one that is highly susceptible to the effects of chemically induced parkinsonism. Therefore, as discussed below, these results for this strain will be of value in future explorations that employ an animal model to examine the putative role of manganese in Parkinson’s disease.

The ICP results of this experiment indicated that a single 100 mg/kg dose MnCl₂ could produce significantly elevated manganese concentration in the basal ganglia within a single day of subcutaneous administration. Furthermore, the significantly elevated striatal manganese concentration observed 1 day after the last dose of the 50 mg/kg multiple-dose regimen, but not 1 day after a single 50 mg/kg dose, suggests that the administered manganese does not simply clear from the striatum in 1 day but accumulates over the multiple doses. Such accumulation is also suggested by the numerically higher striatal manganese levels observed for multiple doses of 50 and 100 mg/kg of MnCl₂ compared with single doses of these concentrations. However, these latter contrasts were not included in the predetermined structure of the post hoc analysis. The numeric trend toward a dose-response relationship between the 50 mg/kg and 100 mg/kg doses, for both regimens, suggests that such a change might prove significant with increased sample size.

As noted, there is a paucity of studies that have examined manganese accumulation in the brain of the C57BL/6 mouse. Three weeks of a 2 mg/kg/day intraperitoneal dose regimen of MnCl₂ failed to change manganese concentration in the basal ganglia of this strain (Baek et al. 2003). However, in CD-1 mice injected with 0.4 mEq/kg of MnCl₂ (equivalent to 39.5 mg/kg of MnCl₂ tetrahydrate), using the same route as the present experiment, the whole brain concentration of manganese was significantly increased by 1 h and monotonically increased to 370% of control by 24 h (Gianutsos et al. 1985). Manganese concentration was also highly elevated, at 250% of control, 3 weeks after a single dose. Using a multiple dose regimen of one dose per week for 3 weeks, manganese concentration showed a monotonic increase over subsequent weeks, reaching 568% of control after the third week. Comparisons between brain manganese concentrations from the present experiment with those cited above are complicated by differences in dose concentration, regimen, and route, as well as differences in strain and brain region examined. However, our data appear fairly similar to those of Gianutsos et al. (1985) who as alluded to earlier used a dose concentration close to our lowest, along with a similar (subcutaneous) route of delivery of MnCl₂, but who used a different strain and examined whole brain. Together, the two studies suggest manganese accumulation in the mouse brain over multiple subcutaneous doses of MnCl₂ and persistence ranging from 24 h to at least 3 weeks. This is concordant with a report by Takeda, Sawashita, and Okada (1995) noting a biological half-life of 51 to 74 days for manganese in adult rat brain. It is also concordant with rat brain data of Dorman, McManus, Marshall et al. (2004a) who examined young and aged animals that inhaled manganese. In aged rats that inhaled 0.5 mg Mn m⁻³ as MnSO₄ for 90 days, striatal manganese concentration was still elevated 45 days after cessation of exposure. It is also interesting to note that manganese cleared more slowly from striatum of aged rats, compared with other brain and body tissues, and clearance from striatum appeared to decline with age. This is significant when considering a putative synergistic link between manganese exposure and Parkinson’s disease, a disease of the aged, and supports our choice of retired breeders for exploring this and future studies of such a link.

Studies of MPTP metabolism in NIH Swiss Webster, NMRI, and C57BL/6 mice, using intravenous, intraperitoneal, or subcutaneous delivery of single 10 to 20 mg/kg doses, revealed that the brain concentration of the neurotoxic MPTP metabolite 1-methyl-4-phenylpyridinium (MPP⁺) reaches a peak within 5 to 60 min and is not detectable in appreciable amounts after several hours (Markey et al. 1984; Nwanze et al. 1995). Both Nwanze et al. (1995) using a single dose of MPTP and Giovanni et al. (1991) using multiple doses found higher concentrations and greater persistence of MPP⁺ in brains of the C57BL/6 mice, compared with other species. Taken together with data from the present experiment, a useful design can be gleaned for future studies of the putative role of manganese preexposure in chemically induced parkinsonism that employ the MPTP-sensitive C57BL/6 mouse. Using the subcutaneous multiple dose regimen of 50 or 100 mg/kg of MnCl₂, or the single dose regimen of 100 mg/kg, would ensure that a significant rise in manganese had taken place in the basal ganglia when a single 10 to 20 mg/kg dose of MPTP was delivered 24 h after the last dose of MnCl₂. It is also likely that significant exposure to the toxic MPTP metabolite MPP⁺ would occur in the basal ganglia while manganese concentrations were still elevated. A single 20 mg/kg intravenous or intraperitoneal dose of MPTP can produce a respective 63% or 49% decrease in striatal dopamine 7 to 14 days after delivery (Nwanze et al. 1995; Dodd and Klein 2003). Such depletions, although substantial, leave room for detecting further decrements possibly induced by synergistic or additive interactions between manganese and MPTP toxicity.

To our knowledge there have been no previous studies of the effects of manganese exposure upon the behavior of the C57BL/6 mouse. Studies in other mouse strains represent a variety of doses, routes of administration, forms of manganese, and specific behavioral tests (Gray and Laskey 1980; Morganti et al. 1985; Komura and Sakamoto 1992; Talavera et al. 1999). All of those studies examined locomotor activity including one that exposed NMRI mice to intraperitoneal MnCl₂ at 5 mg/kg for 5 days/week for 2 or 4 weeks (Talavera et al. 1999). In that study, and all but one of the others (Morganti et al. 1985), there was a decrease in locomotor activity. In the present experiment behavioral analysis revealed that MnCl₂ could also produce a locomotor deficit in the C57BL/6 strain. This effect did not appear to reflect a general poverty of motor behavior because the other behavioral variables examined in this experiment were not significantly affected by the dose level of manganese. The considerable numerical difference in grid crossing frequency between dosing regimens for the vehicle-control mice suggests that the main effect of dose regimen upon horizontal movement in the open field may be more likely to reflect some unidentified aspect of the dosing process, or chance assignment of more active mice to the single-dose group, rather than cumulative effects from multiple doses of manganese. The similar direction of the main effect of dosing regimen upon rearing frequency, in the absence of a main effect of dose, further supports this notion. The absence of a significant change in weight over the course of the multiple dose regimen, for either dose level, suggests it is unlikely that behavioral changes were attributable to a general morbidity induced by manganese treatment.

Considering an LD₅₀ for subcutaneous delivery of MnCl₂ tetrahydrate in mouse of 320 mg/kg (Sigma-Aldrich Material Safety Data Sheet), the present work, using doses of 0, 50, and 100 mg/kg, could be considered a “high-dose” study. Furthermore, organisms are not commonly exposed to subcutaneous delivery of manganese in the environment. However, the major objective of this study was to produce a substantial increase in basal ganglia manganese concentration, in the C57BL/6 mouse, as a precursor to future studies of synergistic interaction between manganese and MPTP neurotoxicities. Therefore, the choice of dose magnitude, route, dosing regimen, and manganese form was guided by this objective. In a review of manganese dosimetry studies, Aschner, Erikson, and Dorman (2005) noted that manganese concentrations in rodent striatum normally range from 4.4 to 18 μM but increase to 23 to 70 μM in manganese-exposed animals. The magnitude of increases in striatal manganese produced in the present study of the C57BL/6 mouse was concordant with such previously observed increases. Nevertheless, the doses used in our study could raise concern over a general toxicity that may have contributed to the observed change in motor behavior. However, our above noted finding of no significant change in four of the five behavioral dependent variables and no significant change in body weight argues against this notion.

In the present experiment MnCl₂ dosing was capable of increasing manganese concentration in the basal ganglia and of producing a deficit in horizontal locomotion. Therefore, it is tempting to attribute the behavioral deficit to interference by manganese with the functional integrity of communication between the highly integrated structures that make up the heterogeneous basal ganglia. However, given the above noted primary objective of this study, manganese levels were only measured in the basal ganglia. This region, particularly the globus pallidus component, is the primary brain focus of manganese accumulation associated with manganese exposure in humans and primates (Olanow et al. 1996; Aschner, Vrana, and Zheng 1999; Kim 2004). However, in rodents, manganese exposure can result in significant accumulation in regions other than the basal ganglia such as the cerebellum and cerebral cortex (Roels et al. 1997; Aschner, Vrana, and Zheng 1999; Sotogaku, Oku, and Takeda 2000; Normandin, Panisset, and Zayed 2002; Normandin et al. 2004; Aschner, Erikson, and Dorman 2005). Therefore, the possibility that the locomotor deficit may be attributable to the action of manganese in motor areas outside the basal ganglia cannot be ruled out. This possibility, or the accumulation of manganese in a basal ganglia region close to but outside the sampled striatum, such as the globus pallidus, could account for the deficit in locomotion observed 24 h after a single dose of 50 mg/kg of MnCl₂. As noted in Results, this dosing failed to produce a significant detectable increase in manganese concentration within the striatum. Experiments by Baek et al. (2003, 2004) suggest that identifiable accumulation of manganese within the basal ganglia of the mouse may not actually be necessary for the induction of pathology in this region following manganese exposure. They have identified gene and protein changes indicative of gliosis within the striatum and globus pallidus of the C57BL/6 mouse following 2 mg/kg/day intraperitoneal dosing with MnCl₂ for 3 weeks. Such astrocytic changes are often used to indicate the onset and locus of neuropathology.

Studies of the effects of MPTP on behavior in the C57BL/6 mouse are fairly numerous and also represent a variety of routes, doses, dosing regimens, and survival times. Given the magnitude of reported decreases in open field locomotion (Sedelis et al. 2000; Crocker et al. 2003; Fernagut et al. 2004; Hayley et al. 2004) and swimming (Donnan et al. 1987), and in light of the magnitude of manganese-induced effects on behavior observed in the present experiment, it appears that even the multiple-dose regimen of 100 mg/kg of MnCl₂ would leave room for detecting potential additive or synergistic effects of combined MnCl₂ and MPTP exposure using the behavioral variables from this experiment. This would be true as long as behavior were assessed 48 hrs or more after the final dose of MPTP (Sedelis et al. 2000).

The findings of this experiment provide guidance for the design of future experiments to assess potential additive or synergistic effects of manganese upon one of the most highly documented animal models of Parkinson’s disease: the MPTP-treated C57BL/6 mouse. Such studies should provide important insights into the potential role of environmental chemicals in neurodegenerative diseases.