Pilot studies were conducted with lower doses (2.0-2.5 mg/kg/day; dissolved in 100% Miglyol 812) to determine the optimal rotenone dose. To determine the optimal rotenone dosing frequency, systemic complex I inhibition was calculated. Male Lewis rats (n = 3 per time-point) were injected with rotenone (2.0 mg/kg, i.p.) and sacrificed at 0, 4, 8, 16, or 32 h after injection. Liver mitochondria were then isolated from rats using differential centrifugation described in the manufacturers protocol (MitoProfile® Benchtop Mitochondria Isolation Kit for Tissue, MitoSciences). Complex I activity was measured in rat liver mitochondria using a protocol adapted from (Janssen et al., 2007). Briefly, liver mitochondrial protein (15μg) was incubated in assay buffer (25 mM potassium phosphate buffer pH 7.8, 3.5 g/L BSA, 70μM DCIP, 6 μM decylubiquinone, 1μM Antimycin A) at 37°C. The reaction was started by adding NADH (200μM). Electron transfer from oxidation of NADH to decylubiquinone to the final electron acceptor 2,6-dichloroindophenol (DCIP) is followed by measuring DCIP reduction spectrophotometrically at 600nm). DCIP reduction was recorded for 20 min. Rotenone (1 μM) sensitive complex I activity was normalized to background DCIP reduction and expressed as μg of DCIP reduced/min/μg of protein.

The rotenone solution was first prepared as a 50× stock in 100% dimethylsulfoxide (DMSO) and diluted in medium-chain triglyceride, Miglyol 812 N (Sasol North America, Inc., Houston, TX, USA; distributed by Warner Graham, Baltimore MD, USA) to obtain a final concentration of 2.75 or 3.0 mg/mL rotenone in 98% Miglyol 812 N, 2% DMSO. Vortexing the solution creates a stable emulsion of the DMSO containing rotenone and Miglyol 812N. The solution was made fresh 2-3 times/week and stored in an amber septa vial protected from light and inverted several times before each injection to eliminate the possibility of settling. The solution was administered at 1 mL/kg and control animals received the vehicle only. Experimental groups were comprised of at least 5-7 animals, with additional animals in the young-adult group for behavioral testing. Rotenone treatment of the three different age groups occurred at different times.

Permanent bilateral denervation of the nigrostriatal dopamine system reproduces the essential motor features of the clinical PD. However, deficits associated with severe dopamine depletion can become debilitating (Cenci et al., 2002; Ungerstedt, 1971). Therefore, animals were monitored twice daily for the appearance of parkinsonian features, including bradykinesia, postural instability/gait disturbances, and rigidity. When the behavioral phenotype became debilitating-limiting mobility, feeding or grooming - animals were euthanized by CO₂ exposure. Brains were rapidly removed and bisected mid-sagittally. One hemisphere was post-fixed in 4% paraformaldehyde for 7 days and then placed in 30% sucrose for at least 3 days, until infiltration was complete. The striatum from the other half of the brain was dissected on ice, flash frozen and then stored at -80°C until processed for neurochemical analysis. Alternatively, for brains wholly dedicated to histological analysis (adult animals), some animals were perfusion-fixed, first with 50 mL ice-cold 0.1 M PBS, and then with 250-350 mL 4% paraformaldehyde. These brains were then post-fixed overnight and transferred to 30% sucrose, where they were stored until fully infiltrated and further processed.

During daily handling, animals were assessed for the emergence of a PD phenotype, which included bradykinesia, postural instability/unsteady gait and rigidity. For all quantified behavioral tests, the experimenter was blinded until the analysis was complete.

When placed in a clear cylinder, rats will engage in exploratory behavior, including rearing (Fleming et al., 2004; Schallert and Tillerson, 2000). During rearing, behavior, the forelimbs will contact the wall of the cylinder. For this test, the rat was placed in a clear plexiglass cylinder (height = 30 cm, diameter = 20 cm) for 5 minutes. The test was conducted under low red-light conditions (10 lux), video-recorded, and during video playback, the number of rears was quantified. To be classified as a rear, the animal had to raise forelimbs above shoulder level and make contact with the cylinder wall with either one or both forelimbs. Removal of both forelimbs from the cylinder wall and contact with the table surface was required before another rear was scored. This test has been successfully used previously to assess behavioral deficits in the rats receiving subcutaneous or intravenous rotenone (Fleming et al., 2004).

Postural instability is a hallmark feature of PD. Recently a test to quantify postural instability was developed in a unilateral 6-OHDA rat PD model (Woodlee et al., 2008). In the current study, the test was conducted as previously reported, except that both forelimbs were averaged together because rotenone produces bilateral symptoms. Briefly, the animal was held vertically, while one forelimb was allowed to contact the table surface, which was lined with medium-grit sand paper. The animal's center of gravity was then advanced until the animal initiated a “catch-up” step. The displacement distance required for the animal to regain the center of gravity was recorded. At each time-point 3 trials for each forelimb were recorded and the average reported.

To determine if observed behavioral deficits were dopamine-dependent, the dopamine agonist apomorphine was administered (1 mg/kg, sc) and behavioral tests were repeated. Apomorphine has previously been found to ameliorate hypokinesia, rigidity and tremor in a bilateral 6-OHDA (a specific dopaminergic neurotoxin) model (Jolicoeur et al., 1991). The rearing trial was conducted 5 minutes after apomorphine administration and the postural instability test was conducted after 10 minutes.

Brains were cut coronally on a frozen sliding microtome at 35 μm and stored in cryoprotectant at -20°C until use. Sections used for single-chromagen development were removed from cryoprotectant, washed in PBS 6× for 5 min each then treated with 3% hydrogen peroxide for 10 minutes followed by 3 PBS washes and blocked for 1 hour in 10% normal donkey serum, containing 0.3% triton X100. For stereological assessment of dopaminergic neurons, the sections were incubated in a primary antibody for tyrosine hydroxylase (1:3000, mouse anti-tyrosine hydroxylase; Millipore #MAB318; Bilerica, MA, USA) for 72 hours at 4°C plus 2 hours at room temperature to obtain optimal antibody penetration. The sections were then washed in PBS 3× 5 min and incubated at room temperature for 1 hour in biotinylated donkey anti-mouse secondary antibody (1:200, Jackson Immuno Research; West Grove, PA, USA). The sections were then washed 3× in PBS and incubated in Advidin-Biotin Complex solution (Vectastain, Vector Labs; Burligame, CA, USA). After washing in PBS, the sections were developed using DAB (Vector Labs). Sections used for stereological analysis, were then washed 3 times in PBS and mounted directly onto slides using Aquamount (Lerner Labs, Pittsburgh, PA, USA). For sections used for general histology, the tissue was mounted onto slides after staining, air dried, dehydrated using graded alcohols and Histoclear (National Diagnostics, Atlanta, GA, USA), followed by mounting with Histomount (National Diagnostics). For immunofluorescence, the staining was done as above except using primary antibodies for α-synuclein (AB5334P, Millipore, Billerica, MA, USA, 1:3000), poly-ubiquitin (Biomol, Plymouth Meeting, PA, USA, PW8805, 1:500), tyrosine hydroxylase, dopamine and cAMP-regulated phosphoprotein (DARPP-32; AB1656, Millipore, 1:500)or pSer19-tyrosine hydroxylase (AB5425, Millipore, 1:3000) and one of the following fluorescent secondary antibodies was used: Cy3-conjugated anti-mouse (Jackson Immuno), Cy5-conjugated anti-rabbit (Jackson Immuno), Alexa 488-conjugated anti-sheep (Molecular Probes) (1:500). These sections were then washed and mounted using gelvatol mounting media. To remove unaggregated protein, some sections were pretreated with proteinase K (2.5 μg/mL, 10 minutes) or formic acid (100%, 10 minutes) before immunohistochemistry.

Cell counting was performed by an experimenter blinded to the treatment group using Stereo Investigator (MBF Bioscience, Williston, VT, USA). One in six sections was used to estimate the number of TH neurons in the entire substantia nigra (including the reticulata portion) of one hemisphere (middle-aged animals). Cells that were clearly stained for TH with a visible nucleus were counted. The counting frame was 45 × 45 × 13 μm (height × width × dissector height) and the sampling grid was 125 × 125 μm. The average final section thickness was 16.1 um, making the average guard zone 1.5 μm for the top and bottom of the section (the dissector was placed in the middle of the section). Stereological parameters were optimized based on a pilot study in both control and treated animals to produce a rigorous estimate of nigral dopamine neurons. The CE Gunderson (m = 1) values were < 0.1 for all animals.

Striatal dopamine and metabolites were assessed for young-adult and middle-aged animals. Adult animals were perfused-fixed for optimal histology and neurochemistry for this group was not assessed. Striatal tissue samples were sonicated on-ice in 0.1 M perchloric acid and centrifuged for 30 min at 16,100 × g. The supernatant was then collected and filtered in Costar Spin-X 0.22 μM nylon membrane polypropylene centrifuge tubes at 1,000 × g and then stored at -80°C. The supernatant was then injected into a Waters 2695 HPLC separation module (Waters Corp., Milford, MA, USA). The samples were injected using an autosampler within the module and the sample temperature was 4 °C. The HPLC mobile phase consisted of 0.06 M sodium phosphate monobasic, 0.03 M citric acid, 8% methanol, 1.075 mM 1-octanesulfonic acid, 0.1 mM ethylenediaminetetraacetic acid, 2 mM sodium chloride, pH 3.5. The flow rate was 1.0 mL/minute. Neurotransmitters were separated on a Waters XBridge C18 4.6 × 150 mm column with a 3.5 μm particle size and detected on a Waters 2465 electrochemical detector with a glassy carbon electrode set at 750 mV referenced to an ISAAC electrode. The separation and detection occurred at 28 °C. Neurotransmitters were quantified using a standard curve generated from injection of standards of the highest available purity.

Mortality occurring within minutes of an injection likely resulted from needle misplacement or settling of rotenone (i.e., accidental overdose). Careful needle placement and vortexing between each injection significantly reduced such mortality. Animals that died within minutes of an injection and that did not experience parkinsonian symptoms were excluded from analysis. Overall mortality related to the injection was low (~ 10% for all treatment groups), was not dose dependent, and decreased with each cohort, as technique improved (vortexing of solution between each injection and the use of 2% DMSO to ensure solubility). Unanticipated mortality from parkinsonian symptoms was eliminated by carefully monitoring animals twice daily; rats were euthanized when the phenotype became debilitating. Severe dopamine depletion (>95%) in the unilateral 6-hydroxydopamine model produces spontaneous behavioral deficits that are apparent with careful examination (Cannon et al., 2005; Cannon et al., 2006). However, bilateral dopamine depletion at even a more modest magnitude (>80%) significantly decreases spontaneous activity and severe (>95%) bilateral depletion can be debilitating causing akinesia, adipsia, and aphagia (Cenci et al., 2002; Deumens et al., 2002; Sakai and Gash, 1994; Schallert et al., 1978; Ungerstedt, 1971). Indeed, once the parkinsonian phenotype presented, it was progressive in nature and eventually became debilitating in all animals.

Daily intraperitoneal rotenone administration (3.0 mg/kg/day) elicits a lesion of the dorsolateral striatal dopamine terminals (Figure 3). The lesions were strikingly similar in size and distribution across animals. High power magnification revealed the presence of hypertrophied (presumably dysfunctional) dopaminergic terminals (Figure 4), both near the lesion edge and also in the dorsolateral striatum of animals that received a lower dose of rotenone (2.75 mg/kg/day) and that had no overt lesion. The lesion was highly selective for dopamine terminals as there was no damage to striatal cell bodies in regions where there was loss of tyrosine hydroxylase positive terminals (Figure 5A-F; cresyl violet staining). Striatal DARPP-32 staining also indicates that there is no overt loss of striatal dopamine-receptive neurons or morphological changes in this cell population (Figure 6). Indeed, high magnification images indicate that in rotenone treated animals (Fig 5Fi), there is no evidence of morphological alterations of striatal neurons or infiltration of inflammatory cells compared to controls (Fig 5Ci).

Loss of dopamine occurred in both young-adult [43.1 ± 9.0 vs. 74.6 ± 6.5 ng/mg protein; rotenone (3.0 mg/kg/day; n = 7) vs. control (n = 6); p<0.05, student's t-test] and middle-aged animals [49.0 ± 12.3 (n = 5) vs. 91.6 ± 7.0 (n = 5); p<0.05] after daily intraperitoneal rotenone administration (Figure 7). Dopamine metabolite turnover [(DOPAC + HVA)/DA) was also increased in young-adult (0.77 ± 0.11 vs. 0.25 vs. 0.01; p<0.001) and middle-aged (0.77 ± 0.09 vs. 0.24 ± 0.02; p<0.001) rotenone-treated animals.

Daily intraperitoneal rotenone resulted in a loss of tyrosine hydroxylase positive neurons of the substantia nigra at both 2.75 and 3.0 mg/kg day in middle-aged rats (Figure 8; p<0.01 at each rotenone dose, Tukey's post hoc test). Because there was no difference in the magnitude of dopamine cell loss between rotenone treatment groups, these groups were merged for further statistical analysis. Overall, unbiased stereology indicated a substantial and significant loss of tyrosine hydroxylase-positive neurons in rotenone-treated rats (9,921 ± 720 vs. 18,010 ± 1,593; # of estimated tyrosine hydroxylase neurons/hemisphere ± S.E.M; p<0.001, student's t-test).