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The numbers of Nisslstained, TH- or MDA-immunoreactive neurons in the substantia nigra pars compacta (SNpc) were counted using the optical fractionator method in the Stereo Investigator (v 4.35) software program (Microbrightfield, Burlington, VT)

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The samples were kept on ice for 10 min and then centrifuged at 12,000 rpm for 5 min at 4 C