Introduction

Parkinson’s disease (PD) is a very common age-related disease affecting more than 1% of the population over 60 years of age. The majority (~ 90%) of PD is sporadic and aging is the major risk factor [1]. PD is one of the most common neurodegenerative diseases and characterized by a selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), a mesencephalic nucleus included in the basal ganglia circuitry, which is responsive for the regulation of voluntary movement. However, the exact cause of the neuronal loss remains unclear [2].

Glutamate excitotoxicity is the pathological process through which neurons are damaged and killed after excess activation of glutamate receptors. Glutamate excitotoxicity is due to intracellular processes such as Ca2+ overload and bioenergetic changes, which induce oxidative stress and apoptosis. This excitotoxicity is known as a final common pathway for neuronal death and is observed in many neurological diseases including PD [3,4,5,6].

In the basal ganglia, the projection from the subthalamic nucleus (STN) to the SNpc is glutamatergic and the SNpc is also innervated from the amygdala via glutamatergic neurotransmitter system [7, 8]. Dopaminergic neurons express glutamate receptors in the SNpc [9]. It has been postulated that excess activation of glutamate receptors on dopaminergic neurons in the SNpc may be involved in pathophysiology of PD [10,11,12,13,14].

The influx of extracellular Ca2+ through N-methyl-D-aspartate (NMDA) receptors plays a crucial role in glutamate excitotoxicity in cortical and hippocampal neurons [15, 16]. Even in dopaminergic neurons where the mechanism of glutamate excitotoxicity is poorly understood, Ca2+ influx through NMDA receptors has been believed to be a trigger for neuronal death [8, 17]. In contrast, it is recognizing that most of the death signaling associated with neurological conditions is mediated by not only Ca2+ but also Zn2+ [18,19,20]. Extracellular Zn2+ influx, which is dynamically linked with Zn2+ release from zincergic neuron, a subclass of glutamatergic neurons, is particularly important in causing the selective and delayed degeneration of hippocampal CA1 pyramidal neurons in transient global ischemia [21]. However, glutamatergic neurons are non-zincergic in the SNpc and do not contain zinc in the presynaptic vesicles [22]. Thus, it is estimated that extracellular Zn2+ concentration is not dynamically increased by glutamatergic synapse excitation in the SNpc unlike the hippocampus.

In human neuroblastoma SH-SY5Y cells, a dopaminergic neuronal cell line, Zn2+-induced neurotoxicity occurs via Zn2+-permeable transient receptor potential melastatin 7 (TRPM7) channel at the micromolar range of extracellular Zn2+ concentration [23]. Yang et al. report that cell death caused by the synergistic effects of micromolar Zn2+ and dopamine is observed in PC12 via a stress sensor gene Gadd45b. They suggest that Zn2+ and dopamine are implicated in the degeneration of dopaminergic neurons [24]. However, the micromolar range of extracellular Zn2+ is improbable in the SNpc in vivo, judging from the estimated basal concentration (~ 10 nM) of extracellular Zn2+ in the hippocampus [25]. On the other hand, Lee et al. report that cytosolic Zn2+ accumulation is observed in degenerating dopaminergic neurons after treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). They suppose that cytosolic Zn2+ accumulation is an indicator of degenerating dopaminergic neurons in animal models of PD [26]. Therefore, it is important to clarify the significance of cytosolic Zn2+ toxicity and its origin in nigrostriatal dopaminergic neurodegeneration.

Here, we report a unique mechanism of nigrostriatal dopaminergic neurodegeneration, in which extracellular Zn2+ influx plays a key role for PD pathogenesis induced with 6-hydroxydopamine (6-OHDA) in rats.

Materials and Methods

Animals and Chemicals

Male Wistar rats (6–12 weeks of age) were purchased from Japan SLC (Hamamatsu, Japan). Rats were housed under the standard laboratory conditions (23 ± 1 °C, 55 ± 5% humidity) and had access to tap water and food ad libitum. All the experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the University of Shizuoka that refers to the American Association for Laboratory Animals Science and the guidelines laid down by the NIH (NIH Guide for the Care and Use of Laboratory Animals) in the USA. The Ethics Committee for Experimental Animals in the University of Shizuoka has approved this work.

ZnAF-2 and ZnAF-2DA, membrane-impermeable and membrane-permeable Zn2+ fluorescence probes, were kindly supplied from Sekisui Medical Co., LTD (Hachimantai, Japan). ZnAF-2DA is taken up into the cells through the cell membrane and is hydrolyzed by esterase in the cytosol to yield ZnAF-2, which cannot permeate the cell membrane [27, 28]. ZnAF-2 is selectively bound to Zn2+, but not bound to other divalent cations such as Ca2+, Mg2+, and Cu2+ [24]. Calcium orange acetoxymethyl ester (AM), a membrane-permeable Ca2+ indicator, was purchased from Molecular Probes, Inc. (Eugene, OR). These fluorescence indicators were dissolved in dimethyl sulfoxide (DMSO) and then diluted to artificial cerebrospinal fluid (ACSF) containing 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 1.0 mM NaH2PO4, 2.5 mM CaCl2, 26.2 mM NaHCO3, and 11 mM D-glucose (pH 7.3).

In Vivo Microdialysis

The rats were anesthetized with chloral hydrate (400 mg/kg) and individually placed in a stereotaxic apparatus. The skull was exposed, a burr hole was drilled, and a microdialysis probe (1-mm membrane, Eicom, Kyoto) was inserted into the right SNpc (5.3 mm posterior to the bregma, 2.0 mm lateral, 7.8 mm inferior to the dura). The SNpc was preperfused with artificial cerebrospinal fluid (ACSF) (127 mM NaCl, 2.5 mM KCl, 1.3 mM CaCl2, 0.9 mM MgCl2, 1.2 mM Na2HPO4, 21 mM NaHCO3, and 3.4 mM D-glucose, pH 7.3) containing 0.1% ascorbic acid at 2.0 μl/min for 120 min to stabilize the region, perfused with ACSF containing 0.1% ascorbic acid or ACSF containing 0.1% ascorbic acid + 50 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist, for 60 min in the same manner to determine the basal concentration of extracellular Zn2+, and then perfused with 0.8 mM 6-OHDA in ACSF containing 0.1% ascorbic acid or 0.8 mM 6-OHDA + 50 μM CNQX in ACSF containing 0.1% ascorbic acid, respectively, for 60 min. The perfusate was collected for 60 min and 1 μM ZnAF-2 (50 μl) was added to aliquot of the perfusate (10 μl). The fluorescence of ZnAF-2 (excitation/emission, 485/535 nm) was measured using a plate reader ARVO sx (Perkin Elmer, USA).

In Vivo Dynamics of Intracellular Zn2+ and Ca2+

The rats were anesthetized with chloral hydrate (400 mg/kg) and individually placed in a stereotaxic apparatus. The skull was exposed, a burr hole was drilled, and injection cannulae (internal diameter, 0.15 mm; outer diameter, 0.35 mm) were carefully and slowly inserted into the right and left SNpc (5.3 mm posterior to the bregma, 2.0 mm lateral, 7.0 mm inferior to the dura) to avoid cellular damages. Thirty minutes after the surgical operation, 6-OHDA (8 mM) or 6-OHDA (8 mM) + N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) (100 μM), a membrane-permeable Zn2+ chelator, in saline containing 0.1% ascorbic acid + ZnAF-2DA (100 μM) or in saline containing 0.1% ascorbic acid + calcium orange AM (50 μM) were bilaterally injected into the SNpc via cannulae at the rate of 0.5 μl/min for 8 min. Ten minutes after injection, the injection cannulas were slowly pulled out the brain in about 10 min and the rats were decapitated. The brain was quickly removed and immersed in ice-cold choline-ACSF containing 124 mM choline chloride, 2.5 mM KCl, 2.5 mM MgCl2, 1.25 mM NaH2PO4, 0.5 mM CaCl2, 26 mM NaHCO3, and 10 mM glucose (pH 7.3) to avoid neuronal excitation. Coronal brain slices (400 μm) were prepared using a vibratome ZERO-1 (Dosaka, Kyoto, Japan) in an ice-cold choline-ACSF and maintained in an ice-cold choline-ACSF for 30 min. The brain slices were immersed in ACSF for 15 min and transferred to a recording chamber filled with ACSF. The fluorescence of ZnAF-2 (laser, 488.4 nm; emission, 500–550 nm) and calcium orange (laser, 561.4 nm; emission, 570–620 nm) was measured with a confocal laser scanning microscopic system (Nikon A1 confocal microscopes, Nikon Corp.). Region of interest was set in the SNpc.

In Vitro Dynamics of Extracellular Zn2+

The brain was quickly removed from rats under anesthesia with chloral hydrate and immersed in ice-cold choline-ACSF. Coronal brain slices (400 μm) were prepared and maintained in ice-cold choline-ACSF for 15 min. To assess intracellular levels of Zn2+, brain slices were placed for 30 min in 10 μM ZnAF-2DA in ACSF, rinsed in choline-ACSF for 20 min, placed in a chamber filled with 8 mM 6-OHDA, 8 mM 6-OHDA + 10 mM CaEDTA, a membrane-impermeable Zn2+ chelator, or 8 mM 6-OHDA + 10 μM CNQX in ACSF containing 10 nM ZnCl2 for 10 min, rinsed in choline-ACSF for 15 min, and transferred to a recording chamber filled with ACSF. The fluorescence of ZnAF-2 was measured in the same manner.

Behavioral Studies

As described in the section of “In vivo dynamics of intracellular Zn2+ and Ca2+”, an injection cannula was inserted into the right SNpc and 6-OHDA (8 mM), 6-OHDA (8 mM) + ZnAF-2DA (200 μM), or 6-OHDA (8 mM) + TREN (100 μM) in saline containing 0.1% ascorbic acid was unilaterally injected into the SNpc via the cannula at the rate of 0.5 μl/min for 8 min. Ten minutes after injection, the injection cannula was slowly pulled out the brain in about 10 min. One and 2 weeks later, the rats were subcutaneously injected with apomorphine (0.5 mg/kg) and turning behavior in response to apomorphine was measured for 30 min after the start of the turning behavior.

Tyrosine Hydroxylase Immunostaining

The rats were anesthetized and perfused with ice-cold 4% paraformaldehyde in PBS after the behavioral studies were finished, followed by removal of the brain and overnight fixation in 4% paraformaldehyde in PBS at 4 °C. Fixed brains were cryopreserved in 30% sucrose in PBS for 2 days and frozen in Tissue-Tek optimal cutting temperature embedding medium. Coronal brain slices (30 μm) were prepared at − 20 °C in a cryostat, picked up on slides, and adhered at room temperature for 30 min. For immunostaining, slides were incubated in blocking solution (3% BSA, 0.1% Triton X-100 in PBS) for 1 h and rinsed with PBS for 5 min followed by overnight incubation with anti-tyrosine hydroxylase antibody (Abcam) at 4 °C. Slides were rinsed with PBS for 5 min and incubated in blocking buffer containing Alexa Fluor 633 goat anti-rabbit secondary antibody (ThermoFisher) for 3 h at room temperature. Following six rinses in PBS for 5 min, slides were mounted with Prolong Gold antifade reagent and placed for 24 h at 4 °C. Alexa Fluor 633 fluorescence was measured in the SNpc and the striatum using a confocal laser scanning microscopic system.

Data Analysis

For statistical analysis, Student’s t test was used for comparison of the means of paired or unpaired data. For multiple comparisons, differences between treatments were assessed by one-way ANOVA followed by post hoc testing using the Tukey test (the statistical software, GraphPad Prism 5). A value of p < 0.05 was considered significant. Data were expressed as means ± standard error. The results of statistical analysis are described in each figure legend.

Results

6-OHDA Increases Extracellular Zn2+ Influx in the SNpc

6-OHDA is a classic animal model of PD [29]. Reactive oxygen species (ROS) derived from 6-OHDA uptake and intraneuronal autooxidation, extracellular 6-OHDA autoxidation, and microglial activation may be involved in the mechanisms responsible for 6-OHDA-induced dopaminergic degeneration [30]. We examined the idea that 6-OHDA-mediated ROS production modifies intracellular Zn2+ dynamics in nigral dopaminergic neurons. 6-OHDA rapidly increased intracellular Zn2+ in the SNpc of brain slices after 10-min incubation with 6-OHDA in ACSF containing 10 nM Zn2+, an estimated basal concentration of extracellular Zn2+ [25], but not in the substantia nigra pars reticulata (SNpr) and other midbrain area (deep mesencephalic nuclei and/or parabrachial pigmented nuclei) (Fig. 1a, b) and this increase was blocked in the presence of CaEDTA, an extracellular Zn2+ chelator and CNQX, an AMPA receptor antagonist (Fig. 1a, c).

Fig. 1
figure 1

6-OHDA increases intracellular Zn2+ concentration in the SNpc in vitro. Brain slices loaded with ZnAF-2DA were immersed in vehicle (control), 8 mM 6-OHDA in ACSF, 8 mM 6-OHDA + 10 mM CaEDTA in ACSF, or 8 mM 6-OHDA + 10 μM CNQX in ACSF for 10 min. a Intracellular ZnAF-2 fluorescence was imaged in the SNpc, SNpr, and other midbrain area. The SN and magnified SN are shown in the coronal brain image (upper side) for better understanding the area of the SNpc where it is surrounded by the white dotted line. b, c Each bar and line represents the ratio of ZnAF-2 fluorescence intensity to the control ZnAF-2 fluorescence intensity, which was expressed as 100%. ***p < 0.001 vs. control, ###p < 0.001 vs. 6-OHDA group (Tukey’s test)

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An in vivo microdialysis experiment indicated extracellular Zn2+ concentration in the perfusate, which was determined with ZnAF-2, was decreased under SNpc perfusion with 6-OHDA, while this decrease was blocked by co-perfusion with CNQX (Fig. 2). Unfortunately, extracellular glutamate concentration was disturbed by the presence of 6-OHDA in the perfusate (data not shown).

Fig. 2
figure 2

6-OHDA decreases extracellular Zn2+ concentration in the SNpc in vivo. The SNpc was perfused with ACSF containing 0.1% ascorbic acid or ACSF containing 0.1% ascorbic acid + 50 μM CNQX for 60 min at 2.0 μl/min to determine the basal concentration of extracellular Zn2+ and then perfused with 0.8 mM 6-OHDA in ACSF containing 0.1% ascorbic acid or 0.8 mM 6-OHDA + 50 μM CNQX in ACSF containing 0.1% ascorbic acid, respectively, for 60 min. Zn2+ concentration in the perfusate was determined with ZnAF-2. Each bar and line represents the ratio of ZnAF-2 fluorescence intensity to the basal ZnAF-2 fluorescence intensity, which was expressed as 100%. *p < 0.001 vs. the basal level, ##p < 0.01 vs. 6-OHDA group (t test)

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Intracellular Zn2+ Chelators Ameliorate 6-OHDA-Induced Neurodegeneration

Turning behavior in response to apomorphine, an index of behavioral abnormality of 6-OHDA-induced PD in rats was ameliorated by co-injection of membrane-permeable Zn2+ chelators, i.e., ZnAF-2DA and TPEN 1 and 2 weeks after 6-OHDA injection (Fig. 3).

Fig. 3
figure 3

Turning behavior to apomorphine after injection of 6-OHDA into the SNpc. Vehicle (control), 6-OHDA (8 mM), 6-OHDA (8 mM) + ZnAF-2DA (200 μM), or 6-OHDA (8 mM) + TPEN (100 μM) in saline containing 0.1% ascorbic acid was unilaterally injected into the SNpc. One (a) and 2 (b) weeks later, the rats were subcutaneously injected with apomorphine (0.5 mg/kg) and turning behavior (the number of times) in response to apomorphine was measured for 30 min after the start of the turning behavior. ***p < 0.001 vs. control, ###p < 0.001 vs. 6-OHDA group (Tukey’s test). ***p < 0.001 vs. control, ###p < 0.001 vs. 6-OHDA group (Tukey’s test)

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6-OHDA-induced loss of nigrostriatal dopaminergic neurons was determined by tyrosine hydroxylase immunostaining after the behavioral test was finished. Staining intensity was drastically reduced in the ipsilateral SNpc (Fig. 4a, c) and the striatum (Fig. 4b, d). However, the reductions were also ameliorated by co-injection of ZnAF-2DA and TPEN.

Fig. 4
figure 4

Neuronal loss in the SNpc and striatum after injection of 6-OHDA into the SNpc. Brain slices were prepared from the rats after the behavioral test was finished and tyrosine hydroxylase immunostaining with Alexa Fluor 633 fluorescence was performed in the SNpc (a) and the striatum (S) where it was surrounded by the white dotted line. Each bar and line represents the ratio of Alexa Fluor 633 fluorescence intensity to Alexa Fluor 633 fluorescence intensity in the control contralateral SNpc (c) and striatum (d), which was expressed as 100%. ***p < 0.001 vs. control, #p < 0.05, ###p < 0.001, vs. 6-OHDA group (Tukey’s test)

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TPEN Blocks 6-OHDA-Induced Increase in Intracellular Zn2+, but not in Intracellular Ca2+

To assess the involvement of the influx of Zn2+ and Ca2+ in behavioral abnormality via 6-OHDA-induced nigrostriatal dopaminergic neurodegeneration, in vivo dynamics of intracellular Zn2+ and Ca2+ was captured 30 min after the start of 6-OHDA injection into the SNpc (Fig. 5a). Both intracellular Zn2+ and Ca2+ were increased in the SNpc, while TPEN blocked the increase in intracellular Zn2+ (Fig. 5b), but not in intracellular Ca2+ (Fig. 5c).

Fig. 5
figure 5

6-OHDA increases intracellular Zn2+ in the SNpc but not intracellular Ca2+ in vivo. Vehicle (control), 6-OHDA (8 mM), or 6-OHDA (8 mM) + TPEN (100 μM) in saline containing 0.1% ascorbic acid was bilaterally injected into the SNpc. (a) Intracellular fluorescence of ZnAF-2 and calcium orange was measured in the SNpc where it is surrounded by the white dotted line. Each bar and line represents the ratio of ZnAF-2 (b) and Ca-orange (c) fluorescence intensity to the control fluorescence intensity, which was expressed as 100%. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; ###p < 0.001 vs. 6-OHDA group (Tukey’s test)

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Discussion

Evidence has pinpointed Ca2+ as the major determinant of ischemic neuronal death, based on Ca2+ imaging experiments that use Ca2+ fluorescence probes or the neuroprotection offered by Ca2+ chelators. However, all of Ca2+ fluorescence probes and Ca2+ chelators are not Ca2+ selective, and they indeed show a higher affinity for Zn2+ [19, 31]. On the other hand, extracellular Zn2+ permeates NMDA receptors and voltage-dependent Ca2+ Channels, while it preferentially passes through Ca2+- and Zn2+-permeable GluR2-lacking AMPA receptors [18, 32]. Ca2+- and Zn2+-permeable GluR2-lacking AMPA receptors are involved in synaptically released Zn2+-mediated neurodegeneration in the hippocampal CA1 and CA3 [33,34,35]. Nigral dopaminergic neurons are not innervated by zincergic neurons unlike hippocampal pyramidal neurons [22]. Thus, extracellular Zn2+ concentration is not significantly increased by glutamatergic neuron excitation in the SNpc. On the other hand, 6-OHDA has been shown to produce endogenously in patients suffering from PD [36, 37] and to increase intracellular Zn2+ release and accumulation via ROS production [38, 39]. Intracellular (cytosolic) Zn2+ concentration is estimated to be considerably less than 1 nM in nigral dopaminergic neurons [40, 41]. We postulated that nigral dopaminergic neurons are sensitive to intracellular Zn2+ dysregulation and tested a unique mechanism of nigrostriatal dopaminergic neurodegeneration, in which extracellular Zn2+ dynamics plays a key role for PD pathogenesis induced with 6-OHDA in rats.

6-OHDA rapidly increased intracellular Zn2+ only in the SNpc of brain slices after 10-min incubation with 6-OHDA in ACSF containing 10 nM Zn2+ while this increase was blocked in the presence of CaEDTA and CNQX, indicating that 6-OHDA rapidly increases extracellular Zn2+ influx via AMPA receptor activation in the SNpc. Extracellular Zn2+ concentration was decreased under in vivo SNpc perfusion with 6-OHDA and this decrease was blocked by co-perfusion with CNQX, supporting 6-OHDA-induced Zn2+ influx via AMPA receptor activation in the SNpc. These results suggest that 6-OHDA-mediated ROS production increases extracellular Zn2+ influx into dopaminergic neurons via AMPA receptor activation in the SNpc. It is likely that 6-OHDA-mediated ROS production increases glutamate release from neuron terminals in the SNpc. 6-OHDA is taken up into dopaminergic neurons through dopamine transporters and rapidly produces ROS in the intracellular compartment, in addition to ROS production by autoxidation in the extracellular compartment [42]. It is possible that both extracellular ROS and ROS in dopaminergic neurons, which is retrogradely transported, are taken up into glutamatergic neuron terminals and induce glutamate release. The data that 6-OHDA-induced increase in Zn2+ influx was selectively observed in the SNpc of brain slices suggest that the rapid Zn2+ influx into dopaminergic neurons is due to ROS produced in dopaminergic neurons rather than extracellular ROS.

Next, we examined whether nigrostriatal dopaminergic neurodegeneration induced with 6-OHDA could be due to the rapid increase in intracellular Zn2+. Interestingly, both 6-OHDA-induced loss of nigrostriatal dopaminergic neurons and turning behavior to apomorphine were ameliorated by co-injection of intracellular Zn2+ chelators, i.e., ZnAF-2DA and TPEN. Extracellular Zn2+ concentration, which is estimated to be approximately 10 nM in the hippocampus [25], was decreased in the SNpc by the rapid Zn2+ influx via 6-OHDA-mediated AMPA receptor activation. The finding suggests that intracellular Zn2+ concentration rapidly reaches ~ 10 nM, resulting in neuronal death. The lethal concentration of intracellular Ca2+ is micromolar (10–20 μM) [43], while the present data indicate that the lethal concentration of intracellular Zn2+ is much low in the SNpc and that nigral dopaminergic neurons are much vulnerable to intracellular Zn2+ dysregulation.

In ischemic neuronal death, acidosis reduces Zn2+ binding to metallothioneins, followed by the increase in intracellular Zn2+ [44]. Furthermore, mitochondrial dysfunction including ROS generation promotes intracellular Zn2+ mobilization, which originates in the mitochondria and metallothioneins [38, 45]. Among metallothionein isoforms, metallothionein III preferentially releases Zn2+ under oxidative condition [46]. ROS-mediated TRPM7 (transient receptor potential cation channel subfamily M member 7) activation releases Zn2+ from intracellular vesicles after Zn2+ overload [47]. In the present study, rapid increase in intracellular Zn2+ induced with 6-OHDA was almost completely blocked in the presence of CaEDTA and CNQX, suggesting that the rapid increase is due to extracellular Zn2+ influx but not Zn2+ release from metallothioneins and/or internal stores. Although it is possible that Zn2+ release from metallothioneins and/or internal stores occurs in the late stage of neurodegeneration, the block of the rapid Zn2+ influx via 6-OHDA-mediated ROS production may be an effective strategy for reducing nigrostriatal dopaminergic neurodegeneration in the SNpc.

Co-injection of TPEN into the SNpc blocked 6-OHDA-induced increase in intracellular Zn2+ but not in intracellular Ca2+. The present study indicates that 6-OHDA-induced rapid increase in extracellular Zn2+ influx into dopaminergic neurons via AMPA receptor activation in the SNpc induces PD via nigrostriatal dopaminergic neurodegeneration. Dopamine is rapidly taken up into dopaminergic neurons via dopamine transporters in the SNpc [48], produces intracellular ROS, and might be metabolized to 6-OHDA [42]. Therefore, 6-OHDA- and dopamine-induced Zn2+ influx may be a trigger for dopaminergic neurodegeneration in the SNpc. Characteristics (easiness) of extracellular Zn2+ influx may be linked with weakened intracellular Zn2+-buffering in the aged dentate gyrus [49, 50], indicating that vulnerability to intracellular Zn2+ dysregulation is promoted in the brain along with aging. Metabolic disorder of synaptic dopamine might induce intracellular Zn2+ dysregulation via ROS production, perhaps followed by pathogenesis of dopaminergic neurodegeneration in the SNpc.