In vivo characterization of somatodendritic dopamine release in the substantia nigra of 6-hydroxydopamine-lesioned rats

We investigated the effect of an injection of 6-hydroxydopamine (6-OHDA) into the rat medial forebrain bundle (MFB) on the degeneration and the function of the dopaminergic cell bodies in the substantia nigra (SN) 3 and 5 weeks after lesioning. After injection of 6-OHDA into the MFB a complete loss of dopamine content was apparent in the striatum 3 weeks after lesioning. In the SN the amount of tyrosine hydroxylase-immunoreactive dopamine cells decreased gradually, with a near-complete lesion (> 90%) obtained only after 5 weeks, indicating that neurodegeneration of the nigral cells was still ongoing when total dopamine denervation of the striatum had already been achieved. Baseline dialysate and extracellular dopamine levels in the SN, as determined by in vivo microdialysis, were not altered by the lesion. A combination of compensatory changes of the remaining neurones and dopamine originating from the ventral tegmental area may maintain extracellular dopamine at near-normal levels. In both intact and lesioned rats, the somatodendritic release was about 60% tetrodotoxin (TTX) dependent. Possibly two pools contribute to the basal dopamine levels in the SN: a fast sodium channel-dependent portion and a TTX-insensitive one originating from diffusion of dopamine. Amphetamine-evoked dopamine release and release after injection of the selective dopamine reuptake blocker GBR 12909 were attenuated after a near-complete denervation of the SN (5 weeks after lesioning). So, despite a 90% dopamine cell loss in the SN 5 weeks after an MFB lesion, extracellular dopamine levels in the SN are kept at near-normal levels. However, the response to a pharmacological challenge is severely disrupted.     

Regulation of dopaminergic neuron firing by heterogeneous dopamine autoreceptors in the substantia nigra pars compacta

Dopamine (DA) receptors generate many cellular signals and play various roles in locomotion, motivation, hormone production, and drug abuse. According to the location and expression types of the receptors in the brain, DA signals act in either stimulatory or inhibitory manners. Although DA autoreceptors in the substantia nigra pars compacta are known to regulate firing activity, the exact expression patterns and roles of DA autoreceptor types on the firing activity are highly debated. Therefore, we performed individual correlation studies between firing activity and receptor expression patterns using acutely isolated rat substantia nigra pars compacta DA neurons. When we performed single-cell RT-PCR experiments, D(1), D(2)S, D(2)L, D(3), and D(5) receptor mRNA were heterogeneously expressed in the order of D(2)L > D(2)S > D(3) > D(5) > D(1). Stimulation of D(2) receptors with quinpirole suppressed spontaneous firing similarly among all neurons expressing mRNA solely for D(2)S, D(2)L, or D(3) receptors. However, quinpirole most strongly suppressed spontaneous firing in the neurons expressing mRNA for both D(2) and D(3) receptors. These data suggest that D(2) S, D(2)L, and D(3) receptors are able to equally suppress firing activity, but that D(2) and D(3) receptors synergistically suppress firing. This diversity in DA autoreceptors could explain the various actions of DA in the brain.     

Neuropathology and behavioral impairments in Wistar rats with a 6-OHDA lesion in the substantia nigra compacta and exposure to a static magnetic field

Studies have sought to assess various potential neuroprotective therapeutics in Parkinson's disease. The aim of this study was to evaluate the effects of static magnetic field stimulation 14 days after a 6-Hydroxydopamine (6-OHDA) substantia nigra compacta (SNc) lesion on motor behavior, as assessed by the rotarod (RR) test and brain tissue morphology. Forty male Wistar rats were used and were divided into five groups: control group, sham group (SG), lesion group (LG), lesion north pole group (LNPG) and lesion south pole group (LSPG). In groups with magnetic stimulation, a 3200-gauss magnet was fixed to the skull. After the experiments, the animals were anesthetized for brain perfusion. Coronal sections of the SNc were stained with Nissl. The RR test showed a decrease in the time spent on the apparatus in the LG compared with all groups. The LNPG and LSPG had significant increases in the time spent when compared to the LG. A morphometric analysis revealed a significant reduction in the number of neurons in the LG, LNPG and LSPG in relation to the SG. There were a higher number of neurons in the LNPG and LSPG than the LG, and a higher number of neurons in the LSPG than the LNPG. We observed that the LG, LNPG and LSPG showed a higher number of glial cells than the SG, and the LNPG and LSPG showed a lower number of glial cells than the LG. Our results demonstrate a potential therapeutic use of static magnetic fields for the preservation of motor behavior and brain morphology in the SNc after 14 days with 6-OHDA lesion.     

Inhibition of thrombin-induced microglial activation and NADPH oxidase by minocycline protects dopaminergic neurons in the substantia nigra in vivo

The present study shows that activation of microglial NADPH oxidase and production of reactive oxygen species (ROS) is associated with thrombin-induced degeneration of nigral dopaminergic neurons in vivo. Seven days after thrombin injection in the rat substantia nigra (SN), tyrosine hydroxylase immunocytochemistry showed a significant loss of nigral dopaminergic neurons. This cell death was accompanied by localization of terminal deoxynucleotidyl transferase-mediated fluorecein UTP nick-end labelling (TUNEL) staining within dopaminergic neurons. This neurotoxicity was antagonized by the semisynthetic tetracycline derivative, minocycline, and the observed neuroprotective effects were associated with the ability of minocycline to suppress NADPH oxidase-derived ROS production and pro-inflammatory cytokine expression, including interleukin-1beta and inducible nitric oxide synthase, from activated microglia. These results suggest that microglial NADPH oxidase may be a viable target for neuroprotection against oxidative damage.     

大鼠黑质多巴胺能神经元的二型性

成年哺乳动物的某些脑区存在性别差异,即二型性,但中脑黑质是否存在性分化目前不清楚。本文旨在探讨成年大鼠中脑黑质是否存在二型性。将60只成年大鼠分成5组：（1）正常雌鼠对照组：（2）正常雄鼠对照组：（3）去卵巢组;（4）去睾丸组;（5）去卵巢后回补雌激素组,该组大鼠在去卵巢后的第7天开始连续3d给予生理剂量的雌激素回补。所有大鼠在右侧黑质埋置记录电极,在清醒和安静的生理状态下连续14d记录黑质的P50听觉诱发电位（P50）,之后作黑质酪氨酸羟化酶（tyrosine hydroxylase,TH）免疫组织化学染色,检查TH阳性（TH^＋）细胞数量和形态变化。结果表明,正常成年雄鼠黑质的TH^＋细胞数量较雌鼠少22．47％（P〈0．05）,P50的T／C值也低34．72％（P〈0．01）,提示正常成年大鼠黑质在结构和功能上存在二型性。与正常雄鼠相比,去睾丸大鼠黑质的TH^＋细胞数量、形态和P50的T／C值无显著性变化（P〉0．05）。与正常雌鼠相比,去卵巢大鼠黑质TH^＋细胞数量减少28．09％（P〈0．01）,P50的T／C值降低30．85％（P〈0．01）。在大鼠去卵巢后的短时间内给予3d生理剂量的雌激素,15-20d后可观察到其黑质TH^＋细胞数量、形态和P50的T／C值基本恢复到去卵巢前水平。结果提示,大鼠中脑黑质的多巴胺能神经元在数量、结构和功能活动上存在性别差异：内源性雌激素在维持黑质多巴胺系统完整性及调节其功能活动中起重要作用。     

Glutamate decarboxylase activity in the substantia nigra and the hippocampus of rats microinjected with inhibitors of the enzyme

Three inhibitors of glutamate decarboxylase (GAD), acting through different mechanisms, as well as pyridoxal phosphate (PLP), were microinjected unilaterally by stereotaxic procedures into the substantia nigra reticulata or the CA₁ area of the hippocampus of the rat. The inhibitors used were thiosemicarbazide (TSC), -glutamyl hydrazide and the PLP-glutamyl-hydrazone (PLPGH) formed by the combination of the latter with PLP. No behavioral alterations were observed after the administration of any of the drugs used, in any of the two brain regions studied. When measured in the absence of exogenous PLP, GAD activity in the substantia nigra injected with TSC was diminished by about 35%, and no changes were observed with the other drugs. In the CA₁ hippocampal area both TSC and PLPGH inhibited GAD by more than 50%, and this inhibition was not reversed by PLP added in vitro. The results are discussed in terms of the possible explanation for the differences between the drugs used and for the lack of effects of GAD inhibition on the behavior of the animals.Special issue dedicated to Dr. Eugene Roberts.     

Relationship between microglial activation and dopaminergic neuronal loss in the substantia nigra: a time course study in a 6-hydroxydopamine model of Parkinson's disease

Cellular interactions between activated microglia and degenerating neurons in in vivo models of Parkinson's disease are not well defined. This time course study assesses the dynamics of morphological and immunophenotypic properties of activated microglia in a 6-hydroxydopamine (6-OHDA) model of Parkinson's disease. Neurodegeneration in the substantia nigra pars compacta (SNc) was induced by unilateral injection of 6-OHDA into the medial forebrain bundle. Activated microglia, identified using monoclonal antibodies: clone of antibody that detects major histocompatibility complex (MHC) class II antigens (OX6) for MHC class II, clone of antibody that detects cell surface antigen-cluster of differentiation 11b – anti-complement receptor 3, a marker for complement receptor 3 and CD 68 for phagocytic activity. Activation of microglia in the lesioned SNc was rapid with cells possessing amoeboid or ramified morphology appeared on day 1, whilst antibody clone that detects macrophage-myeloid associated antigen immunoreactivity was observed at day 3 post-lesion when there was no apparent loss of tyrosine hydroxylase (TH)+ve dopaminergic (DA) SNc neurons. Thereafter, OX6 and antibody clone that detects macrophage-myeloid associated antigen activated microglia selectively adhered to degenerating axons, dendrites and apoptotic (caspase 3+ve) DA neurons in the SNc were observed at day 7. This was followed by progressive loss of TH+ve SNc neurons, with the peak of TH+ve cell loss (51%) being observed at day 9. This study suggests that activation of microglia precedes DA neuronal cell loss and neurons undergoing degeneration may be phagocytosed prematurely by phagocytic microglia.     

Simvastatin prevents the inflammatory process and the dopaminergic degeneration induced by the intranigral injection of lipopolysaccharide

Anti-inflammatory strategies have attracted much interest for their potential to prevent further deterioration of Parkinson's disease. Recent experimental and clinical evidence indicate that statins – extensively used in medical practice as effective lipid-lowering agents – have also anti-inflammatory effects. In this study, we investigated the influence of simvastatin on the degenerative process of the dopaminergic neurons of the rat following intranigral injection of lipopolysaccharide (LPS), a potent inductor of inflammation that we have previously used as an animal model of Parkinson's disease. We evaluated TH positive neurons, astroglial, and microglial populations and found that simvastatin prevented the inflammatory processes, as the induction of interleukin-1β, tumor necrosis factor-α, and iNOS and the consequent dopaminergic degeneration induced by LPS. Moreover, simvastatin produced the activation of the neurotrophic factor BDNF, along with the prevention of the oxidative damage to proteins. Moreover, it also prevents the main changes produced by LPS on different mitogen-activated protein kinases, featured as increases of P-c-Jun N-terminal protein kinase, P-extracellular signal-regulated kinase, p-38, and P-glycogen synthase kinase and the decrease of the promotion of cell survival signals such as cAMP response element-binding protein and Akt. Our results suggest that statins could delay the progression of dopaminergic degeneration in disorders involving inflammatory processes.     

Neuromelanin associated redox-active iron is increased in the substantia nigra of patients with Parkinson's disease

Degeneration of dopaminergic neurones during Parkinson's disease is most extensive in the subpopulation of melanized-neurones located in the substantia nigra pars compacta. Neuromelanin is a dark pigment produced in the dopaminergic neurones of the human substantia nigra and has the ability to bind a variety of metal ions, especially iron. Post-mortem analyses of the human brain have established that oxidative stress and iron content are enhanced in association with neuronal death. As redox-active iron (free Fe2+ form) and other transition metals have the ability to generate highly reactive hydroxyl radicals by a catalytic process, we investigated the redox activity of neuromelanin (NM)-aggregates in a group of parkinsonian patients, who presented a statistically significant reduction (- 70%) in the number of melanized-neurones and an increased non-heme (Fe3+) iron content as compared with a group of matched-control subjects. The level of redox activity detected in neuromelanin-aggregates was significantly increased (+ 69%) in parkinsonian patients and was highest in patients with the most severe neuronal loss. This change was not observed in tissue in the immediate vicinity of melanized-neurones. A possible consequence of an overloading of neuromelanin with redox-active elements is an increased contribution to oxidative stress and intraneuronal damage in patients with Parkinson's disease.     

Subtype selective antagonism of substantia nigra pars compacta Group I metabotropic glutamate receptors protects the nigrostriatal system against 6-hydroxydopamine toxicity in vivo

Evidence suggests that increased glutamatergic input to the substantia nigra pars compacta as a result of hyperactivity of subthalalmic nucleus output pathways may contribute to the progressive degeneration of nigral dopaminergic neurones in Parkinson's disease (PD), a debilitating neurodegenerative disorder which affects approximately 1% of people aged over 65. Substantial electrophysiological evidence suggests that the excitation of nigral dopaminergic neurones is regulated by the activation of Group I metabotropic glutamate receptors (mGluR), comprising mGluR1 and mGluR5 subtypes. As activation of these receptors by endogenous glutamate may promote multiple cascades leading to excitotoxic neuronal death, it may be hypothesised that functional antagonism of Group I mGluR should be neuroprotective and could form the basis of a novel neuroprotective treatment for PD. To investigate this hypothesis, the neuroprotective potential of the selective competitive mGlu1 antagonist (+)-2-methyl-4-carboxyphenylglycine ((S)-(+)-alpha-amino-4-carboxy-2-methlybenzeneacetic acid; LY367385) and the selective allosteric mGlu5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) was tested in a rodent 6-hydroxydopamine (6-OHDA) model of PD in vivo. Both acute and subchronic intranigral administration of either LY367385 or MPEP resulted in significant neuroprotection of nigral tyrosine hydroxylase immunoreactive cell bodies, which correlated closely with prevention of striatal monoamine depletion following 6-OHDA lesioning. This neuroprotective action of LY367385 and MPEP displayed a clear concentration-dependent effect, suggesting a receptor-mediated mechanism of action. LY367385 produced robust neuroprotection at all concentrations tested (40, 200 and 1000 nmol in 4 microL), whilst MPEP displayed a bell-shaped neuroprotective profile with significant neuroprotection at low concentrations (2 and 10 nmol in 4 microL) but not at higher concentrations (50 nmol). Importantly, subchronic intranigral administration of MPEP and LY367385 appeared to slow the degeneration of remaining nigral dopaminergic neurones and prevented further striatal dopamine depletion in animals with established 6-OHDA induced nigrostriatal lesions, suggesting that these compounds may significantly influence disease progression in this model.     

Striatal dopamine-NMDA receptor interactions in the modulation of glutamate release in the substantia nigra pars reticulata in vivo: opposite role for D1 and D2 receptors

To investigate the antioxidative capacities of oligodendrocytes, rat brain cultures enriched for oligodendroglial cells were prepared and characterized. These cultures contained predominantly oligodendroglial cells as determined by immunocytochemical staining for the markers galactocerebroside and myelin basic protein. If oligodendroglial cultures were exposed to exogenous hydrogen peroxide (100 micro m), the peroxide disappeared from the incubation medium following first order kinetics with a half-time of approximately 18 min. Normalization of the disposal rate to the protein content of the cultures by calculation of the specific hydrogen peroxide detoxification rate constant revealed that the cells in oligodendroglial cultures have a 60% to 120% higher specific capacity to dispose of hydrogen peroxide than cultures enriched for astroglial cells, microglial cells or neurones. Oligodendroglial cultures contained specific activities of 133.5 +/- 30.4 nmol x min(-1) x mg protein(-1) and 27.5 +/- 5.4 nmol x min(-1) x mg protein(-1) of glutathione peroxidase and glutathione reductase, respectively. The specific rate constant of catalase in these cultures was 1.61 +/- 0.54 min(-1) x mg protein(-1). Comparison with data obtained by identical methods for cultures of astroglial cells, microglial cells and neurones revealed that all three of the enzymes which are involved in hydrogen peroxide disposal were present in oligodendroglial cultures in the highest specific activities. These results demonstrate that oligodendroglial cells in culture have a prominent machinery for the disposal of hydrogen peroxide, which is likely to support the protection of these cells in brain against peroxides when produced by these or by surrounding brain cells.     

The chemical characterization of melanin contained in substantia nigra of human brain

The pigment of substantia nigra human brain has been extracted by a mild procedure consisting of washes with phosphate buffer, methanol and incubation with SDS-proteinase. Pyrolisis gas chromatography mass spectrometry infrared spectrometry, termogravimetric analysis and elemental analysis were the techniques used for the chemical characterization. An indole moiety bound to a sulfur containing amino acid and to palmitic acid were the main aspects found in the structure. The presence of a 7% inorganic component was observed. This probably contains Fe, Cu, Zn and Cr which are also relevant, for the formation and the role of melanin in substantia nigra neurons. The fatty acid moiety is chemically bound to the indole structure as it was not eliminated by repeated methanol washing. The same situation occurs for the sulfur containing gropu. Considering these data and the most abundant molecules present in substantia nigra the precursor of neuromelanin seems to be a cysteinyl-cethecol, to which is then bound a palmityl group.     

A one-carbon modification of protein lysine associated with elevated oxidative stress in human substantia nigra

We describe for the first time a naturally occurring lysine modification that is converted to methyllysine by reduction with sodium borohydride. This modification is approximately 1.7 times as abundant in soluble proteins from human substantia nigra pars compacta as in proteins from other brain regions, possibly as a result of elevated oxidative stress in the nigra. Proteins from cultured PC12 cells exposed to oxidative stress conditions also contain elevated levels of this lysine modification. The abundance of the naturally occurring modification is roughly 0.08 nmoles/mg protein in either unstressed brain or PC12 cells. Modification levels remain stable in isolated proteins incubated for 2 h at 37 degrees C in pH 7 buffer. We propose that the endogenous modification is the lysine Schiff base, epsilon-N-methylenelysine, and that lysine modifications may result from a reaction with formaldehyde in vivo. Rat brain contains approximately 60 nmoles/g wet weight of formaldehyde, which probably includes both free and reversibly bound forms. Adding approximately 35 microm HCHO to PC12 cell growth medium introduces methylenelysine modifications in cell proteins and impairs cell viability. The existence of this post-translational modification suggests new mechanisms of oxidative stress that may contribute to tissue degeneration, including loss of nigral dopamine neurons during normal aging and in Parkinson's disease.     

Oxidative post-translational modifications of alpha-synuclein in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease

Structural and functional alterations of alpha-synuclein is a presumed culprit in the demise of dopaminergic neurons in Parkinson's disease (PD). Alpha-synuclein mutations are found in familial but not in sporadic PD, raising the hypothesis that effects similar to those of familial PD-linked alpha-synuclein mutations may be achieved by oxidative post-translational modifications. Here, we show that wild-type alpha-synuclein is a selective target for nitration following peroxynitrite exposure of stably transfected HEK293 cells. Nitration of alpha-synuclein also occurs in the mouse striatum and ventral midbrain following administration of the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Conversely, beta-synuclein and synaptophysin were not nitrated in MPTP-intoxicated mice. Our data demonstrate that alpha-synuclein is a target for tyrosine nitration, which, by disrupting its biophysical properties, may be relevant to the putative role of alpha-synuclein in the neurodegeneration associated with MPTP toxicity and with PD.     

Iron, neuromelanin and ferritin content in the substantia nigra of normal subjects at different ages: consequences for iron storage and neurodegenerative processes

Information on the molecular distribution and ageing trend of brain iron in post‐mortem material from normal subjects is scarce. Because it is known that neuromelanin and ferritin form stable complexes with iron(III), in this study we measured the concentration of iron, ferritin and neuromelanin in substantia nigra from normal subjects, aged between 1 and 90 years, dissected post mortem. Iron levels in substantia nigra were 20 ng/mg in the first year of life, had increased to 200 ng/mg by the fourth decade and remained stable until 90 years of age. The H‐ferritin concentration was also very low (29 ng/mg) during the first year of life but increased rapidly to values of ≈ 200 ng/mg at 20 years of age, which then remained constant until the eighth decade of life. L ‐Ferritin also showed an increasing trend during life although the concentrations were ≈ 50% less than that of H‐ferritin at each age point. Neuromelanin was not detectable during the first year, increased to ≈ 1000 ng/mg in the second decade and then increased continuously to 3500 ng/mg in the 80th year. A Mössbauer study revealed that the high‐spin trivalent iron is probably arranged in a ferritin‐like iron?oxyhydroxide cluster form in the substantia nigra. Based on this data and on the low H‐ and L‐ferritin content in neurones it is concluded that neuromelanin is the major iron storage in substantia nigra neurones in normal individuals.     

Phosphoproteome profiling of substantia nigra and cortex regions of Alzheimer's disease patients

The induction of ischemic tolerance by preconditioning provides a platform to elucidate endogenous mechanisms of stroke protection. In these studies, we characterize the relationship between hypoxia‐inducible factor (HIF), sphingosine kinase 2 (SphK2), and chemokine (C–C motif) ligand 2 (CCL2) in models of hypoxic or pharmacological preconditioning‐induced ischemic tolerance. A genetics‐based approach using SphK2‐ and CCL2‐null mice showed both SphK2 and CCL2 to be necessary for the induction of ischemic tolerance following preconditioning with hypoxia, the hypoxia‐mimetic cobalt chloride, or the sphingosine‐1‐phosphate (S1P) agonist FTY720. A pharmacological approach confirmed the necessity of HIF signaling for all three preconditioning stimuli, and showed that the SphK/S1P pathway transduces tolerance via the S1P₁ receptor. In addition, our data suggest significant cross‐talk between HIF and SphK2‐produced S1P signaling, which together act to up‐regulate CCL2 expression. Overall, HIF, SphK, S1P, and CCL2 participate in a signaling cascade to induce the gene expression responsible for the stroke‐tolerant phenotype established by hypoxic and FTY720 preconditioning. The identification of these common molecular mediators involved in signaling the genomic response to multiple preconditioning stimuli provides several targets for therapeutic manipulation.