Single section and disector counts in evaluating neuronal loss from the substantia nigra in patients with Parkinson's disease

In order to investigate the correlation between single section (SS) and disector (DS) counts for estimating the pigmented neurons in the pars compacta of the substantia nigra (SNpc), 12 patients with Parkinson's disease (PD) and seven controls were studied. The SS counts were obtained at the level of the exit of the third cranial nerve in the SNpc. The DS counts were made from the whole volume of the SNpc. There was a significant correlation between SS and DS counts (r=0.94, P<0.001), suggesting that the methods closely agree. Comparison of SS and DS counts in controls and PD patients showed that the pigmented neurons in PD were decreased by 75% (P<0.001) and 55% (P<0.001) in the SS and DS counts, respectively. This study suggests that loss of pigmented neurons from the SNpc in PD can be estimated by either method.     

Synaptic connections between pars compacta and pars reticulata neurones: electrophysiological evidence for functional modules within the substantia nigra

Intracellular recordings were performed in vitro from both pars compacta and pars reticulata neurones of the substantia nigra, and their postsynaptic responses to electrical stimulation within the nucleus were analysed. Intracellular staining by biocytin was used to reveal the morphology and location of the recorded neurone and its position and distance to the stimulating electrode. Inhibitory postsynaptic potentials in pars compacta neurones were evoked exclusively from a specific region of stimulation within the pars reticulata; this field could be mapped out as a cone-shaped region surrounding the apical dendrite of the cell and perpendicular to the plane of the pars compacta. Furthermore, hemitransection, prior to the experiments which eliminated the most likely source of extrinsic inhibition (the GABAergic striatonigral pathway) affected neither the generation of inhibitory postsynaptic potentials nor its topographic pattern during pars reticulata stimulation. In contrast to the response of dopaminergic pars compacta neurones, pars reticulata neurones responded to stimulation over wide areas of substantia nigra, without any clear site-specific selectivity. It is concluded that within the substantia nigra, dopaminergic neurones are arranged in functional modules such that most inhibition is derived from a highly local circuit with the collaterals of adjacent pars reticulata cells. This module is intrinsic to the substantia nigra and may represent the basic functional unit of the nucleus.     

Cell counts in the substantia nigra: a comparison of single section counts and disector counts in patients with Parkinson's disease and in controls

The disector (DS) is a method by which it is possible to estimate the density of cells in a volume of tissue, and when associated with Cavalieri's principle. the total number of cells of a neural nucleus. Since the introduction of this method, there has been some uncertainty about the value of cell counts estimated using single section (SS). To compare these two methods, we counted the pigmented neurons in the pars compacta of the substantia nigra (SNpc) in seven normal controls and in four patients with Parkinson's disease (PD), using both single histological sections and the DS. The calculations were performed at two different levels of the substantia nigra. Level I was cut through the colliculus superior and caudal red nucleus, and level II showed the exit of the third cranial nerve in all cases. As methods, the two counts were in close agreement (Pearson's correlation coenicient at levels I and II were r=0.97 and r=1.00, respectively, P<0.001). The correspondence was different in the controls and in the parkinsonian patients, however, as can be judged from the slopes of the average regression lines (0.20 for controls, 0.31 for parkinsonian patients). It is nevertheless evident that once the regression line has been defined, DS counts can be estimated Gom SS counts when the pigmented neuron numbers are counted in the SNpc. The results suggest that comparisons of pigmented neuron counts made from SS are relevant if the cutting level of the section is well defined, and if section thickness is kept constant.     

Neuronal damage of the substantia nigra in HIV-1 infected brains

Extrapyramidal motor disorders are frequently noted in HIV-1-infected patients. In the present study, the substantia nigra was analyzed morphometrically to detect neuronal changes which might contribute to the pathogenetic mechanisms causing extrapyramidal motor dysfunction in HIV-1-infected patients. The numerical density and the size of pigmented, non-pigmented small, and non-pigmented large neurons in four nuclei of the substantia nigra pars compacta (antero-medial, antero-intermediolateral, postero-lateral, and postero-medial nuclei) in HIV-1-infected patients and in age-matched normal controls were determined. In HIV-1-infected brains, the numerical density of total neurons (i.e., pigmented and non-pigmented) as well as of pigmented neurons was significantly decreased, whereas that of non-pigmented neurons was not significantly changed in all investigated nuclei of the substantia nigra as compared to normal controls. A specific pattern of increase and decrease of non-pigmented large and non-pigmented small neurons was observed. The size of total neurons (pigmented and non-pigmented neurons) and of pigmented neurons was significantly reduced in all investigated nuclei of HIV-1-infected brains. The results suggest that neuronal degeneration in the substantia nigra commonly occurs and may be related to extrapyramidal symptoms in HIV-1-infected patients. Received: 22 February 1999 / Revised, accepted: 12 August 1999     

Dopamine transporter messenger RNA in Parkinson's disease and control substantia nigra neurons

Dopamine transporter messenger RNA (mRNA) expression was assessed by in situ hybridization over individual pigmented neurons from the substantia nigra pars compacta in midbrain sections from 7 parkinsonian and 7 age-matched, neurologically normal patients. In the normal control brains, high levels of expression of dopamine transporter mRNA were noted over pigmented neurons in the substantia nigra pars compacta; neurons in the adjacent nucleus paranigralis of the ventral tegmental area displayed less hybridization. Nigra compacta neurons surviving in brains of patients with Parkinson's disease displayed only 57% of the dopamine transporter mRNA hybridization intensity displayed by nigral neurons in normal control brains. The disease-related decrease in the apparent level of dopamine transporter mRNA expression in remaining neurons could reflect neuronal dysfunction. Conceivably, it might also reflect differential vulnerability of those neurons that initially expressed higher levels of this transporter to the insult of parkinsonism.     

Nitric oxide synthase inhibition and MPTP-induced toxicity in the common marmoset

Nitric oxide, produced following activation of N-methyl-D-aspartate (NMDA) receptors, may be involved in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity since NMDA receptor antagonists have been shown to prevent MPTP induced nigral cell loss in primates. Common marmosets were treated with either saline or MPTP or L-N^Gnitro arginine methyl ester (L-NAME) or MPTP and L-NAME. MPTP-treated common marmosets showed motor deficits including bradykinesia, rigidity, and tremor accompanied by a marked loss of tyrosine hydroxylase-immunoreactive neurones in the substantia nigra pars compacta and of [³H]-mazindol binding in the caudate-putamen. MPTP treatment also caused an increase in glial fibrillary acidic protein (GFAP) staining in the substantia nigra compared to controls. However, MPTP treatment did not alter the number of constitutive nitric oxide synthase-immunoreactive neurones in the caudate-putamen. Furthermore, neurones or glial cells immunoreactive for inducible nitric oxide synthase were not observed in the substantia nigra pars compacta following MPTP treatment. L-NAME treatment alone did not produce any behavioural changes in marmosets and did not alter the number of tyrosine hydroxylase-immunoreactive cells in the substantia nigra pars compacta, the number of constitutive nitric oxide synthase-immunoreactive neurones or [³H]-mazindol binding in the caudate-putamen compared to saline-treated control animals. Furthermore, L-NAME did not affect the motor deficits, loss of tyrosine hydroxylase-immunoreactive neurones in the substantia nigra pars compacta, loss of [³H]-mazindol binding in the caudate-putamen, or the increase in GFAP staining in the substantia nigra induced by MPTP treatment of common marmosets. The failure of L-NAME to protect against MPTP-induced toxicity in the marmoset suggests that nitric oxide does not play a major role in such toxicity and casts doubt over the involvement of the NMDA:nitric oxide system in neurodegeneration in MPTP-treated primates. Synapse 26:301–316, 1997. © 1997 Wiley-Liss, Inc.     

Unbiased morphological measurements show no neuronal loss in the substantia nigra in Alzheimer's disease

This is the first study to use the unbiased stereological method, the disector, to estimate the total number of pigmented neurons in the pars compacta of the substantia nigra (SNpc) in Alzheimer's disease (AD) patients as compared to healthy controls. The right half of the SNpc of 11 AD patients and 24 controls was studied. We also used single sections to determine the neuronal number and area in different subregions of the SNpc. The results showed that there was no significant difference in the total number of pigmented neurons in the SNpc (154,415+/-13,593 for AD and 160,163+/-8027 for controls) or in the volume of the SNpc between the patients with AD and controls. Studies on single sections revealed that even subregionally there was no significant difference in the neuronal number or area in the SNpc between AD patients and controls.     

The pedunculopontine nucleus in Parkinson's disease

This study demonstrated a significant loss of neurons within the lateral part of the pedunculopontine nucleus pars compacta in individuals with idiopathic Parkinson's disease and in individuals with combined Parkinson's and Alzheimer's disease. We also examined the extent of neuronal loss within the substantia nigra pars compacta, locus ceruleus, dorsal raphe nucleus, and nucleus basalis of Meynert. The number of pedunculopontine nucleus pars compacta neurons in the patients with Parkinson's or Parkinson's and Alzheimer's disease was reduced (average, 40%) in comparison with the number in control subjects or patients with Alzheimer's disease (p less than 0.01). This finding correlated significantly with the extent of loss of substantia nigra pars compacta neurons (p less than 0.01).     

Nigral degeneration in acquired immune deficiency syndrome (AIDS)

Summary Using stereological techniques we have estimated the volume density of melanin and counted the number of pigmented and non-pigmented neuronal cell bodies in the pars compacta of the substantia nigra of 12 autopsied patients with acquired immune deficiency syndrome (AIDS) who did not have inflammation or necrosis of the midbrain or clinical parkinsonism. The total number of neuronal cell bodies was 25% lower in AIDS (P<0.01) than in 12 age-matched controls, although the volume density of neuronal melanin did not differ from that of controls because the percentage of pigmented cell bodies was higher (P<0.01) and the cell bodies were more fully packed with melanin in AIDS. Also, the expected increase with age of the volume density of neuronal melanin (P<0.02) and the percentage of pigmented neurons (P<0.01) occurred in the controls but not in AIDS patients. Importantly, our histopathological examination showed unequivocal nigral degeneration with neuronal loss, small neuronal cell bodies packed with melanin, reactive astrocytosis and extra-cellular melanin in the AIDS patients but not in controls. Our study shows that a subclinical nigral degeneration is common in AIDS and could possibly explain the heightened suspectibility of some patients to drug-induced parkinsonism.Supported in part by grants from the National Research Institute of Neurology and Psychology and the Lena Price Memorial Fund. Presented in part at the Annual Meeting of the American Academy of Neurology, Chicago, Ill., April 10, 1989     

Soma size of substantia nigra neurons increases after a prenatal neocortical lesion in cats

Seeking an explanation for an increase in volume of the caudate nucleus in adult cats that had sustained a fetal unilateral neocortical lesion, we investigated possible morphological changes in the reciprocally interconnected substantia nigra. In fetal-lesioned cats the cross-sectional area of neuronal somata in substantia nigra, pars reticulata was 33% larger than in control cats (P<0.05), while in pars compacta there was a marked tendency to an increase (25%, P<0.06). This size increase might have caused the survival of a larger number of caudate nucleus neurons during development, and thus contributed to the reported increase in caudate nucleus volume.     

The electrophysiological properties of substantia nigra pars compacta neurones recorded from 6-hydroxydopamine lesioned guinea-pigs in vitro

Summary Intracellular recordings were made from substantia nigra pars compacta neurones in vitro from animals with partial unilateral 6-hydroxydopamine lesions of the nigrostriatal tract. Lesions were assessed and grouped according to the severity of the strital dopamine depletion. No differences were seen between neurones from control and lesioned side nigrae as regards their membrane properties, firing rates, burst activity or percentage of quiescent neurones in any of the lesioned categories. It is concluded that following partial lesioning, the remaining substantia nigra zona compacta neurones in vitro, are functioning normally.     

Atypical dopa responsive parkinsonism in a patient with megalencephaly, midbrain Lewy body disease, and some pathological features of Hallervorden-Spatz disease.

A 38 year old patient with megalencephaly, mental retardation, and lifelong tremor developed levodopa responsive parkinsonism in his mid-30s followed by the appearance of dyskinesiae, motor fluctuations, hallucinations, and dementia. Brain MRI showed, as well as other changes, iron deposition in the globus pallidus, substantia nigra, and the pulvinar of the thalamus. Postmortem examination disclosed depigmentation of the substantia nigra pars compacta with neuronal loss, gliosis, and Lewy body formation. Axonal dystrophic spheroids, neuronal loss, calcification, and iron deposition were found in the substantia nigra pars reticulata. Less severe changes without neuronal loss were seen in the globus pallidus. This combination of megalencephaly with neuroaxonal changes predominantly in the pars reticulata and Lewy body degeneration isolated to the substantia nigra pars compacta has not been previously reported.     

New pathologic observations in juvenile onset parkinsonism with dystonia

A patient with hereditary juvenile onset parkinsonism with dystonia died at age 39. There were Lewy bodies and regionally selective neuronal damage in the substantia nigra pars compacta. These changes closely resemble those seen in Parkinson's disease, and emphasize the selective vulnerability of the ventral tier of the pars compacta in these degenerations.     

Marked microglial reaction in normal aging human substantia nigra: correlation with extraneuronal neuromelanin pigment deposits

Multiple reports have documented an age-related loss, estimated at about 10% per decade, of the pigmented neurons in the substantia nigra. This is associated with motor dysfunction, including bradykinesia, stooped posture and gait disturbance. As microglia are activated by cell death and neuromelanin pigment, we hypothesized that there should be a significant microglial reaction in normal aging human substantia nigra. Sections of substantia nigra from elderly subjects (N = 15; mean 81.3; SD 7.0) and younger subjects (N = 7; mean 30.3; SD = 8.7), all of which had no specific neurologically or neuropathologically defined disorders, were stained immunohistochemically for MHC Class II and the area occupied by microglia was quantified in substantia nigra pars compacta. All elderly subjects showed a pronounced microglial reaction in the substantia nigra, with frequent, intensely stained hypertrophic microglia, while immunoreactive nigral microglia were much less frequent in the younger subjects. Quantification showed that in older subjects, the percentage of substantia nigra area occupied by microglial bodies and processes was significantly greater than for younger subjects (mean 19.6 vs. 3.6; P = 0.005). Extraneuronal neuromelanin deposits were present in all the older subjects but were absent or rare in the younger subjects. The neuromelanin deposit abundance score in the older subjects correlated significantly with the area occupied by immunoreactive microglia. The marked microglial reaction in normal aging human substantia nigra, together with the previously reported 35–80% pigmented neuron loss, indicates the presence of a powerful pathologic process that may be additive with specific age-related neurodegenerative diseases, including Parkinson’s disease.     

Nuclear factor kappa-B p50 and p65 subunits expression in dementia with Lewy bodies

Dementia with Lewy bodies (DLB) is the second most common cause of neurodegenerative dementia after Alzheimer's disease (AD). Parkinsonism in DLB is mainly caused by neuronal loss with Lewy bodies (LBs) in the substantia nigra, thereby inducing degeneration of the nigrostriatal dopaminergic pathway similar to that in Parkinson's disease (PD). To clarify the pathogenesis of DLB, it is important to investigate the mechanisms involved in the degenerative process of LB-bearing neurones. Several reports suggest a role for nuclear factor kappa-B (NFkappaB) in the manifestation of neurodegenerative conditions such as AD and PD. The aim of the present study was to investigate whether NFkappaB subunits are involved in the pathogenesis of neurodegeneration in DLB by measuring tyrosine hydroxylase (TH), NFkappaB p65 and p50 protein expression in frontal cortex and substantia nigra pars compacta of DLB and control human brains. An increase, although not statistically significant, in nigral TH expression in DLB cases was observed. There were no differences in the cortical and nigral expression levels of NFkappaB p65 subunit between control and DLB cases. Western blots of the frontal cortex showed no differences in the expression levels of NFkappaB p50 subunit. However, NFkappaB p50 levels were significantly decreased (P < 0.05) in the pars compacta of the substantia nigra in the DLB cases in comparison with controls. The decrease in the expression of the p50 subunit in the substantia nigra of DLB cases achieved in the present study may increase the vulnerability of the dopaminergic neurones to a possible neurotoxic effect of p65 subunit. Thus, normal levels of NFkappaB p65 might be toxic in neurones with a low expression of the NFkappaB p50 subunit.     

Low numbers and no loss of melanized nigral neurons with increasing age in normal human brains from India

The prevalence of Parkinson's disease (PD) is higher in whites than in nonwhites and it increases with advancing age. The pathological hallmarks of PD are loss of pigmented neurons in the substantia nigra pars compacta (SNpc) and presence of Lewy bodies. With increasing age, a similar loss of pigmented neurons in the SNpc has been reported. Hence, age and race possibly play a role in the pathogenesis of PD. The objectives of this study were to count the number of melanized neurons in the SNpc in normal human brains from India and study the change in neuronal count with advancing age and to compare the neuronal counts from this Indian population with counts reported in normal brains from the United Kingdom. Melanized neurons in the SNpc were counted in 84 normal human brains (age range, 5–84 years) in a single 7-μm section at the level of emergence of the oculomotor nerve. In the brains from India, there was no loss of melanized nigral neurons with advancing age. The absolute number of these melanized neurons was about 40% lower than the brains from UK. Despite a low number of melanized nigral neurons in the brains from India, individuals function normally and have dopamine levels comparable with their Western counterparts, suggesting that it is not the absolute number of melanized nigral neurons but the percent loss of nigral neurons that results in dopaminergic deficiency in PD. There is no significant loss of pigmented nigral neurons with age, suggesting that the loss seen in PD is exclusively due to the disease process itself. Indians have a lower prevalence of PD despite having a low count of melanized nigral neurons, suggesting that better protective mechanisms may be present in the Indians to prevent the loss of nigral neurons.     

The influence of striatal stimulation and putative neurotransmitters on identified neurones in the rat substantia nigra

The response of two populations of neurones in the substantia nigra (nigro-striatal compacta neurones and reticulata neurones) to microelectrophoretically administered putative neurotransmitters and stimulation of the ipsilateral striatum has been investigated in anaesthetized rats. There were marked differences between compacta and reticulata neurones in respect to their action potential configurations, spontaneous firing rates and their responses to striatal stimulation. However, both compacta and reticulata neurones were excited and/or inhibited by striatal stimulation, although inhibition was usually the predominant response in both neuronal populations. Compacta neurones were strongly inhibited by noradrenaline (NA) and dopamine (DA) but were unaffected by acetylcholine (ACh) and 5-hydroxytryptamine (5-HT). Reticulata neurones were excited by ACh and showed mixed responses to 5-HT, DA and NA. Excitant amino acids overdepolarized compacta neurones preventing them from firing rapidly, but induced large increases in reticulata neurone firing rate; effects that were readily antagonized by D-alpha-aminoadipate. Compacta neurones were less sensitive than reticulata neurones to GABA and glycine. The action of these inhibitory amino acids were selectively and reversibly antagonized by bicuculline methochloride and strychnine, respectively. The striatal-evoked inhibition of both compacta and reticulata neurones was reversibly reduced by bicuculline methochloride and irreversibly reduced by tetanus toxin, but was unaffected by strychnine. These results demonstrate that nigrostriatal-compacta neurones and reticulata neurones are physiologically and pharmacologically distinct neuronal populations and both receive inhibitory GABAergic and excitatory striatal inputs.     

Activation of cerebral peroxisome proliferator-activated receptors γ (PPARγ) reduces neuronal damage in the substantia nigra after transient focal cerebral ischaemia in the rat

M. Zuhayra, Y. Zhao, C. von Forstner, E. Henze, P. Gohlke, J. Culman and U. Lützen (2011) Neuropathology and Applied Neurobiology 37, 738–752 Activation of cerebral peroxisome proliferator‐activated receptors γ (PPARγ) reduces neuronal damage in the substantia nigra after transient focal cerebral ischaemia in the rat Aim: The function of brain (neuronal) peroxisome proliferator‐activated receptor(s) γ (PPARγ) in the delayed degeneration and loss of neurones in the substantia nigra (SN) was studied in rats after transient occlusion of the middle cerebral artery (MCAO). Methods: The PPARγ agonist, pioglitazone, or vehicle was infused intracerebroventricularly over a 5‐day period before, during and 5 days after MCAO (90 min). The neuronal degeneration in the SN pars reticularis (SNr) and pars compacta (SNc), the analysis of the number of tyrosine hydroxylase‐immunoreactive (TH‐IR) neurones and the expression of the PPARγ in these neurones were studied by immunohistochemistry and immunofluorescence staining. The effects of PPARγ activation on excitotoxic and oxidative neuronal damage induced by glutamate and 6‐hydroxydopamine were investigated in primary cortical neurones expressing PPARγ. Results: Pioglitazone reduced the total and striatal infarct size, neuronal degeneration in both parts of the ipsilateral SN, the loss of TH‐IR neurones in the SNc and increased the number of PPARγ‐positive TH‐IR neurones. Pioglitazone protected primary cortical neurones against oxidative and excitotoxic damage, prevented the loss of neurites and supported the formation of synaptic networks in neurones exposed to glutamate or 6‐hydroxydopamine by a PPARγ‐dependent mechanism. Conclusions: Activation of cerebral PPARγ confers neuroprotection after ischaemic stroke by preventing both, neuronal damage within the peri‐infarct zone and delayed degeneration of neurones and neuronal death in areas remote from the site of ischaemic injury. Pioglitazone and other PPARγ agonists may be useful therapeutic agents to prevent progression of brain damage after cerebral ischaemia.     

Dopaminergic and non-dopaminergic neurons in substantia nigra: Differential response to bromocriptine

Summary Bromocriptine reduces the spontaneous firing rate of neurons in the pars compacta of the substantia nigra but does not change the electrical activity of the neurons located in the pars reticulata. On the other hand, bromocriptine induces contralateral circling behaviour in rats with unilateral 6-hydroxydopamine nigral lesion. This increased motor activity follows an initial period of hypomotility. The decrease of the neuronal firing rate in the pars compacta of the substantia nigra coincides with the hypomotility observed in the lesioned rats.     

蛋白酶体抑制剂诱导大鼠黑质变性伴包涵体形成

目的 观察蛋白酶体抑制剂Lactacystin诱导大鼠黑质变性伴包涵体形成及运动行为学的改变,探讨蛋白酶体功能下降在帕金森病(PDl发病机制中的作用. 方法 24只SD大鼠采用随机数字表法分为kactacystin实验组和生理盐水组.每组12只,Lactacystin实验组将蛋白酶体抑制剂Lactacystin立体定向注射人大鼠左侧黑质致密部(SNc).生理盐水组注射等体积生理盐水;观察大鼠自主行为和阿朴吗啡(APO)诱导的旋转行为的改变;Nissl染色法观察SNc病理改变;免疫组化法观察SNc及纹状体酪氨酸羟化酶(TH)和SNc中α-共核蛋白的表达;透射电镜观察SNc超微结构的改变. 结果 Lactacystin实验组大鼠给药7 d后出现自发性活动减少、动作缓慢、震颤、且症状逐步加重.APO可诱导出向健侧的旋转运动;Nissl染色发现Lactacystin实验组左侧SNc神经元数量减少,尼氏体结构松散;免疫组化结果表明21 d后Lactacystin实验组左侧SNc出现变性,TH免疫阳性神经元数量减少,α-共核蛋白表达增强,纹状体内TH免疫阳性纤维数量减少;电镜观察到蛋白质聚集形成的包涵体. 结论 Lactacystin单侧SNc注射可以诱导大鼠黑质变性伴包涵体形成及大鼠行为改变.蛋白酶体功能下降可能在PD发病机制中起重要作用.