Dopamine transporter mRNA: dense expression in ventral midbrain neurons.

Oligonucleotides and a full-length cDNA encoding a functional dopamine transporter (DAT1) hybridize to a 3.7 kb mRNA that is concentrated in mRNA prepared from midbrain and absent in specimens from cerebellum or cerebral cortex. In situ hybridization reveals substantial hybridization densities overlying neurons of the substantia nigra, pars compacta, and the parabrachialis pigmentosus region of the ventral tegmental area (VTA). Neurons in the linear and paranigral VTA regions display lower levels of expression. Preliminary studies in arcuate neurons suggest modest hybridization. Different dopaminergic cell groups display different levels of DAT1 dopamine transporter expression.     

Dopaminergic mRNA expression in the intact sebstantia nigra of unilaterally 6-OHDA-lesioned and grafted rats: an in situ hybridization study

Summary. The present study was performed to investigate the influence of intrastriatal fetal mesencephalic grafts on dopaminergic mRNA expression in the non-lesioned substantia nigra pars compacta of unilaterally 6- hydroxydopamine-lesioned rats. The expression of dopamine transporter mRNA, synaptic vesicular monoamine transporter mRNA and tyrosine hydroxylase mRNA was assessed in adjacent cryostat sections using in situ hybridization. Rotational behavior induced by apomorphine and amphetamine as well as hybridization of striatal sections cut at the grafting coordinates were used to prove the functional recovery and the presence of grafted cells, respectively. After grafting, the number of rotations was decreased and hybridization signals overlying cells in the grafted striatum were detected. Mean grain densities overlying labeled neurons in the substantia nigra pars compacta of grafted rats were compared to those of shamgrafted rats and revealed differential expression of dopamine transporter mRNA, whereas synaptic vesicular monoamine transporter mRNA and tyrosine hydroxylase mRNA expression showed no difference. The results will be discussed in relation to previous in vitro and in vivo studies suggesting a reduction of functional dopamine transporter molecules in the contralateral striatum. Received April 25, 2000; accepted August 17, 2000     

Expression of ErbB4 in substantia nigra dopamine neurons of monkeys and humans

Abnormal neuregulin-1 signaling through its receptor (ErbB4) might be associated with schizophrenia, although their neuropathological contribution remains controversial. To assess the role of neuregulin-1 in the dopamine hypothesis of schizophrenia, we used in situ hybridization and immunoblotting to investigate the cellular distribution of ErbB4 mRNA in the substantia nigra of Japanese monkeys (Macaca fuscata) and human postmortem brains. In both monkeys and humans, significant signal for ErbB4 mRNA was detected in substantia nigra dopamine neurons, which were identified by melanin deposits. The expression of ErbB4 mRNA in nigral dopamine neurons was confirmed with an independent RNA probe, as well as with combined tyrosine hydroxylase immunostaining. Immunoblotting appeared to support the observation of in situ hybridization. Immunoreactivity for ErbB4 protein was much more enriched in substantia nigra pars compacta containing dopamine neurons than in neighboring substantia nigra pars reticulata. These observations suggest that ErbB4 is expressed in the dopaminergic neurons of primate substantia nigra and ErbB4 abnormality might contribute to the dopaminergic pathology associated with schizophrenia or other brain diseases.     

Microtopography of methionine-enkephalin, dopamine and noradrenaline in the ventral mesencephalon of human control and Parkinsonian brains

The topographical distributions of Met-enkephalin, dopamine and noradrenaline were determined in serial frontal sections of human substantia nigra (pars compacta and pars reticulata) and ventral tegmental area. Met-enkephalin was identified by Biogel and thin layer chromatography and assayed by a specific radioimmunoassay. In the substantia nigra (pars compacta and pars reticulata), the levels of Met-enkephalin increased progressively from the rostal to the caudal part of the structure. This pattern closely resembled that of dopamine levels, particularly in the pars compacta. Noradrenaline levels in the substantia nigra and those of Met-enkephalin, dopamine, and noradrenaline in the ventral tegmental area, exhibited only limited fluctuations from the anterior to the posterior part of each structure.Highly significant decreases in Met-enkephalin, dopamine and noradrenaline levels were observed in the substantia nigra and ventral tegmental area of Parkinsonian brains. This observation, together with the close topographical association of dopamine and Met-enkephalin in the substantia nigra, further supports the likely existence of important functional relationships between dopaminergic and enkephalinergic neurons in the human brain.     

Subpopulations of mesencephalic dopaminergic neurons express different levels of tyrosine hydroxylase messenger RNA.

Subpopulations of mesencephalic dopamine containing neurons possess different electrophysiological, pharmacological, biochemical, and anatomical properties. In order to determine whether such differences are related to the regulation of tyrosine hydroxylase, the rate limiting enzyme in the synthesis of catecholamines, the regional distribution of tyrosine hydroxylase messenger RNA in these neurons was examined using in situ hybridization histochemistry. In the mouse, labelling for tyrosine hydroxylase messenger RNA associated with individual neurons was significantly less in the lateral substantia nigra pars compacta than in the medial substantia nigra pars compacta and the ventral tegmental area. A similar pattern of labelling was observed in the rat. Labelling for tyrosine hydroxylase messenger RNA was significantly less in the lateral substantia nigra pars compacta than in medial pars compacta (a densely cellular region), the area dorsal to the medial substantia nigra pars compacta (a less cell dense region), and the ventral tegmental area. Differences in levels of labelling for messenger RNA in mesencephalic dopamine neurons were not related to differences in cell size as measured in sections processed for tyrosine hydroxylase immunohistochemistry. The results suggest that tyrosine hydroxylase messenger RNA is differentially regulated in subpopulations of mesencephalic dopamine neurons, supporting the view that these neurons are physiologically distinct.     

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     

ALDH1 mRNA: presence in human dopamine neurons and decreases in substantia nigra in Parkinson's disease and in the ventral tegmental area in schizophrenia

Dopamine (DA) neurons degenerate in Parkinson's disease and dopamine neurotransmission may be affected in psychotic states seen in schizophrenia. Understanding the regulation of enzymes involved in DA metabolism may therefore lead to new treatment strategies for these severe conditions. We investigated mRNA expression of the cytosolic aldehyde dehydrogenase (ALDH1), presumably involved in DA degradation, by in situ hybridization in DA neurons of human postmortem material. Parallel labeling for GAPDH, neuron-specific enolase, tyrosine hydroxylase, dopamine transporter, and dopamine beta-hydroxylase was used to ensure suitability of tissue specimen and to identify all dopamine neurons. ALDH1 was found to be expressed highly and specifically in DA cells of both substantia nigra (SN) and the ventral tegmental area (VTA) of controls. A marked reduction of ALDH1 expression was seen in surviving neurons of SN pars compacta but not of those in the VTA in Parkinson's disease. In patients suffering from schizophrenia we found ALDH1 expression at normal levels in DA cells of SN but at significantly reduced levels in those of the VTA. We conclude that ALDH1 is strongly and specifically expressed in human mesencephalic dopamine neurons and that low levels of ALDH1 expression correlate with DA neuron dysfunction in the two investigated human conditions.     

An altered histaminergic innervation of the substantia nigra in Parkinson's disease

The central histaminergic system is one of the subcortical aminergic projection systems involved in several regulatory functions. The central dopaminergic and histaminergic systems interact extensively, but little is known about the histaminergic system in diseases affecting the dopaminergic neurons. The distribution of histaminergic fibers in the substantia nigra (SN) in postmortem brain samples from patients suffering from Parkinson's disease (PD) and normal controls was examined with a specific immunohistochemical method. Direct connections between dopaminergic neurones and histaminergic fibers were observed. Histamine in human SN was stored in fibers and varicosities. Sites of histamine formation were examined by l-histidine decarboxylase in situ hybridization. In both normal and PD brains HDC mRNA was found only in posterior hypothalamus and not in SN. The presence of histaminergic innervation of the human substantia nigra pars compacta (SNc) and reticulata (SNr), paranigral nucleus, radix of oculomotor nerve, and parabrachial pigmented nucleus was demonstrated. The density of histaminergic fibers in the middle portion of SNc and SNr was increased in brains with PD. In PD the morphology of histaminergic fibers was also altered; they were thinner than in controls and had enlarged varicosities. An increase of histaminergic innervation may reflect a compensatory event due to deficiency of, e.g., dopamine or a putative fiber growth inhibitory factor. Whether the changes seen in histaminergic fibers in PD are primary or secondary remains to be investigated.     

Regional dopamine transporter gene expression in the substantia nigra from control and Parkinson's disease brains

OBJECTIVE—To test thehypothesis that differential regional dopamine transporter (DAT) geneexpression may underlie the selective vulnerability of certain nigraldopaminergic neurons in Parkinson's disease, DAT mRNA expression wasexamined in neuronal subpopulations of human postmortem ventralmesencephalon from patients with Parkinson's disease and controls.
METHODS—Radioactivein situ hybridisation histochemistry using a polymerase chain reactionderived ribonucleotide probe for DAT was performed on sections ofventral mesencephalon from the brains of five donors with no history ofneurological illness and from five patients with pathologicallyestablished Parkinson's disease. The number of silver grains overlyingmelanised neurons from the paranigral nucleus, dorsal and ventral tier,and pars lateralis of the substantia nigra pars compacta were comparedwith each other and to background labelling by using a one wayfactorial analysis of variance (ANOVA) with a significance level of5%.
RESULTS—In controlbrains, there was intense DAT mRNA expression in the ventral midbrainwith no significant difference in mRNA concentrations among the four regions studied. In theParkinson's disease brains, there was an overall decrease in theintensity of DAT mRNA expression in the surviving dopaminergic neurons.There were no significant differences in signal between regions ineither the control or parkinsonian brains.
CONCLUSION—Takentogether, these findings do not support the hypothesis thatdifferential regional DAT gene expression underlies the selectivevulnerability of certain nigral dopaminergic neurons in Parkinson'sdisease, as the vulnerable neurons of the substantia nigra parscompacta do not express more DAT mRNA than the resistant paranigral neurons.

     

肝性脑病大鼠γ氨基丁酸转运体mRNA的表达

目的 研究肝性脑病模型大鼠γ氨基丁酸转运体(GAT)mRNA在各脑区的表达.方法 雄性SD大鼠12只按随机数字表法分为模型组和对照组,每组6只,采用硫代乙酰胺腹腔注射制作大鼠急性肝性脑病模型.对照组注射等量生理盐水.采用经地高辛标记的针对GAT-1的寡核苷酸探针,应用原位杂交方法 检测大鼠基底核、皮层、黑质网状部和致密部及海马各区GAT-1mRNA的表达. 结果 与对照组比较,模型组大鼠基底核、黑质网状部和致密部、海马各区GAT-1 mRNA表达均降低,差异有统计学意义(P<0.05),而大脑皮层其表达无明显变化. 结论 肝性脑病的发生可能与GAT mRNA的表达改变有关.     

Subsets of midbrain dopaminergic neurons in monkeys are distinguished by different levels of mRNA for the dopamine transporter: Comparison with the mRNA for the D2 receptor,tyrosine hydroxylase and calbindin immunoreactivity

The midbrain dopamine system can be divided into two groups of cells based on chemical characteristics and connectivity. The dorsal tier neurons, which include the dorsal pars compacta and the ventral tegmental area, are calbindin positive, and project to the shell of the nucleus accumbens. The ventral tier neurons are calbindin-negative and project to the sensonmotor striatum. This study examined the distribution of the mRNAs for the dopamine transporter molecule (DAT) and the D₂ receptor in the midbrain of monkeys by using in situ hybridization. The distribution patterns were compared to that of tyrosine hydroxylase and calbindin immunohistochemistry. The results show that high levels of hybridization for DAT and the D₂ receptor mRNA are found in the ventral tier, calbindin-negative neurons and relatively low levels are found in the dorsal, calbindin-positive tier. Within the dorsal tier, the dorsal substantia nigra pars compacta has the least amount of both messages. These results show that in monkeys, the ventral tegmental area and the dorsal pars compacta form a dorsal continuum of dopamine neurons which express lower levels of mRNA for DAT and D₂ receptor than the ventral tier. DAT has been shown to be involved in the selective neurotoxicity of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Different levels of DAT mRNA and calbindin may explain the differential effects of MPTP neurotoxicity. © 1995 Wiley-Liss Inc.     

Transient increase of brain derived neurotrophic factor mRNA expression in substantia nigra reticulata after partial lesion of the nigrostriatal dopaminergic pathway

By using non-isostopic in situ hybridization we have demonstrated a transient increase of BDNF mRNA in the lateral subregion of the substantia nigra pars reticulata 1 week after intrastriatal application of 6-OH-DA. These changes correlate with a partial reduction of dopamine (DA) content in the striatum but with a normal tyrosine hydroxylase immunoreactivity in substantia nigra pars compacta. Our data suggest that non-DA, BDNF expressing cells in substantia nigra pars reticulata may play a role in neuronal protection after partial lesions of the DA nigrostriatal pathway.     

Does cholecystokinin colocalize with dopamine in the human substantia nigra?

In situ hybridization was used to examine the distribution of neurons containing cholecystokinin (CCK) mRNA in human, monkey and rat brain. In rat and monkey brain CCK mRNA was visualized in the substantia nigra pars compacta and in the ventral tegmental area. The dopaminergic cell bodies in the human substantia nigra did not however show detectable amounts of CCK mRNA. Low levels of CCK mRNA were observed in the nucleus paranigralis, the human equivalent of the rodent ventral tegmental area. High levels of CCK mRNA were seen in other regions of the same brains including the cortex and the hippocampus. Thus, the adult human substantia nigra dopaminergic cells, in contrast to primate and rodent substantia nigra, do not express CCK. These results question the hypothesis of an involvement of CCK in the regulation of dopaminergic neurons and help to explain the absence of decreased CCK levels in the caudate and putamen of Parkinson's disease victims.     

Environmental enrichment in adulthood eliminates neuronal death in experimental Parkinsonism

Idiopathic Parkinson's disease (PD) affects 2% of adults over 50 years of age. PD patients demonstrate a progressive loss of dopamine neurons in the substantia nigra pars compacta (SNpc). One model that recapitulates the pathology of PD is the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Here we show that exposure to an enriched environment (EE) (a combination of exercise, social interactions and learning) or exercise alone during adulthood, totally protects against MPTP-induced Parkinsonism. Furthermore, changes in mRNA expression would suggest that increases in glia-derived neurotrophic factors, coupled with a decrease of dopamine-related transporters (e.g. dopamine transporter, DAT; vesicular monoamine transporter, VMAT2), contribute to the observed neuroprotection of dopamine neurons in the nigrostriatal system following MPTP exposure. This non-pharmacological approach presents significant implications for the prevention and/or treatment of PD.     

Neurofilament mRNA is reduced in Parkinson's disease substantia nigra pars compacta neurons

Lewy bodies are filamentous neuronal inclusions characteristic of Parkinson's disease, and neurofilament triplet proteins are the major components of the filaments in Lewy bodies. Since the neurofilament proteins found in Lewy bodies are abnormally phosphorylated and partially degraded, the formation of Lewy bodies may be due to the defective metabolism of these proteins, and this could lead to impairments in the structure and function of neurofilament rich neuronal processes (i.e., large caliber axons). To gain further insights into the metabolism of neurofilaments in Parkinson's disease, we evaluated neurofilament mRNA levels by semiquantitative in situ hybridization histochemistry in postmorten tissues from Parkinson's disease and control subjects. Substantia nigra pars compacta neurons were examined with digoxigenin-UTP labeled cRNA probes to the heavy and light neurofilament mRNAs. The relative abundance of these mRNAs was measured by videodensitometric image analysis of chromogenic reaction product. Using this approach, we demonstrated that the levels of both heavy and light neurofilament mRNAs were reduced in Parkinson's disease substantia nigra pars compacta neurons. Additionally, the levels of heavy neurofilament mRNA were lowest in Lewy body containing neurons in the Parkinson's disease cases. These results suggest that the formation of neurofilament-rich Lewy bodies in substantia nigra pars compacta neurons is associated with reduced levels of the heavy and light neurofilament mRNAs in Parkinson's disease. Thus, it is possible that the accumulation of abnormal neurofilament proteins in Lewy bodies and diminished neurofilament mRNAs contribute to the degeneration of substantia nigra pars compacta neurons in Parkinson's disease. © 1993 Wiley-Liss, Inc.     

Age-related decline in striatal dopamine content and motor performance occurs in the absence of nigral cell loss in a genetic mouse model of Parkinson's disease

Dopamine cytotoxicity is thought to contribute towards the selective loss of substantia nigra pars compacta dopamine neurons and disease progression in Parkinson's disease. However, the long-term toxicity of dopamine in vivo has not previously been established. The vesicular monoamine transporter 2 (VMAT2) sequesters monoamines into synaptic vesicles, a process that, in addition to being important in normal transmission, may also act to keep intracellular levels of monoamine neurotransmitters below potentially toxic thresholds. The homozygous VMAT2-hypomorphic mouse has an insertion in the VMAT2 gene (Slc18a2). Consequently, VMAT2-deficient mice (VD(-/-)) have an approximately 95% reduction in VMAT2 expression and an equivalent level of dopamine depletion in the striatum which results in moderate motor impairment. Here, we show that L-DOPA induces locomotor hyperactivity in VD(-/-) mice and reverses the deficit in motor coordination and balance as tested with the rotarod. We report that evidence for cytosolic accumulation of dopamine in substantia nigra neurons in these mice is two-fold: firstly, there is reduced phosphorylation of tyrosine hydroxylase at the residue associated with catechol feedback inhibition; and, secondly, there are increased rates of dopamine turnover at 6, 12 and 24 months of age. These animals exhibit a progressive decline in striatal monoamine levels and rotarod performance with increasing age. However, despite these data, there was no loss of nigral dopamine neurons as estimated by quantification of tyrosine hydroxylase-immunoreactive cells in the substantia nigra pars compacta of old VD(-/-) mice (24-month-old), implying that these age-dependent manifestations may be due to senescence alone.     

Dopaminergic neurons in rat ventral midbrain express brain-derived neurotrophic factor and neurotrophin-3 mRNAs

Studies of the trophic activities of brain-derived neurotrophic factor and neurotrophin-3 indicate that both molecules support the survival of a number of different embryonic cell types in culture. We have shown that mRNAs for brain-derived neurotrophic factor and neurotrophin-3 are localized to specific ventral mesencephalic regions containing dopaminergic cell bodies, including the substantia nigra and ventral tegmental area. In the present study, in situ hybridization with ³⁵S-labeled cRNA probes for the neurotrophin mRNAs was combined with neurotoxin lesions or with immunocytochemistry for the catecholamine-synthesizing enzyme tyrosine hydroxylase to determine whether the dopaminergic neurons, themselves, synthesize the neurotrophins in adult rat midbrain. Following unilateral destruction of the midbrain dopamine cells with 6-hydroxydopamine, a substantial, but incomplete, depletion of brain-derived neurotrophic factor and neurotrophin-3 mRNA-containing cells was observed in the ipsilateral substantia nigra pars compacta and ventral tegmental area. In other rats, combined in situ hybridization and tyrosine hydroxylase immunocytochemistry demonstrated that the vast majority of the neurotrophin mRNA-containing neurons in the substantia nigra and ventral tegmental area were tyrosine hydroxylase immunoreactive. Of the total population of tyrosine hydroxylase-positive cells, double-labeled neurons constituted 25–50% in the ventral tegmental area and 10–30% in the substantia nigra pars compacta, with the proportion being greater in medial pars compacta. In addition, tyrosine hydroxylase/neurotrophin mRNA coexistence was observed in neurons in other mesencephalic regions including the retrorubral field, interfascicular nucleus, rostral and central linear nuclei, dorsal raphe nucleus, and supramammillary region. The present results demonstrate brain-derived neurotrophic factor and neurotrophin-3 expression by adult midbrain dopamine neurons and support the suggestion that these neurotrophins influence dopamine neurons via autocrine or paracrine mechanisms. These data raise the additional possibility that inappropriate expression of the neurotrophins by dopaminergic neurons could contribute to the neuropathology of disease states such as Parkinson's disease and schizophrenia. © 1994 Wiley-Liss, Inc.     

Differential expression of kainate receptors in the basal ganglia of the developing and adult rat brain

Glutamate is the principal excitatory transmitter of the mammalian brain and plays a particularly important role in the physiology of the basal ganglia structures responsible for movement regulation. Using in situ hybridization with oligonucleotide probes, we examined the expression patterns of the five known kainate type glutamate receptor subunit genes, KA1, KA2 and GluR5–7, in the basal ganglia of adult and developing rat brain. In the adult rat, a highly organized and selective pattern of expression of the kainate subunits was observed in the basal ganglia and associated structures as well as in other regions of the brain. KA2 mRNA was abundant in the striatum, nucleus accumbens, subthalamic nucleus and substantia nigra pars compacta, and was present at lower levels in the globus pallidus and substantia nigra pars reticulata. Neither KA1 nor GluR5 expression was observed in the basal ganglia of adult rats, although these messages were present in other regions. GluR6 was highly expressed in the striatum and subthalamic nucleus and to a lesser extent in the substantia nigra pars reticulata, while no hybridization signal was detectable in the large, presumably dopaminergic neurons of the substantia nigra pars compacta. In contrast, GluR7 was strongly expressed in the substantia nigra pars compacta, was present at lower levels in the striatum, globus pallidus and substantia nigra pars reticulata, and was not detectable in the subthalamic nucleus. During postnatal development, expression of the kainate receptor subunits was characteristically highest on postnatal day 1 and declined to adult levels by day 20; however, in the globus pallidus we did observe the transient expression of KA1 and GluR5 between day 1 and day 10. These results demonstrate that the neuronal structures comprising the basal ganglia express a distinct combination of kainate receptor subunit genes, suggesting that the pharmacological properties of the resultant glutamate receptors are likely to be regionally specific. The organization of expression of these genes is established early in life, which is consistent with the important role they may play in establishing the functions of the motor system.