Local support cells promote survival of substantia nigra dopaminergic neurons in culture.

Recent studies suggest that brain neurons require extracellular signals for continued survival during maturity as well as development. However, factors underlying the survival of specific populations of central neurons remain to be defined. To examine the regulation of neuronal survival, we have studied the substantia nigra (SN) dopaminergic (DA) system, in dissociated cell culture. DA neuron number was monitored immunocytochemically with antibody to tyrosine hydroxylase (TH), the DA biosynthetic enzyme. Initially, mixed cultures were grown at low, medium, and high densities in serum-containing media. After 7 days, the number of neuron-specific enolase (NSE)-positive cells, a measure of total neuron number, was proportional to cell plating density. In contrast, high density culture elicited a marked, disproportionate increase in TH-immunopositive cells, suggesting that high density conditions selectively enhanced the DA subpopulation. To define the role of cellular interactions in the selective increase in DA cells, virtually pure neuron cultures were compared to support cell-neuron cocultures, in fully defined medium. In support cell-neuron cocultures, SN support cells evoked a four-fold increase in TH cells, while NSE number did not differ from controls. Moreover, local support cells elicited a greater increase in TH cell number than support cells derived from other brain regions. To determine whether increased TH cell number reflected enhanced survival, or possibly expression of TH by new populations, we monitored the time course of this effect. TH cell number remained constant after 3 days in cocultures, while declining fourfold in controls. In parallel studies, support cells were added to SN dissociates at zero time or after 3 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human midbrain dopamine neurons express serotonin 2A receptor: an immunohistochemical demonstration

We demonstrated intense serotonin (5-HT) 2A receptor immunoreactivity in the human ventral tegmental area (VTA) using by a recently raised antibody against 5-HT2A receptor. The substantia nigra (SN) neurons also showed 5-HT2A receptor immunoreactivity. Double immunohistochemistry of 5-HT2A receptor and tyrosine hydroxylase (TH) revealed many neurons doubly labeled by 5-HT2A receptor and TH in the VTA and SN. It is suggested that activity of human midbrain dopaminergic neurons might be strongly regulated via 5-HT2A receptors at the level of their originating nuclei.     

Modulation of glutamate neurotoxicity on mesencephalic dopaminergic neurons in primary cultures by the presence of striatal target cells

Glutamate toxicity was compared in substantia nigra (SN)/striatum (STR) and SN/cerebellum (CRB) co-cultures on both the entire neuronal population (neuron specific enolase (NSE) immunopositive cells) and dopaminergic neurons (tyrosine hydroxylase (TH) immunopositive cells). In SN/CRB co-cultures NSE- and TH-positive cells were more sensitive to glutamate-induced toxicity than in SN/STR co-cultures. Moreover, in SN/STR co-cultures as compared to SN/CRB and SN cultures, glutamate toxicity was prevented to a larger extent by TCP, a non-competitive NMDA antagonist. These results suggest that target cells induce a differential expression of the different glutamate receptor subtypes in mesencephalic dopaminergic cells. Alternatively, the presence of target cells may induce the selective development of a subpopulation of dopaminergic neurons expressing predominantly NMDA receptors.     

Bcl-2 and GDNF delivered by HSV-mediated gene transfer act additively to protect dopaminergic neurons from 6-OHDA-induced degeneration

Previous studies have demonstrated that either the neurotrophin glial-derived neurotrophic factor (GDNF) or the antiapoptotic peptide Bcl-2 delivered into striatum by a viral vector protects dopaminergic neurons of the substantia nigra in vivo from degeneration induced by the administration of the neurotoxin 6-hydroxydopamine (6-OHDA). In this study we used recombinant, replication-incompetent, genomic herpes simplex virus-based vectors to deliver the genes coding for Bcl-2 and GDNF into rat substantia nigra (SN) 1 week prior to 6-OHDA injection into the striatum. Vector-mediated expression of either Bcl-2 or GDNF alone each resulted in a doubling in cell survival as measured by retrograde labeling with fluorogold (FG) and a 50% increase in tyrosine hydroxylase-immunoreactive (TH-IR) neurons in the lesioned SN compared to the unlesioned side. Gene transfer of Bcl-2 and GDNF were equivalent in this effect. Coadministration of the Bcl-2-expressing vector with the GDNF-expressing vector improved the survival of lesioned SN neurons as measured by FG labeling by 33% and by the expression of TH-IR by 15%. These results suggest that the two factors delivered together act in an additive fashion to improve DA cell survival in the face of 6-OHDA toxicity.     

Sex steroids promote neurite growth in mesencephalic tyrosine hydroxylase immunoreactive neurons in vitro

The influence of steroid hormones on the differentiation of catecholaminergic and serotonergic (5-HT) neurons was studied in dissociated cell cultures from embryonic day 14 (E14) rat diencephalon, mesencephalon and metencephalon treated for 6 days with 17 beta-estradiol (E), testosterone (T), 5 alpha-dihydrostestosterone (DHT), progesterone (P), dexamethasone (DEX), or E + T. The effects of these hormones on morphologic differentiation were determined by morphometric measurements of total length of neurites of immunocytochemically identified neurons in culture, which were stained with antisera against tyrosine hydroxylase (TH) or 5-HT. A significant increase in neurite length was observed in cultures of TH-immunoreactive (TH-IR) neurons from the mesencephalon treated with E, T, E + T, but not with P, DHT or DEX. Based on labeling with [3H]dopamine (DA) uptake and competition with specific inhibitors, these mesencephalic TH-IR cells appear to represent DA neurons of the A8-A10 groups (which includes the substantia nigra). No statistically significant effects of these steroids were observed on TH-IR neurons from the diencephalon (assumed to be precursors of the tuberoinfundibular and incertohypothalamic dopaminergic groups). The 5-HT neurons of the raphe nuclei (metencephalon) showed no statistically significant response to steroids. We conclude that during the early fetal period, sex steroids can affect the morphologic differentiation of mesencephalic DA neurons in vitro, indicating that these hormones are capable of selectively influencing the development of a specific population of monoamine neurons during this critical period.     

Mesolimbic dopaminergic neurons innervating the hippocampal formation in the rat: a combined retrograde tracing and immunohistochemical study

A major mesolimbic projection towards the hippocampal formation (HF) has been extensively described, but no clear evidence of its dopaminergic content has been demonstrated. In order to evaluate the percentage of dopaminergic (DA) cells of ventral tegmental area (VTA-A10) and adjacent substantia nigra (SN-A9) projecting to the HF, the retrograde neuronal tracer technique was combined with the tyrosine hydroxylase (TH) immunocytochemistry. Fluoro-gold (FG) was injected in several areas (subiculum, CA1, CA3, dentate gyrus) of either septal and temporal HF. Sections containing retrogradely FG labeled neurons were either mounted directly as controls or incubated with TH antiserum and revealed with rhodamine. The quantitative evaluation of retrogradely labeled and TH-IR stained cells showed that both VTA and SN projections towards the HF are partially (15–18%) dopaminergic. Ten percent of the DA neurons of the VTA projected to contralateral HF, whereas none did in the SN. In conclusion, the temporal HF (mainly subiculum and adjacent CA1) appears to receive the main DA afferents from both VTA cells and medial half of SN, pars compacta, whereas the septal HF (particularly CA1) receives its DA input from neurons located in the ventral half and in the upper and lower borders of the VTA.     

Altered distribution of dopaminergic neurons in the brain of L1 null mice

Dopaminergic neurons of the mouse mesencephalon originate in the ventricular zone and migrate radially along radial glia then tangentially along nerve fibers that express the neural cell adhesion molecule L1 to form the substantia nigra (A9 group) and ventral tegmental area (VTA) (A10 group). The role of L1 in migration of dopaminergic neuronal precursors was investigated in L1 knockout mice by tyrosine hydroxylase (TH) immunostaining. An altered rostrocaudal distribution of dopaminergic neurons was observed within the substantia nigra and VTA of L1-minus mice. In L1-minus mice at postnatal day 0, TH-positive cells were present abnormally in the dorsomedial mesencephalon, suggesting impaired migration. Axons projecting from the substantia nigra to the caudate putamen also exhibited an abnormal targeting pattern. There was no evidence of dopaminergic cell loss in the mutant SN. Abnormal localization of dopaminergic neurons in L1-minus mice was also evident in the zona incerta of the thalamus (A13 group), and the arcuate (A12) and periventricular nucleus (A14) of the hypothalamus. Cell bodies and axons in the substantia nigra, VTA, and hypothalamus of wild type mouse embryos expressed L1. These results suggested that L1 plays an important developmental role in the organization of dopaminergic neuronal cell groups in the mesencephalon and diencephalon.     

Inflammatory process as a determinant factor for the degeneration of substantia nigra dopaminergic neurons

Summary. The specific degeneration of dopaminergic neurons in the substantia nigra (SN) is a pathological hallmark of Parkinsons disease (PD). Although the cause of chronic nigral cell death in PD and its underlying mechanisms remain elusive, substantial involvement of inflammatory events has been postulated since inflammatory features have been described in parkinsonians CNS tissue. We have developed an animal model of dopaminergic neurons degeneration by the single intranigral injection of lipopolysaccharide (LPS), an inflammatory compound. This single injection produced the induction of inflammatory process with the activation of microglia along with the specific degeneration of dopaminergic neurons in the SN without affecting neither other neurotransmitter systems nor other structures of the CNS. Dexamethasone, a potent anti-inflammatory drug preventing many of the features characterizing pro-inflammatory glial activation, prevented the loss of dopaminergic cells. We also discuss other inductors of inflammatory process in relationship to the dopaminergic degeneration in the SN.     

Lateromedial Gradient of the Susceptibility of Midbrain Dopaminergic Neurons to Neonatal 6-Hydroxydopamine Toxicity

The topography-dependent vulnerability of midbrain dopaminergic neurons to neonatal intracranial exposure to 6-hydroxydopamine (6-OHDA) was investigated at adult age by the quantitative analysis of cell counts of tyrosine hydroxylase-immunopositive neurons. In all cases of intracisternal 6-OHDA treatment, A9 dopaminergic neurons in the substantia nigra (SN) were much more vulnerable to death than more medially located A10 dopaminergic neurons. Moreover, within each cell group, there were also lateromedial topographic gradients. In the A9 neuronal group, cells located in the pars lateralis of the SN and the lateral part of the pars compacta of the SN were more susceptible to 6-OHDA toxicity than those located more medially. In the A10 neuronal group, cells located in the medial part of the ventral tegmental area were more resistant to toxicity than those located more laterally, and dopaminergic cells in the midline midbrain areas (interfascicular nucleus and rostral linear nucleus of raphe) were completely spared from 6-OHDA toxicity. These findings revealed that 6-OHDA is not equally toxic to all midbrain dopaminergic neurons in neonates and that the lateromedial vulnerability pattern shows similarities to those reported in Parkinson's disease.     

Human neuromelanin induces neuroinflammation and neurodegeneration in the rat substantia nigra: implications for Parkinson's disease

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by a selective loss of dopaminergic neurons in the substantia nigra (SN). It has been suggested that microglial inflammation augments the progression of PD. Neuromelanin (NM), a complex polymer pigment found in catecholaminergic neurons, has sparked interest because of the suggestion that NM is involved in cell death in Parkinson's disease, possibly via microglia activation. To further investigate the possible role of NM in the pathogenesis of PD, we conducted in vivo experiments to find out whether microglial cells become activated after injection of human neuromelanin (NM) into (1) the cerebral cortex or (2) the substantia nigra to monitor in this PD-relevant model both microglial activation and possible neurodegeneration. In this study, adult male Wistar rats received an intracerebral injection of either NM, bacterial lipopolysaccharide (LPS, positive control), phosphate-buffered saline (PBS, negative control) or colloidal gold suspension (negative particular control). After different survival times (1, 8 or 12 weeks), brain slices from the cerebral cortex or substantia nigra (SN, 1 week) were stained with Iba-1 and/or GFAP antibody to monitor microglial and astrocytic reaction, and with tyrosine hydroxylase (TH) to monitor dopaminergic cell survival (SN group only). The injection of LPS induced a strong inflammatory response in the cortex as well in the substantia nigra. Similar results could be obtained after NM injection, while the injection of PBS or gold suspension showed only moderate or no glial activation. However, the inflammatory response declined during the time course. In the SN group, there was, apart from strong microglia activation, a significant dopaminergic cell loss after 1 week of survival time. Our findings clearly indicate that extracellular NM could be one of the key molecules leading to microglial activation and neuronal cell death in the substantia nigra. This may be highly relevant to the elucidation of therapeutic strategies in PD.     

S-100 beta and insulin-like growth factor-II differentially regulate growth of developing serotonin and dopamine neurons in vitro.

To study the phenotypic specificity of S-100 beta and insulin-like growth factor II (IGF-II) for developing monoamine neurons, serotonin (5-HT) neurons from the embryonic day 14 (E14) rostral raphe or dopamine (TH) neurons from the substantia nigra/ventral tegmental area were cultured for 3 days in vitro (3 DIV) in the presence of these factors. Neuronotrophic effects were analyzed by computer-assisted morphometry of 5-HT and TH-immunoreactive neurons. S-100 beta and IGF-II differentially regulated the growth of 5-HT and TH neurons but did not affect their survival. S-100 beta significantly increased several parameters of neurite outgrowth by 5-HT neurons but inhibited the spatial extent (field area) of TH neurites. IGF-II promoted growth of cell bodies of both phenotype, but only stimulated neurite outgrowth by TH neurons. S-100 beta and IGF-II differentially affected the number of GFAP immunoreactive cells from raphe and substantia nigra, but these effects did not correlate with the specificity of neuronotrophic effects. S-100 beta and IGF-II immunoreactivities were expressed in glial cultures derived from the same brain regions, raising the possibility that these factors have autocrine effects on glia as well as paracrine actions on neurons. The results of this study suggest that specificity of neurotrophic factors for particular embryonic neurons may be correlated with their neurotransmitter phenotype.     

5-HT1D receptors in the guinea pig brain: pre- and postsynaptic localizations in the striatonigral pathway

The caudate-putamen, globus pallidus and substantia nigra pars reticulata of the guinea pig contain high densities of the 5-HT1D receptor subtype. The cellular localization of these sites in the striatonigral pathway was investigated using receptor autoradiography and selective neurotoxin lesions. In guinea pigs with unilateral 6-hydroxydopamine lesions of the nigral dopaminergic cells, no significant decrease was observed in any of the components of the striatonigral pathway. In contrast, when quinolinic acid was injected in the caudate-putamen, marked reductions in [3H]5-HT binding were seen in the caudate-putamen, the globus pallidus and the substantia nigra pars reticulata, on the side ipsilateral to the lesion. These data, which are comparable to previous results in human pathologies where similar cell populations are known to degenerate (Parkinson disease and Huntington's chorea), indicate a presynaptic localization of 5-HT1D receptors on the terminals of the striatal neurons projecting to the pars reticulata of the substantia nigra. In addition, these receptors could be located on the cell bodies or dendrites of these neurons in the striatum, postsynaptically to serotoninergic fibers.     

HIV-1 gp120 neurotoxicity proximally and at a distance from the point of exposure: Protection by rSV40 delivery of antioxidant enzymes

Toxicity of HIV-1 envelope glycoprotein (gp120) for substantia nigra (SN) neurons may contribute to the Parkinsonian manifestations often seen in HIV-1-associated dementia (HAD). We studied the neurotoxicity of gp120 for dopaminergic neurons and potential neuroprotection by antioxidant gene delivery. Rats were injected stereotaxically into their caudate-putamen (CP); CP and (substantia nigra) SN neuron loss was quantified. The area of neuron loss extended several millimeters from the injection site, approximately 35% of the CP area. SN neurons, outside of this area of direct neurotoxicity, were also severely affected. Dopaminergic SN neurons (expressing tyrosine hydroxylase, TH, in the SN and dopamine transporter, DAT, in the CP) were mostly affected: intra-CP gp120 caused approximately 50% DAT+ SN neuron loss. Prior intra-CP gene delivery of Cu/Zn superoxide dismutase (SOD1) or glutathione peroxidase (GPx1) protected SN neurons from intra-CP gp120. Thus, SN dopaminergic neurons are highly sensitive to HIV-1 gp120-induced neurotoxicity, and antioxidant gene delivery, even at a distance, is protective.     

Effects of dopaminergic cell degeneration on electrophysiological characteristics and GAD65/GAD67 expression in the substantia nigra: different action on GABA cell subpopulations.

The motor disturbances occurring in Parkinson's disease have been partially attributed to a hyperactivity of gamma-aminobutyric acid (GABA)-ergic nigral cells largely in the substantia nigra pars reticulata (SNr) secondary to the degeneration of dopaminergic nigrostriatal neurons. However, some aspects of this response remain unclear. In this work, different electrophysiological and neurochemical parameters were studied in GABAergic cells of the SN after unilateral nigrostriatal dopaminergic lesion using 6-hydroxydopamine injection in rats. Our data showed that 1) the SN under normal conditions contains different subsets of GABAergic cells according to their firing pattern and glutamic acid decarboxylase mRNA levels, and 2) the response of these GABAergic cell subgroups was different after the ipsi- and contralateral dopaminergic cell degeneration. These findings indicate a complex regulation of nigral GABAergic activity after nigrostriatal dopaminergic degeneration that probably involves local mechanisms, the nigro-striato-nigral loop, as well as interhemispheric mechanisms whose anatomical basis remains unstudied.     

Improved survival of embryonic porcine dopaminergic neurons in coculture with a conditionally immortalized GDNF-producing hippocampal cell line

Transplantation of embryonic nigral tissue is used as an experimental therapy for patients with Parkinson's disease but is hampered by a limited survival rate of dopaminergic neurons. Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for nigrostriatal dopaminergic neurons, and the present in vitro study aimed at improving the survival of dopaminergic neurons in porcine mesencephalic brain slice cultures by adding transfected, immortalized, temperature-sensitive GDNF-releasing HiB5 cells (HiB5-GDNF). Embryonic (E27/28) porcine ventral mesencephalic brain slices were placed on membrane inserts in six-well plates with serum-containing medium, and HiB5-GDNF, nontransfected HiB5 cells (HiB5-control), or green fluorescent protein-producing HiB5 cells (HiB5-GFP) were seeded onto each tissue slice. The concentration of GDNF in the coculture medium was 0.49 +/- 0.13 ng/ml at day 9 and 0. 22 +/- 0.05 ng/ml at day 19 (mean +/- SEM) as measured by GDNF ELISA. The decrease in release of GDNF over time was paralleled by a gradual reduction in the number of HiB5-GFP cells expressing the reporter gene (EGFP). At day 12, HPLC analysis revealed that medium from HiB5-GDNF cocultures contained 2.0 times more dopamine than medium from HiB5-control cocultures. At day 21 there was 1.6 times more dopamine. Similar results were obtained for the dopamine metabolite 3,4-dihydroxyphenylacetic acid. At day 21, cell counts showed that HiB5-GDNF cocultures contained 1.5 times more tyrosine hydroxylase immunoreactive neurons than HiB5-control cocultures, which must be compared with a 1.8 fold increase after chronic treatment with rhGDNF (10 ng/ml). In conclusion, the better survival of HiB5-GDNF cocultures is promising for the generation of effective cell lines for local delivery of neurotrophic factors to intracerebral nigral grafts.     

6-Hydroxydopamine induced apoptosis of dopaminergic cells in the rat substantia nigra

The pathologic hallmark of Parkinson's disease is the dopaminergic cell death in the substantia nigra (SN). The cause of the cell death is, however, unknown. Even the question on whether the cells die by apoptosis or necrosis has not been answered with certainty. In 6-Hydroxydopamine induced Parkinsonian rats, the present study observed apoptotic nuclei from 1 day to 14 days after lesioning, using the TdT(terminal deoxynucleotidyl transferase)-mediated dUTP-biotin nick-end labeling method. Tyrosine hydroxylase immunohistochemistry and haematoxylin staining further revealed that these apoptotic cells are dopaminergic cells in the substantia nigra. The results suggest that dopaminergic cells in SN undergo apoptosis in the rat model of Parkinson's disease.     

Trisialoganglioside GT1b induces in vivo degeneration of nigral dopaminergic neurons: role of microglia

We recently showed that trisialoganglioside (GT1b) induces cell death of dopaminergic neurons in rat mesencephalic cultures (Chung et al., Neuroreport 12:611-614, 2001). The present study examines the in vivo neurotoxic effects of GT1b on dopaminergic neurons in the substantia nigra (SN) of Sprague-Dawley rats. Seven days after GT1b injection into the SN, immunocytochemical staining of SN tissue revealed death of nigral neurons, including dopaminergic neurons. Additional immunostaining using OX-42 and OX-6 antibodies showed that GT1b-activated microglia were present in the SN where degeneration of nigral neurons was found. Western blot analysis and double-labeled immunohistochemistry showed that inducible nitric oxide synthase (iNOS) was expressed in the SN, where its levels were maximal at 8 h post-GT1b injection, and that iNOS was localized exclusively within microglia. GT1b-induced loss of dopaminergic neurons in the SN was partially inhibited by N(G)-nitro-L-arginine methyl ester hydrochloride, an NOS inhibitor. Our results indicate that in vivo neurotoxicity of GT1b against nigral dopaminergic neurons is at least in part mediated by nitric oxide released from activated microglia. Because GT1b exists abundantly in central nervous system neuronal membranes, our data support the hypothesis that immune-mediated events triggered by endogenous compounds such as GT1b could contribute to the initiation and/or the progression of dopaminergic neuronal cell death that occurs in Parkinson's disease.     

Selenium deficiency potentiates methamphetamine-induced nigral neuronal loss; comparison with MPTP model

The present study was designed to understand the role of an antioxidant, selenium (Se) on methamphetamine (MA)-induced dopaminergic cell damage in the substantia nigra (SN). Male C57BL/6J mice were fed either selenium-deficient (<0.01 ppm Se) or selenium-replete (0.2 ppm Se) diet for 90 days. Se-deficiency potentiates MA-induced reductions of tyrosine hydroxylase-like immunoreactivity (TH-IR), dopamine (DA) and its metabolites, 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) in the SN. These dopaminergic toxicities were comparable to that induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). By contrast, Se-repletion significantly blocked dopaminergic toxicity after MA treatments. These results suggest that Se-deficient MA-treated mouse is a relevant model of Parkinsonism, and that optimal level of Se plays a crucial role in preventing nigral dopaminergic toxicity induced by MA. However, different mechanisms in the thermoregulation mediated by MA or MPTP remain to be further determined.