Neurodegeneration of substantia nigra accompanied with macrophage/microglia infiltration after intrastriatal hemorrhage

Intrastriatal hemorrhage in rats causes neurodegenaration of the substantia nigra (SN) followed by the appearance of ED1(+) cells (macrophage/microglia). ED1(+) cells were observed for at least 8 weeks after hemorrhage. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) was shown in ED1(+) cells with the expression of both brain-derived neurotrophic factor (BDNF) mRNA and BDNF, suggesting that activated-p38 MAPK(+)/ED1(+) cells would produce BDNF and may exhibit trophic effect on the degenerating neurons in the SN. However, in ELISA, BDNF protein decreased significantly in ipsilateral SN at 7 days after hemorrhage, which may be due to a dramatic decrease of BDNF immunoreactive neurons in pars compacta. Data suggest that activation of p38 MAPK in ED1(+) cells infiltrating in ipsilateral SN after hemorrhage may produce BDNF, but that the amount of BDNF produced from ED1(+) cells is insufficient for the rescue of degenerating neurons.     

多巴胺受体在大鼠嗅球的表达及其在帕金森病大鼠嗅觉障碍中的作用

目的 观察多巴胺受体在大鼠嗅球（OB）的表达与分布,探讨左旋多巴（L-DOPA）治疗对帕金森病（PD）大鼠嗅觉的影响。 方法 采用免疫印迹、免疫荧光等方法观察多巴胺受体在大鼠OB中的表达;6-羟多巴胺（6-OHDA）双侧注射建立PD大鼠模型,检测L-DOPA治疗对PD大鼠嗅觉功能及谷氨酸脱羧酶（GAD）和脑源性神经营养因子（BNDF）表达的影响。 结果 嗅球内D1和D2两种多巴胺受体亚型表达含量高。D1和D2在颗粒细胞层（GCL）内GAD阳性的γ-氨基丁酸（GABA）能神经元上大量表达,被酪氨酸羟化酶（TH）阳性神经纤维终末包绕。PD大鼠OB内GCL层TH蛋白表达明显下降（0.05±0.01 vs 0.01±0.00,P<0.001）。L-DOPA治疗后,PD大鼠找寻食物小球时间显著降低［（624.4±113.4）s vs(312.4±79.35)s,P<0.05］,OB内BDNF表达显著升高（0.02±0.01 vs 0.07±0.01,P<0.01）。 结论 D1和D2在GCL层GABA能神经元大量表达。L-DOPA治疗可缓解PD大鼠嗅觉障碍,可能与激活OB内GABA能神经元上的D1和D2复合体,进而改善BDNF表达有关。     

Recovery of type I fiber regionalization in gastrocnemius medialis of the rat after reinnervation along original and foreign paths,with and without muscle rotation

Tyrosine hydroxylase (TH) and brain-derived neurotrophic factor (BDNF), expressed in normal astrocytes, were used in combination for the treatment of Parkinson's disease (PD) symptoms in a rat model. Normal neonatal rat astrocytes were co-transfected with a vector expressing BDNF (AAVBDNF) and a retroviral vector expressing TH (termed TH-BDNF-DA(+) cells), and then implanted into the striatum of PD rats induced by 6-hydroxydopamine. TH-BDNF-DA(+) cells compensated for a severe insufficiency of endogenous dopaminergic neurons in the PD rats, resulting in a significant improvement of PD symptoms. The decrease in the rotational rate of PD rats implanted with TH-BDNF-DA(+) cells was more marked than that in PD rats implanted with normal astrocytes expressing either TH or BDNF alone (termed TH(+) and BDNF(+) cells, P<0.01 and 0.001, respectively), and suggested a synergistic effect between TH and BDNF. In contrast, the rotational rate was not altered from the baseline in PD rats without treatment or implanted with parental rat astrocytes alone (P>0.05). BDNF protected the dopaminergic neurons from apoptosis induced by 6-hydroxydopamine, and significantly increased the long-term survival of TH-positive cells in the striatum.Our data indicate that the combined use of TH and BDNF has a synergistic therapeutic effect, and is more efficient for the treatment of PD than a single gene therapy using either TH or BDNF alone.     

Mechanisms of dopamine activation of fast-spiking interneurons that exert inhibition in rat prefrontal cortex

Prefrontal cortical dopamine (DA) modulates pyramidal cell excitability directly and indirectly by way of its actions on local circuit GABAergic interneurons. DA modulation of interneuronal functions is implicated in the computational properties of prefrontal networks during cognitive processes and in schizophrenia. Morphologically and electrophysiologically distinct classes of putative GABAergic interneurons are found in layers II-V of rat prefrontal cortex. Our whole cell patch-clamp study shows that DA induced a direct, TTX-insensitive, reversible membrane depolarization, and increased the excitability of fast-spiking (FS) interneurons. The DA-induced membrane depolarization was reduced significantly by D1/D5 receptor antagonist SCH 23390, but not by the D2 receptor antagonist (-)sulpiride, D4 receptor antagonists U101958 or L-745870, alpha1-adrenoreceptor antagonist prazosin, or serotoninergic receptor antagonist mianserin. The D1/5 agonists SKF81297 or dihydrexidine, but not D2 agonist quinpirole, also induced a prolonged membrane depolarization. Voltage-clamp analyses of the voltage-dependence of DA-sensitive currents, and the effects of changing [K(+)](O) on reversal potentials of DA responses, revealed that DA suppressed a Cs(+)-sensitive inward rectifier K(+) current and a resting leak K(+) current. D1/D5, but not D2 agonists mimicked the suppressive effects of DA on the leak current, but the DA effects on the inward rectifier K(+) current were not mimicked by either agonist. In a subgroup of FS interneurons, the slowly inactivating membrane outward rectification evoked by depolarizing voltage steps was also attenuated by DA. Collectively, these data showed that DA depolarizes FS interneurons by suppressing a voltage-independent 'leak' K(+) current (via D1/D5 receptor mechanism) and an inwardly rectifying K(+) current (via unknown DA mechanisms). Additional suppression of a slowly inactivating K(+) current led to increase in repetitive firing in response to depolarizing inputs. This D1-induced increase in interneuron excitability enhances GABAergic transmission to PFC pyramidal neurons and could represent a mechanism via which DA suppresses persistent firing of pyramidal neurons in vivo.     

Expression of angiotensinogen and receptors for angiotensin and prorenin in the monkey and human substantia nigra: an intracellular renin–angiotensin system in the nigra

We have previously obtained in rodents a considerable amount of data suggesting a major role for the brain renin–angiotensin system (RAS) in dopaminergic neuron degeneration and potentially in Parkinson’s disease. However, the presence of a local RAS has not been demonstrated in the monkey or the human substantia nigra compacta (SNc). The present study demonstrates the presence of major RAS components in dopaminergic neurons, astrocytes and microglia in both the monkey and the human SNc. Angiotensin type 1 and 2 and renin–prorenin receptors were located at the surface of dopaminergic neurons and glial cells, as expected for a tissular RAS. However, angiotensinogen and receptors for angiotensin and renin–prorenin were also observed at the cytoplasm and nuclear level, which suggests the presence of an intracrine or intracellular RAS in monkey and human SNc. Although astrocytes and microglia were labeled for angiotensin and prorenin receptors in the normal SNc, most glial cells appeared less immunoreactive than the dopaminergic neurons. However, our previous studies in rodent models of PD and studies in other animal models of brain diseases suggest that the RAS activity is significantly upregulated in glial cells in pathological conditions. The present results together with our previous findings in rodents suggest a major role for the nigral RAS in the normal functioning of the dopaminergic neurons, and in the progression of the dopaminergic degeneration.     

D2 receptors inhibit the secretory process downstream from calcium influx in dopaminergic neurons: implication of K+ channels

Dopaminergic (DAergic) neurons possess D2-like somatodendritic and terminal autoreceptors that modulate cellular excitability and dopamine (DA) release. The cellular and molecular processes underlying the rapid presynaptic inhibition of DA release by D2 receptors remain unclear. Using a culture system in which isolated DAergic neurons establish self-innervating synapses ("autapses") that release both DA and glutamate, we studied the mechanism by which presynaptic D2 receptors inhibit glutamate-mediated excitatory postsynaptic currents (EPSCs). Action-potential evoked EPSCs were reversibly inhibited by quinpirole, a selective D2 receptor agonist. This inhibition was slightly reduced by the inward rectifier K(+) channel blocker barium, largely prevented by the voltage-dependent K(+) channel blocker 4-aminopyridine, and completely blocked by their combined application. The lack of a residual inhibition of EPSCs under these conditions argues against the implication of a direct inhibition of presynaptic Ca(2+) channels. To evaluate the possibility of a direct inhibition of the secretory process, spontaneous miniature EPSCs were evoked by the Ca(2+) ionophore ionomycin. Ionomycin-evoked release was insensitive to cadmium and dramatically reduced by quinpirole, providing evidence for a direct inhibition of quantal release at a step downstream to Ca(2+) influx through voltage-dependent Ca(2+) channels. Surprisingly, this effect of quinpirole on ionomycin-evoked release was blocked by 4-aminopyridine. These results suggest that D2 receptor activation decreases neurotransmitter release from DAergic neurons through a presynaptic mechanism in which K(+) channels directly inhibit the secretory process.     

Induction of inducible nitric oxide synthase expression in activated microglia following domoic acid (DA)-induced neurotoxicity in the rat hippocampus

Neuronal degeneration followed by glial activation (microglia and astrocytes) and nitric oxide synthase (NOS) expression in the hippocampus was investigated at 3 months after domoic acid (DA) administration and compared with DA treated rats at 5 days time interval which was reported earlier. Massive degeneration with complete absence of neurons in the hippocampal CA1 and CA3 regions and hypertrophied microglial cells showing intense immunoreaction with the antibody OX-42 was observed at 3 months after DA administration. Sparsely distributed OX-42 positive microglial cells were observed in the hippocampus of control rats at 3 months after saline treatment No apparent changes could be observed in the immunoreactivity of GFAP at 3 months after saline and DA administration. Neuronal nitric oxide synthase immunoreactive neurons were completely absent in the hippocampus at 3 months after DA administration. In contrast, nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemical analysis revealed absence of NADPH-d reactivity in the neurons, but positive reactivity in the microglial cells of CA1-CA3 regions in the hippocampus after DA treatment. Double immunofluorescense revealed co-expression of inducible nitric oxide synthase with immunoreactive OX-42 positive microglial cells in the hippocampal subfields at 3 months after DA administration. The microglia-produced NO appears to be a secondary phenomenon in the prolonged inflammatory process following DA-induced neuronal degeneration.     

Chemically defined sequential culture media for TH+ cell derivation from human embryonic stem cells

During the past few years several differentiation protocols to derive midbrain dopamine (DA) neurons from human embryonic stem (hES) cells have been developed, but the production of sufficient amounts of the 'right' therapeutic DA cells has not yet been accomplished. The aim of this study was to efficiently generate tyrosine hydroxylase (TH)-positive cells in vitro from our hES cells using a chemically defined culture system. At the end of differentiation, the vast majority of cells (>90%) were positive for both TH and beta-tubulin isotype III (TuJ1). Other markers of dopaminergic cells, like dopamine transporter (DAT) and Nurr1 were also detected by immunofluorescence or RT-PCR. The functions of these cells were confirmed by measurements of DA release in vitro and by transplantation of derived cells into Parkinson's disease (PD) rats in vivo. We found these cells were able to release DA when depolarized by high K(+). Moreover, 4 weeks after transplantation, the hES-derived cells could survive and reduce the apomorphine-induced rotation behaviour of the rats. In conclusion, the experimental system presented here provided a reliable protocol to produce a large number of hES-derived TH(+) cells which may be used in cell therapy for PD in future.     

Dopamine D(2) receptor modulation of K(+) channel activity regulates excitability of nucleus accumbens neurons at different membrane potentials

The nucleus accumbens (NAc) is a forebrain area in the mesocorticolimbic dopamine (DA) system that regulates many aspects of drug addiction. Neuronal activity in the NAc is modulated by different subtypes of DA receptors. Although DA signaling has received considerable attention, the mechanisms underlying D(2)-class receptor (D(2)R) modulation of firing in medium spiny neurons (MSNs) localized within the NAc remain ambiguous. In the present study, we performed whole cell current-clamp recordings in rat brain slices to determine whether and how D(2)R modulation of K(+) channel activity regulates the intrinsic excitability of NAc neurons in the core region. D(2)R stimulation by quinpirole or DA significantly and dose-dependently decreased evoked Na(+) spikes. This D(2)R effect on inhibiting evoked firing was abolished by antagonism of D(2)Rs, reversed by blockade of voltage-sensitive, slowly inactivating A-type K(+) currents (I(As)), or eliminated by holding membrane potentials at levels in which I(As) was inactivated. It was also mimicked by inhibition of cAMP-dependent protein kinase (PKA) activity, but not phosphatidylinositol-specific phospholipase C (PI-PLC) activity. Moreover, D(2)R stimulation also reduced the inward rectification and depolarized the resting membrane potentials (RMPs) by decreasing "leak" K(+) currents. However, the D(2)R effects on inward rectification and RMP were blocked by inhibition of PI-PLC, but not PKA activity. These findings indicate that, with facilitated intracellular Ca(2+) release and activation of the D(2)R/G(q)/PLC/PIP(2) pathway, the D(2)R-modulated changes in the NAc excitability are dynamically regulated and integrated by multiple K(+) currents, including but are not limited to I(As), inwardly rectifying K(+) currents (I(Kir)), and "leak" currents (I(K-2P)).     

血管活性肠肽对帕金森病大鼠黑质胶质细胞活化及相关炎性因子释放的影响

目的探讨血管活性肠肽(VIP)对帕金森病(PD)大鼠模型中脑黑质胶质细胞活化及相关炎性因子表达的影响。方法将6-羟多巴胺(6-OHDA)定向注入大鼠右侧纹状体制备PD模型。32只制备成功的PD大鼠随机分为VIP组和模型组,VIP组大鼠腹腔注射VIP 1ml(20μg/L),另10只正常大鼠为对照组。分别采用免疫组织化学、Western blotting、RT-PCR方法观察大鼠中脑黑质多巴胺(DA)能神经元、小胶质细胞、星形胶质细胞的数量和形态变化以及肿瘤坏死因子α(TNF-α)和环氧化酶2(COX-2)的表达变化。结果模型组大鼠黑质损毁侧小胶质细胞即白细胞分化抗原11b(CD11b)阳性细胞,数量较对照组明显增加(P0.05)并呈阿米巴样改变,星形胶质细胞(GFAP阳性细胞)数量明显增加(P0.05),炎性因子表达水平也明显上升(P0.05),DA能神经元数量较对照组明显下降(P0.05);与模型组相比,VIP组大鼠损毁侧黑质小胶质细胞和星形胶质细胞数量明显下降(P0.05),炎性因子表达水平显著降低(P0.05),DA能神经元数量较模型组增加(P0.05)。结论 VIP对帕金森病大鼠黑质小胶质细胞和星形胶质细胞的活化具有抑制作用,并可减少相关炎症因子的表达,从而保护黑质DA能神经元。     

Dopamine D1/D5 receptor activation modulates a persistent sodium current in rat prefrontal cortical neurons in vitro

The effects of dopamine (DA) on a persistent Na(+) current (I(NaP)) in layer V-VI prefrontal cortical (PFC) pyramidal cells were studied using whole cell voltage-clamp recordings in rat PFC slices. After blocking K(+) and Ca (2+) currents, a tetrodotoxin-sensitive I(NaP) was activated by slow depolarizing voltage ramps or voltage steps. DA modulated the I(NaP) in a voltage-dependent manner: increased amplitude of I(NaP) at potentials more negative than -40 mV, but decreased at more positive potentials. DA also slowed the inactivation process of I(NaP). The D1/D5 dopamine receptor agonists SKF 38393, SKF 81297, and dihydrexidine (3-10 microM), but not the dopamine D2/D3 receptor agonist qiunpirole (1-20 microM), mimicked the effects of DA on I(NaP). Modulation of I(NaP) by D1/D5 agonists was blocked by the D1/D5 antagonist SCH23390. Bath application of specific protein kinase C inhibitor, chelerhythrine, or inclusion of the specific protein kinase C inhibiting peptide([19-36]) in the recording pipette, but not protein kinase A inhibiting peptide([5-24]), blocked the effect of D1/D5 agonists on I(NaP). In current-clamp recordings, D1/D5 receptors activation enhanced the excitability of cortical pyramidal cells. Application of the D1/D5 agonist SKF 81297 induced a long-lasting decrease in the first spike latency in response to depolarizing current ramp. This was associated with a shift in the start of nonlinearity in the slope resistance to more negative membrane potentials. We proposed that this effect is due to a D1/D5 agonist-induced leftward shift in the activation of I(NaP). This enables DA to facilitate the firing of PFC neurons in response to depolarizing inputs.     

NGF、BDNF及受体trkA、trkB、trkC在正常猴脊髓的表达

采用免疫组织化学方法观察了神经生长因子 (NGF) ,脑源性神经营养因子 (BDNF)以及 NGF家族因子受体 trk A、trk B、trk C的免疫阳性反应在正常猴脊髓的分布。结果表明 :NGF免疫反应阳性的神经元在脊髓灰质各层中均有分布 ,灰、白质内也可见较多的 NGF免疫反应阳性的胶质细胞。 BDNF在脊髓各型神经无有明显的表达 ,特别是前角运动神经元。 trk A、trk B、trk C的免疫阳性反应产物主要分布在灰质的神经元及胶质细胞。本实验结果揭示了在正常猴脊髓中神经营养因子 (NGF、BDNF )及受体 trk A、trk B、trk C的表达状况 ,提示这些神经营养因子及受体在维持猴脊髓神经元的正常生理功能中具有重要作用。     

Regulation of glycinergic and GABAergic synaptogenesis by brain-derived neurotrophic factor in developing spinal neurons

Brain-derived neurotrophic factor (BDNF) effects on the establishment of glycinergic and GABAergic transmissions in mouse spinal neurons were examined using combined electrophysiological and calcium imaging techniques. BDNF (10 ng/ml) caused a significant acceleration in the onset of synaptogenesis without large effects on the survival of these neurons. Amplitude and frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) associated to activation of glycine and GABA(A) receptors were augmented in neurons cultured with BDNF. The neurotrophin effect was blocked by long term tetrodotoxin (TTX) addition suggesting a dependence on neuronal activity. In addition, BDNF caused a significant increase in glycine- and GABA-evoked current densities that partly explains the increase in synaptic transmission. Presynaptic mechanisms were also involved in BDNF effects since triethylammonium(propyl)-4-(2-(4-dibutylamino-phenyl)vinyl)pyridinium (FM1-43) destaining with high K(+) was augmented in neurons incubated with the neurotrophin. The effects of BDNF were mediated by receptor tyrosine kinase B (TrkB) and mitogen-activated protein kinase kinase (MEK) activation since culturing neurons with either (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'- kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester (K252a) or 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) blocked the augmentation in synaptic activity induced by the neurotrophin.     

Postnatal X-ray irradiation effects on glomerular layer of rat olfactory bulb: quantitative and immunocytochemical analysis

Summary In the rat olfactory bulb, the majority of interneurons in the glomerular layer (GL) are supposed to be generated during first postnatal week. Low and repeated doses of X-rays (200 rad x 4 and 200 rad x 6) were used during this period to impair the development of interneurons. The resulting effects on olfactory bulb neurons were examined stereologically and immunocytochemically in animals of 4 and 12 weeks of age. Quantitative analysis showed that, 1) the volume of the GL decreased to 55% (1200 rad) – 70% (800 rad) of control, 2) numerical cell densities in GL decreased to 40% (1200 rad) – 60% (800 rad) of control, thus resulting in 3) a decrease of the total cell number in GL to 20% (1200 rad) – 40% (800 rad) of control in irradiated olfactory bulbs of animals 4 weeks old. In comparison, mitral cells, which are generated prenatally, were much less affected (total cell number: 70–80% of control), indicating a selective loss of cells generated during the first postnatal week in GL. Effects on somata and processes immunoreactive for GABA, tyrosine hydroxylase (TH), calbindin D-28K and parvalbumin (PV) were examined in irradiated bulbs of both 4 and 12 week-old rats. All of these immunoreactive elements showed a drastic decrease in all layers. Semiquantitative analysis showed that in the GL, calbindin D-28K immunoreactive (calbindin D-28K(+)) neurons decreased more extensively than TH immunoreactive (TH(+)) and GABA-like immunoreactive (GABA(+)) neurons; that is, TH(+) and GABA(+) neurons decreased to 20% (1200 rad) – 40% (800 rad) of control, whereas calbindin D-28K(+) neurons decreased to 10% (1200 rad) – 30% (800 rad) of control in the GL of irradiated bulbs. These findings indicated that larger proportions of calbindin D-28K(+) neurons might be generated during the first postnatal week than those of GABA(+) and TH(+) neurons. Furthermore, in irradiated bulbs the proportion of GABA(-)TH(+) cells in TH(+) cells increased to about twice of control, and the estimated total numbers of GABA(-)TH(+) cells in irradiated rats were 95% (800 rad) and 40% (1200 rad) of control. These observations suggest that the majority of GABA(-)TH(+) neurons were less affected by X-ray irradiation during the first postnatal week and thus that they might be generated in the prenatal period. Since during the first 2 postnatal weeks, neurons showing GABA(-)TH(+) were not seen in GL (Kosaka et al. 1987a), the majority of GABA(-)TH(+) neurons in adult olfactory bulb were assumed to change their phenotype at some postnatal developmental period.     

Down-regulation of CXCR4 expression on human CD8+ T cells during peripheral differentiation

Multi-color flow cytometric analysis on human CD8(+) T cell subsets revealed that CXCR4 is predominantly expressed on CD8(+) T cells with the naive CD27(+)CD28(+)CD45RA(+) phenotype, and is down-regulated during differentiation into those with an effector phenotype. The down-regulation of CXCR4 expression during peripheral differentiation was supported by the fact that the expression of CXCR4 on CD8(+) T cells was negatively correlated with that of perforin. The analysis of CCR5, CCR7, and CXCR4 co-expression further showed that CD8(+) T cells expressing a high level of CXCR4 are CCR7(+)CCR5(-) naive or central memory subsets, and those expressing a low level of CXCR4 were included in the CCR7(-)CCR5(+/-) memory/effector and effector subsets. Epstein Barr virus-specific CD8(+) T cells, which mostly express the memory phenotype, expressed CXCR4, while human cytomegalovirus-specific CD8(+) T cells, which mostly express the effector phenotype, partially expressed this receptor, showing that the expression of CXCR4 is also down-regulated during differentiation of viral antigen-specific CD8(+) T cells. The classification of human CD8(+) T cells based on the expression of these chemokine receptors should prove useful for studies that clarify the differentiation of human CD8(+) T cells.     

Impaired CD4 and CD8 T cell phenotype and reduced chemokine secretion in recent-onset type 1 diabetic children

Although the role of the T cell-mediated autoimmune reaction in type 1 diabetes (T1D) is conclusive, studies including data from human circulating CD4(+) and CD8(+) lymphocytes subsets during the disease onset and posterior development are scarce. Further, chemokines and chemokine receptors are key players in the migration of pathogenic T cells into the islets of non-obese diabetic mice developing T1D, but few studies have investigated these markers in human T1D patients. We studied the expression of T helper 1 (Th1)- and Th2-associated chemokine receptors, and the two isoforms of CD45 leucocyte antigen on CD4(+) and CD8(+) lymphocytes from T1D and healthy children, as well as the secretion of chemokines in cell supernatants in peripheral blood mononuclear cells. Our results showed increased expression of CCR7 and CD45RA and reduced CD45RO on CD8(+) cells among recent-onset T1D patients. The percentages of CD4(+) cells expressing CXC chemokine receptor 3 (CXCR3), CXCR6 and CCR5, and the secretion of interferon-gamma-induced protein-10, monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta was lower among diabetics. Low expression of Th1-associated receptors and secretion of chemokines, together with an increased amount of CD8(+) cells expressing CD45RA and CCR7 in T1D patients therefore might represent suboptimal Th function in T1D, leading to impaired T cytotoxic responses or alternatively reflect a selective recruitment of Th1 cells into the pancreas.     

Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection

Molecular differences between dopamine (DA) neurons may explain why the mesostriatal DA neurons in the A9 region preferentially degenerate in Parkinson's disease (PD) and toxic models, whereas the adjacent A10 region mesolimbic and mesocortical DA neurons are relatively spared. To characterize innate physiological differences between A9 and A10 DA neurons, we determined gene expression profiles in these neurons in the adult mouse by laser capture microdissection, microarray analysis and real-time PCR. We found 42 genes relatively elevated in A9 DA neurons, whereas 61 genes were elevated in A10 DA neurons [> 2-fold; false discovery rate (FDR) < 1%]. Genes of interest for further functional analysis were selected by criteria of (i) fold differences in gene expression, (ii) real-time PCR validation and (iii) potential roles in neurotoxic or protective biochemical pathways. Three A9-elevated molecules [G-protein coupled inwardly rectifying K channel 2 (GIRK2), adenine nucleotide translocator 2 (ANT-2) and the growth factor IGF-1] and three A10-elevated peptides (GRP, CGRP and PACAP) were further examined in both alpha-synuclein overexpressing PC12 (PC12-alphaSyn) cells and rat primary ventral mesencephalic (VM) cultures exposed to MPP+ neurotoxicity. GIRK2-positive DA neurons were more vulnerable to MPP+ toxicity and overexpression of GIRK2 increased the vulnerability of PC12-alphaSyn cells to the toxin. Blocking of ANT decreased vulnerability to MPP+ in both cell culture systems. Exposing cells to IGF-1, GRP and PACAP decreased vulnerability of both cell types to MPP+, whereas CGRP protected PC12-alphaSyn cells but not primary VM DA neurons. These results indicate that certain differentially expressed molecules in A9 and A10 DA neurons may play key roles in their relative vulnerability to toxins and PD.     

MPTP诱导的小鼠PD模型中相关脑区小胶质细胞反应性差异研究

为了探讨Parkinson病(PD)小鼠相关脑区的小胶质细胞反应性是否存在差异,本研究采用MPTP诱导的小鼠PD模型,用GSA I-B4-HRP组织化学染色方法对黑质致密部(SNc)、尾壳核(CPu)、腹侧被盖区(VTA)和额叶联合皮层(FrA)四个部位中小胶质细胞的形态和数量进行了观察;同时用免疫荧光染色检测了SNc和VTA中TH阳性神经元的数量,用Western blot方法检测CPu和FrA内TH蛋白含量的变化情况。结果显示:SNc中TH阳性神经元的数量和CPu中TH蛋白的含量在MPTP腹腔注射后1 d即大幅度降低(降幅约为68%);在MPTP腹腔注射后3 d起SNc中TH阳性神经元的数量和CPu中TH蛋白的含量逐步增加;小胶质细胞在MPTP腹腔注射后1～3 d的反应明显,然后逐渐减弱。VTA中TH阳性神经元的数量和FrA中TH蛋白的含量在MPTP腹腔注射后降幅不明显(降幅约在15%),小胶质细胞在MPTP腹腔注射后1 d的反应较为明显,其它时段的变化不显著。本研究结果提示:PD模型小鼠SNc和CPu中的小胶质细胞反应明显强于VTA和FrA区,反应性小胶质细胞在SNc中的多巴胺(DA)能神经元损伤过程中扮演了重要角色。