胎脑黑质脑内移植矫正PD鼠纹状体内脑啡肽mRNA过度表达

用６－ＯＨＤＡ损毁大鼠一侧黑质—纹状体通路可引起ＰＤ模型鼠脑纹状体内多巴胺匿乏．同时,亦可导致脑啡肽ｍＲＮＡ过度表达。我们用地高辛标记的ｃＲＮＡ探针对纹状体内脑啡肽ｍＲＮＡ含量进行了斑点杂交定量研究,发现损伤侧脑啡肽ｍＲＮＡ含量较健侧增高８０％,胎中脑移植到失神经支配的纹状体内,脑啡肽ｍＲＮＡ过度表达得以矫正至正常水平,说明胎多巴胺能神经元脑内移植能够调节脑啡肽基因表达,提供了移植物能与宿主发生神经整合、建立突触联系的间接证据。     

胚鼠黑质细胞分别移植于帕金森病模型鼠纹状体和侧脑室的比较研究

胚鼠黑质细胞悬液分别移植于帕金森病（ＰＤ）鼠纹状体和侧脑室。移植后两组动物的Ａｐｏｍｏｒｐｈｉｎｅ诱导旋转行为均得到极明显改善,移植细胞生长发育良好。移植细胞和宿主细胞间的信息联系,在纹状体内可能以突触传递方式为主。侧脑室内移植的黑质细胞,相当于人工放置的“接触脑脊液神经元”,可能主要通过非实触传递方式而发挥作用。     

黑质内微量注射P物质对大鼠旋转行为的影响

研究表明 ,帕金森病 (ＰＤ)的发生是因中脑黑质多巴胺 (ＤＡ)能神经元变性 ,导致纹状体内ＤＡ释放量减少所致。目前有补充ＤＡ ,应用Ｍ受体阻断剂 ,脑内刺激及基因移植等治疗方法 ,但不能缓解所有症状。有人证明帕金森病时 ,黑质内Ｐ物质和ＧＡＢＡ的浓度明显低于正常 ,且降低幅度与发病的严重程度呈平行关系。说明帕金森病的发生可能还与黑质 纹体 黑质之间的其它神经递质作用失衡有关。黑质是Ｐ物质和ＧＡＢＡ含量很高的核团之一 ,对黑质的功能有重要的调节作用。本研究采用黑质网状部微量注射方法 ,观察不同浓度的Ｐ物质对ＡＰＯ诱发的…     

骨髓间充质干细胞黑质内移植对帕金森病大鼠的治疗作用

目的探索骨髓间充质干细胞（BMSCs）移植到帕金森病（Parkinson’s disease,PD）大鼠毁损侧黑质内,PD模型大鼠的姿势不对称性和黑质及纹状体内酪氨酸羟化酶（tyrosinehy droxylase,TH）表达的改变,以及BM—SCs在大鼠脑内的存活、分化情况。方法黑质、前脑内侧束两点法注射6一羟多巴胺（6-OHDH）并行为学分析筛选PD模型大鼠。将PD模型大鼠随机分为移植组和对照组。BMSCs移植术后4周和8周,观察大鼠姿势不对称性,免疫组织化学及免疫荧光显色方法检测黑质和纹状体酪氨酸羟化酶（tyrosine hydroxylase,TH）的表达变化以及BMSCs在大鼠体内的存活、迁移及分化情况。结果BMSCs黑质内移植可使PD模型大鼠的转动频率由（10．62±2．97）r／min降至（4．65±1．08）r／min（P〈0．01）,显著增加毁损侧黑质TH阳性细胞数量和纹状体内TH阳性纤维密度。BMSCs在大鼠黑质内可以存活至少8周,部分细胞分化为神经干细胞、神经元和神经胶质细胞。结论黑质内移植BMSCs对PD模型大鼠有一定的治疗作用。     

外源性TH基因在帕金森氏病鼠脑内的表达——行为和TH免疫组化的研究

用成年大鼠７５只,给右侧黑质区注射６－羟基多巴胺（６－ＯＨＤＡ）,损毁黑质多巴胺能神经元,制备偏侧帕金森氏病（ＰＤ）鼠模型。四周后,注射阿朴吗啡（ＡＰＯ）诱发大鼠向左侧旋转。旋转数为每分钟７次以上的３５只ＰＤ鼠作实验用。其中实验组１５只,对照组２０只。向实验组ＰＤ鼠右侧纹状体多点植入含大鼠酪氨酸羟化酶ｃＤＮＡ（ＴＨｃＤＮＡ）的真核表达载体ｐＳＶＫ３－ＴＨ和脂质体Ｌｉｐｏｆｅｃｔｉｎ混合的基因转染复     

胚中脑黑质移植入震颤麻痹模型大鼠脑内的实验研究

将6 - 羟基多巴胺(6 - OHDA) 分两点注入SD大鼠左侧中脑黑质内侧端,制成震颤麻痹模型,毁损后6 ～8 周,用胚龄14～16 天同种胚鼠中脑黑质细胞悬液植入模型鼠尾壳核。实验分正常对照组,模型对照组,单纯胚中脑黑质移植组(MS组) ,含层粘蛋白Laminin 胚中脑黑质移植组(LMS组) ,含雪旺氏细胞胚中脑黑质移植组(SMS组) 。于移植后2 ～4 个月分别测试旋转试验,MS组、LMS组及SMS组与模型组间均有显著差异(P< 0-01) 。动物经测试后处死。脑切片观察发现,损伤侧中脑黑质DA神经元95% 以上死亡,移植组织位于尾壳核背侧或突入侧脑室,LMS组移植区> SMS组移植区> MS组移植区。三个移植区均见TH、GABA 免疫阳性细胞,其形态及大小与正常对照侧中脑黑质所见相似。LMS组与SMS组多巴胺阳性细胞突起较MS组多且长。本实验证明中脑黑质移植物Laminin 与雪旺氏细胞均能促进DA能神经元突起生长。     

用微囊化转基因细胞治疗PD猴的初步研究

用海藻酸钠／多聚赖氨酸（ＡＬＧ／ＰＬＬ）方法制备了包裹转 酪氨酸羟化酶基因的大鼠在肌细胞微囊。在在体外,微囊内的细胞能长时间存活及生长,并能产生ＴＨ蛋白。将这种微囊移植于正常猴的纹状体,１个月后仍能在微囊内观察到活细胞,而且移植处凶胶质细胞明显增生,在此基础上,将这种微囊移植于患帕金森病（ＰＤ）的模型猴的纹状体,初步观察到的其病理性旋转行为有明显改善。     

大鼠实验性帕金森病的基因治疗

将稳定转染了大鼠酪氨酶羟化酶（Ｔｙｒｏｓｉｎｅｈｙｄｒｏｘｙｌａｓｅ,ＴＨ）基因的大鼠成肌细胞移植于帕金森病大鼠模型的纹状体,进行基因治疗研究。ＲＴ－ＰＣＲ和免疫组织化学检测都证明转基因细胞可在纹状体内存活并表达ＴＨ,动物的不对称旋转行为明显改善,而且疗效可维持半年以上。     

大鼠实验性帕金森病的基因治疗

将稳定转染了大鼠酪氨酶羟化酶基因的大鼠成肌细胞移植于帕金森病大鼠模型模型的纹状体,进行基因治疗研究,ＲＴ－ＰＣＲ和免疫组织化学检测都证明转基因细胞可在纹状体内存活并表达ＴＨ,动物的不对称旋转行为明显改善,而且疗效可维持半年以上。     

INCREASED PHOSPHORYLATION OF DARPP-32 BY D_1 AGONISTIC ACTION OF l-STEPHOLIDINE IN THE 6-OHDA-LESIONED RAT STRIATUM

为了进一步阐明ＳＰＤ对大鼠纹状体突触后Ｄ１受体的激动作用特性,本文应用反磷酸化在体内测定及放射配体结合方法,分别观察ＳＰＤ对６ＯＨＤＡ损毁大鼠纹状体ＤＡＲＰＰ３２体内磷酸化作用及突触后Ｄ１受体密度的影响。结果表明：皮下给予ＳＰＤ（２０,４０ｍｇ／ｋｇ,２１ｄ）,损毁侧纹状体ＤＡＲＰＰ３２体外［３２Ｐ］的掺入量较健侧下降５０％（Ｐ＜００１）。换言之,损毁侧纹状体内ＤＡＲＰＰ３２的磷酸化程度增加了。然而,ＳＰＤ使损毁导致Ｄ１受体上调的作用减弱（Ｂｍａｘ从３８５０±２６１ｆｍｏｌ／ｍｇ降至３１９７±２０１ｆｍｏｌ／ｍｇ水平）。因此,ＳＰＤ激动Ｄ１受体,使６ＯＨＤＡ损毁大鼠纹状体内ＤＡＲＰＰ３２磷酸化作用加强,而受体密度减少。这是ＳＰＤ调节脑内Ｄ１受体信号转导功能的重要机制。     

Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model

Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson''s disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.     

6-OHDA制备的帕金森病大鼠黑质神经元的超微结构改变

目的观察6-羟多巴胺(6-OHDA)单侧注射制备的帕金森病(PD)大鼠多巴胺(DA)能神经元的超微结构改变。方法单侧微量注射6-OHDA制备PD大鼠模型,用免疫荧光组织化学方法观察正常侧与6-OHDA注射侧黑质酪氨酸羟化酶(TH)阳性神经细胞及神经纤维的变化;并利用免疫电镜技术观察大鼠正常侧与注射侧黑质致密部DA能神经元的超微结构。结果免疫荧光法显示注射侧黑质致密部TH阳性细胞数和网状部TH阳性纤维面积与正常侧的百分比平均值分别为21.83%,23.19%。免疫电镜显示:TH免疫反应阳性产物表达于PD大鼠正常侧DA能神经元的高尔基复合体质膜面及胞质内,电子密度较高,注射侧很少见或几乎未见,且注射侧线粒体嵴有不同程度的溶解,呈空泡样变或髓样变,粗面内质网脱颗粒。结论6-OHDA可引起DA能神经元发生凋亡的超微结构改变。     

Neuronal activity regulates expression of tyrosine hydroxylase in adult mouse substantia nigra pars compacta neurons

Striatal delivery of dopamine (DA) by midbrain substantia nigra pars compacta (SNc) neurons is vital for motor control and its depletion causes the motor symptoms of Parkinson's disease. While membrane potential changes or neuronal activity regulates tyrosine hydroxylase (TH, the rate limiting enzyme in catecholamine synthesis) expression in other catecholaminergic cells, it is not known whether the same occurs in adult SNc neurons. We administered drugs known to alter neuronal activity to mouse SNc DAergic neurons in various experimental preparations and measured changes in their TH expression. In cultured midbrain neurons, blockade of action potentials with 1?μM tetrodotoxin decreased TH expression beginning around 20?h later (as measured in real time by green fluorescent protein (GFP) expression driven off TH promoter activity). By contrast, partial blockade of small-conductance, Ca(2+) -activated potassium channels with 300?nM apamin increased TH mRNA and protein between 12 and 24?h later in slices of adult midbrain. Two-week infusions of 300?nM apamin directly to the adult mouse midbrain in vivo also increased TH expression in SNc neurons, measured immunohistochemically. Paradoxically, the number of TH immunoreactive (TH+) SNc neurons decreased in these animals. Similar in vivo infusions of drugs affecting other ion-channels and receptors (L-type voltage-activated Ca(2+) channels, GABA(A) receptors, high K(+) , DA receptors) also increased or decreased cellular TH immunoreactivity but decreased or increased, respectively, the number of TH+ cells in SNc. We conclude that in adult SNc neurons: (i) TH expression is activity-dependent and begins to change ～20?h following sustained changes in neuronal activity; (ii) ion-channels and receptors mediating cell-autonomous activity or synaptic input are equally potent in altering TH expression; and (iii) activity-dependent changes in TH expression are balanced by opposing changes in the number of TH+ SNc cells.     

Biochemical Evidence for Alteration of Neostriatal Dopaminergic Function by 5,7-Dihydroxytryptamine

Unilateral injection of 5,7-dihydroxytryptamine (DHT) into the rat neostriatum markedly reduced not only striatal tryptophan hydroxylase (TPH) activity but also striatal tyrosine hydroxylase (TH) activity and dopamine (DA) concentration measured 10--15 days later. The decrease in striatal TH activity was dose related over the range of 8--32 micrograms of DHT; a dose of 16 micrograms reduced striatal TH activity to 40--50% of control, DA concentration to 38% of control, and TPH activity to 5--20% of control. Intrastriatal injection of 16 micrograms of DHT reduced TH activity in the ipsilateral substantia nigra to 51% of control. Pretreatment with amfonelic acid, a potent DA uptake inhibitor, significantly reduced the effect of DHT on striatal and nigral TH activity and striatal DA concentration without affecting the DHT-induced decrease in striatal TPH activity. Desmethylimipramine (5 and 25 mg/kg) had no effect on the DHT-induced decrease in striatal TH activity. Striatal choline acetyltransferase and glutamic acid decarboxylase activities were not decreased by 16 micrograms of DHT. The results indicate that DHT can alter dopaminergic function in the rat neostriatum through a direct effect of the drug on DA neurons.     

Pitx3 regulates tyrosine hydroxylase expression in the substantia nigra and identifies a subgroup of mesencephalic dopaminergic progenitor neurons during mouse development

Recent studies of mouse mutant aphakia have implicated the homeobox gene Pitx3 in the survival of substantia nigra dopaminergic neurons, the degeneration of which causes Parkinson's disease. To directly investigate a role for Pitx3 in midbrain DA neuron development, we have analysed a line of Pitx3-null mice that also carry an eGFP reporter under the control of the endogenous Pitx3 promoter. We show that the lack of Pitx3 resulted in a loss of nascent substantia nigra dopaminergic neurons at the beginning of their final differentiation. Pitx3 deficiency also caused a loss of tyrosine hydroxylase (TH) expression specifically in the substantia nigra neurons. Therefore, our study provides the first direct evidence that the aphakia allele of Pitx3 is a hypomorph and that Pitx3 is required for the regulation of TH expression in midbrain dopaminergic neurons as well as the generation and/or maintenance of these cells. Furthermore, using the targeted GFP reporter as a midbrain dopaminergic lineage marker, we have identified previously unrecognised ontogenetically distinct subpopulations of dopaminergic cells within the ventral midbrain based on their temporal and topographical expression of Pitx3 and TH. Such an expression pattern may provide the molecular basis for the specific dependence of substantia nigra DA neurons on Pitx3.     

Dopamine turnover is upregulated in the caudate/putamen of asymptomatic MPTP-treated rhesus monkeys

In Parkinson's disease (PD) and experimental parkinsonism, losses of up to 60% and 80%, respectively, of dopaminergic neurons in substantia nigra, and dopamine (DA) in striatum remain asymptomatic. Several mechanisms have been suggested for this functional compensation, the DA-mediated being the most established one. Since this mechanism was recently challenged by striatal DA analysis in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys, we present data on several DAergic parameters in three groups of rhesus monkeys: MPTP-treated asymptomatic animals; symptomatic MPTP-treated animals with stable parkinsonism; and untreated sex and age matched controls. We determined ratios of striatal and nigral 3,4-dihydroxyphenyl acetic acid (DOPAC) to DA levels and tyrosine hydroxylase (TH) enzyme activity to DA levels, in addition to the commonly used homovanillic acid (HVA)/DA ratios which, as such, might be less reliable under the conditions of partial denervation. We found that in the asymptomatic MPTP monkeys the DOPAC/DA ratios in putamen and caudate nucleus were shifted with high statistical significance 1.9-5.8-fold, as compared to controls, the shifting of the ratios being in the same range as the 2.6-5.4-fold shifts in the symptomatic animals. Also TH/DA ratios were significantly increased in both, the asymptomatic and the symptomatic MPTP-treated monkeys, with shifts in the putamen and caudate nucleus of 3- and 2.7-7.0-fold, respectively. In the substantia nigra, DOPAC levels and TH activity were strongly decreased after MPTP (-77 to -97%), but the ratios DOPAC/DA and TH/DA were not changed in this brain region. Collectively, our findings support the concept of DAergic compensation of the progressive striatal DA loss in the presymptomatic stages of the parkinsonian disease process.     

Dopamine neuron transplants: electrophysiological unit activity of intrastriatal nigral grafts in freely moving cats

Fetal transplants of substantia nigra dopamine (DA)-containing neurons into the caudate nucleus in cats produced functional synaptic connections, as measured by behavioral analysis. Electrophysiological activity of single units from the intrastriatal nigral grafts revealed that these neurons displayed some of the characteristic electrophysiological parameters of DA neurons, i.e., long duration action potentials and tri-phasic waveform. However, these neurons discharged at a faster rate and never displayed the characteristic decremental burst pattern seen in intact, non-grafted animals. These data are the first electrophysiological recordings of grafted DA neurons in freely moving animals.     

Synaptic connections of substance P-immunoreactive nerve terminals in the substantia nigra of the rat

Summary A monoclonal antibody that recognises the C-terminal part of substance P was used to study immunoreactive structures in the substantia nigra by the unlabeled antibody, peroxidase-antiperoxidase procedure. Immunoreactivity was present in nerve fibres in all parts of the substantia nigra, particularly in the pars reticulata and pars lateralis. Electron microscopically two types of bouton immunoreactive for substance P were found: Type 1 contained large electron-lucent vesicles, occasional large granulated vesicles and formed symmetrical synapses with dendrites. Type 2 boutons contained smaller, round electron-lucent vesicles, many large granular vesicles and formed asymmetrical synapses (having prominent postjunctional dense bodies) with dendrites and perikarya.Immunoreactive fibres with varicosities that had been identified light microscopically were studied in serial sections in the electron microscope. Each identified varicosity contained synaptic vesicles and formed a single synapse. An individual fibre formed boutons of only one kind (type 1 or type 2) and could form multiple synapses with the same neuron. Thus, an identified fibre in the pars compacta had eight varicosities, each of which was in synaptic contacts (type 2) with the dendrites or soma of the same neuron.The results are consistent with the concept that substance P is a synaptic transmitter in the substantia nigra and indicate that neurons in this region may receive a significant input from substance P-containing afferents, and that there are at least two types of such afferent fibres.     

Effects of Chronic Striatal Kainate Lesions on Some Dopaminergic Parameters and Enkephalin Immunoreactive Neurons in the Basal Ganglia

Abstract: The chronic effects of kainate-induced lesions of the neostriatum have been evaluated in rats 12 months following the injection of kainic acid. Light microscopical analysis revealed marked disappearance of nerve cells in the neostriatum, with some cells remaining within the medial and lateral zone of the neostriatum and in the most ventral part. The rest of the markedly atrophied neostriatum was mainly made up of densely packed myelinated nerve bundles. Tyrosine hydroxylase immunoreactivity was used as a marker for dopamine neurons and revealed that tyrosine hydroxylase immunoreactive nerve terminals remained between the axon bundles in the striatum and that tyrosine hydroxylase immunoreactive nerve cell bodies in the substantia nigra seemed intact. Studies on enkephalin immunoreactive neurons revealed a marked disappearance of such nerve cells and nerve terminals within the neostriatum. Neurochemical analysis showed a clearcut reduction in the number of dopamine receptors as evaluated by studies on both [³H]spiperone binding and on [³H]ADTN binding. Dopamine levels remained unchanged while choline acetyltransferase activity was reduced significantly. Taken together, the present findings demonstrate that the chronically kainate lesioned striatum is characterized by a substantial loss of enkephalin immunoreactive and cholinergic nerve cells and a marked reduction in the number of dopamine receptors. These findings are discussed in relation to neurochemical and therapeutic aspects of Huntington's disease.     

Distribution of dopamine immunoreactive systems in brain stem and spinal cord of the chameleon

An immunohistochemical method, using glutaraldehyde fixation and a highly specific monoclonal antibody recently synthetized against dopamine (DA)-glutaraldehyde protein conjugate, permitted direct visualization of DA structures in the brainstem and spinal cord of a reptile (Chameleon). DA-immunoreactive cell bodies occurred in some contiguous areas of the midbrain tegmentum. The first one was located in the ventral tegmental area. Some somata intermingled with the oculomotor nucleus. The second group was the large round or oval DA-Immunostained neurons located in the substantia nigra. More caudally, a third group of round or fusiform DA-cell bodies was seen in an homologous area of so called mammalian A8 and were continuous with the substantia nigra group. In the medulla oblongata, the DA-containing cells were shown in the nucleus of solitary tract and in the dorsal lateral part of the dorsal motor nucleus of the vagus. The density of this DA-Immunoreactive neurons decreased more caudally. At the medullo-spinal level and upper cervical spinal cord, a few labelled cells were distinguished near the central canal. In the spinal cord DA-immunopositive cell bodies were observed in the vicinity of the central canal and formed a continuous column that extended throughout the rostral spinal cord. The apical processes of these neurons seemed to be in contact with the lumen of the central canal. This study constitute the first visualization of the immunoreactive DA-cell bodies at the medullo-spinal level which were already described, as TH immunoreactive in other species of reptiles.