大鼠全脑缺血再灌注损伤后海马CA1区神经元酪氨酸羟化酶和多巴胺-β-羟化酶表达的变化

目的　研究大鼠全脑缺血再灌注损伤后酪氨酸羟化酶 (TH)和多巴胺 β 羟化酶 (DβH)表达的改变及意义。方法　利用改良四血管闭塞法 ,建立大鼠全脑缺血模型。于缺血再灌注后 1,3,5d断头取脑 ,行免疫组化染色 ,在光镜下观察海马CA1区神经元TH及DβH表达的变化 ,并计数海马CA1区正常神经元。结果　全脑缺血再灌注后 1d ,TH及DβH的表达呈阴性 ,海马CA1区神经元未见显著的病理形态学改变 ;全脑缺血再灌注后 3d ,TH及DβH呈阳性表达 ,海马CA1区可见部分神经元死亡 ;全脑缺血再灌注后 5d ,TH及DβH呈明显阳性表达 ,海马CA1区可见大部分神经元死亡。结论　全脑缺血再灌注后 ,由于TH及DβH异常表达 ,使得儿茶酚胺生物合成增加 ,这可能是短暂性脑缺血损伤的机理之一。     

多巴胺β羟化酶基因多态性和首发精神分裂症的关联分析

有研究发现多巴胺和肾上腺素异常在精神分裂症发病机制中起重要作用[1]。多巴胺β羟化酶(dopamine beta-hydroxy-lase,DBH)是催化多巴胺向去甲肾上腺素转化的核心酶[2]。有研究已报道DBH活动和水平的异常被看做精神分裂症发病的生物标志物[3]。而DBH基因5′端19 bp插入/缺失(位于位于染色体9q34)调控启动子活动进而调节血浆和脑脊液DBH     

大鼠全脑缺血再灌注损伤后海马CAl区神经元酪氨酸羟化酶和多巴胺—β—经化酶表达的变化

目的 研究大鼠全脑缺血再灌注损伤后酪氨酸羟化酶(TH)和多巴胺—β—羟化酶(DβH)表达的改变及意义。方法 利用改良四血管闭塞法，建立大鼠全脑缺血模型。于缺血再灌注后1，3，5d断头取脑，行免疫组化染色，在光镜下观察海马CA1区神经元TH及DβH表达的变化，并计数海马CAl区正常神经元。结果 全脑缺血再灌注后1d，TH及DβH的表达呈阴性，海马CA1区神经元末见显著的病理形态学改变；全脑缺血再灌注后3d，TH及DβH呈阳性表达，海马CA1区可见部分神经元死亡；全脑缺血再灌注后5d，TH及DβH呈明显阳性表达，海马CA1区可见大部分神经元死亡。结论 全脑缺血再灌注后，由于TH及DβH异常表达，使得儿茶酚胺生物合成增加，这可能是短暂性脑缺血损伤的机理之一。     

2004/低频脉冲电场对PC12细胞酪氨酸羟化酶和多巴胺合成的影响

运用Western 印迹和HPLC分别测定不同时间电场刺激和刺激后不同培养时间条件下,PC12细胞内酪氨酸羟化酶（TH）和细胞培养液中多巴胺（DA）含量的变化。结果显示,受到短时间（5、10 min）脉冲电场刺激的PC12 细胞,经较短时间（2天）的培养后,细胞内TH的含量和培养液中DA的含量均比对照组有所提高,但随着培养时间的延长（3~5天）,TH和DA的含量均明显下降。然而,长时间（15、20、30 min）脉冲电场刺激组则先表现为TH和DA的合成受到抑制,但随着培养时间的延长,其合成则被逐渐激活。采用蛋白激酶A（PKA）特异性抑制剂H-89和有丝分裂原活化蛋白激酶的激酶（MEK1/2）特异性抑制剂U0126,研究脉冲电场刺激所激活的与TH和DA合成相关的信号通路。结果表明,在没有神经生长因子（NGF）存在的情况下,PC12细胞主要通过PKA通路来激活TH的合成,低频脉冲电场刺激也主要激活PKA通路,因为抑制这条信号通路能显著抑制电场刺激所诱导的TH合成。     

死亡相关蛋白小分子干扰RNA对帕金森病大鼠黑质酪氨酸羟化酶表达的影响

目的:利用动物模型探讨死亡相关蛋白(FADD)小分子干扰RNA(siRNA)对帕金森病(PD)大鼠黑质酪氨酸羟化酶(TH)表达的影响。方法:40只雄性Wistar大鼠随机分为4组。经立体定向定位黑质,PD组:注入6-羟基多巴(6-OHDA);siRNA组:注入FADD siRNA2次后再注入6-OHDA;SiRNA阴性对照组:注入FADD siRNA阴性对照物2次后再注入6-OHDA;正常对照组:注入抗坏血酸生理盐水。各组大鼠术后4周处死,取鼠的术侧中脑黑质,利用原位杂交检测FADD、TH mRNA表达。结果:PD组与正常对照组比较FADD mRNA表达显著增加(P<0.01);siRNA组与PD组比较FADD mRNA表达显著减少(P<0.05);siRNA阴性对照组与正常对照组比较FADD mRNA表达量无显著差异(P>0.05)。PD组与正常对照组比较TH mRNA表达显著减少(P<0.01);siRNA组与PD组比较TH mRNA表达显著增加(P<0.05);siRNA阴性对照组与正常对照组比较TH mRNA表达量差异无统计学意义(P>0.05)。结论:FADDsiRNA可能通过抑制凋亡对PD大鼠模型有一定的保护和治疗作用。     

多巴胺β羟化酶基因内含子5区A1/A2多态与帕金森病的相关研究

帕金森病 (ＰＤ)作为一种危及人类生存生活质量的老年神经退行性疾病 ,其多种临床表现均与脑区黑质纹状体多巴胺浓度的降低有关 ,故与多巴胺系统代谢相关的基因已成为ＰＤ临床分子遗传学研究的热点。本研究主要探讨多巴胺β羟化酶 (ＤＢＨ)基因内含子 5区Ａ1/Ａ2多态在ＰＤ发病风险中的意义 ,并试图为ＰＤ的早期诊断提供可靠的生物学标记。1 研究对象 :原发性ＰＤ患者 35例 (男 16例 ,女 19例 ) ,年龄 4 3～ 79岁 ,平均 (6 2 3± 14 1)岁 ,源于我院神经内科门诊病人 ,诊断符合全国第一届锥体外系疾病会议制定标准。对照组 5 0例 (男 2 3例…     

小脑性共济失调大鼠模型的行为学和病理学研究

目的：评价海人酸（KA）损毁单侧小脑顶核制备的小脑性共济失调大鼠模型。方法：实验大鼠分为小脑性共济失调模型组（KA模型组）和假手术组。KA模型组采用经立体定向仪定位大鼠左侧小脑顶核注射KA毁损制备单侧小脑性共济失调模型;假手术组在相同部位注入等量生理盐水。用平衡木实验及旋转杠实验检测两组大鼠的行为学改变,并在光学显微镜下观察两组大鼠小脑顶核及小脑的病理改变。结果：KA模型组模型大鼠在平衡木上的得分减少、通过平衡木的时间延长、在旋转杠上持续运动的时间减少,与假手术组相比,差异有统计学意义（P〈0.05）。光学显微镜下病理学观察KA注射位置准确,大鼠小脑左侧顶核神经元细胞破坏,其他周围组织未受影响。在1个月的检测期内,大鼠生存能力好。结论：用KA制备的大鼠小脑性共济失调模型适用于后续的治疗研究。     

大鼠D1多巴胺受体墓因和cDNA的比较发现5’端有不同的序列

我们克隆了大白鼠Ｄ１多巴胺基因。序列分析表明，表型克隆的５’端与Ｍｏｎｓｍａ等报告的大鼠纹状体Ｄ１多巴胺受体的ｃＤＮＡ克隆有所不同。我们又应用聚合酶链反应（ＰＣＲ）法检查了这种差别是否系由于Ｄ１受体基因的不同剪接所造成，但实验结果并不支持这种设想。因此可以认为，Ｍｏｎｓｍａ等克隆的Ｄ１受体５’端序列值得进一步研究。     

老年大鼠黑质致密部多巴胺能神经元的变化

目的探讨老年大鼠黑质致密部多巴胺(DA)能神经元的变化。方法采用逆转录PCR(RT-PCR)、免疫印迹法、免疫组化染色和电镜技术,观察老年大鼠(≥24月龄)黑质致密部DA能神经元酪氨酸羟化酶(TH)mRNA、蛋白表达水平和TH阳性神经元数及其超微结构,并与成年大鼠(4～5月龄)比较。结果老年组大鼠黑质致密部THmRNA水平(0.66±0.12)明显低于成年组(1.09±0.08)(P<0.05);TH蛋白水平及TH阳性神经元数与成年组差异均无统计学意义(均P>0.05);老年组大鼠黑质致密部尾侧段TH阳性神经元数比成年组明显减少(P<0.05),其超微结构显示细胞器少、脂褐素多、核固缩、线粒体形态异常且数量减少。结论老年大鼠黑质致密部DA能神经元THmRNA水平下降,尾侧DA能神经元明显减少,并呈衰老性改变,这可能是老年黑质纹状体功能降低的生物学基础。     

外源性kynurenic acid对大鼠黑质多巴胺能神经元损伤的保护作用研究

目的观察评价预先应用谷氨酸(Glu)受体拮抗剂kynurenic acid(KYNA)对黑质多巴胺(DA)能神经元及神经传导纤维损伤的保护性作用. 方法雌性SD大鼠40只,随机分为4组,每组10只,应用江湾I型C立体定向仪,在单侧黑质致密部及中脑被盖腹侧部, A组注射生理盐水,B组注射KYNA,C组注射KYNA和6-羟基多巴胺(6-OHDA), KYNA先于6-OHDA 30 min, D组注射6-OHDA.注射药物3 d后,进行症状观察,4周后处死大鼠.切片HE染色观察黑质细胞的形态特点,冰冻切片免疫组化特殊染色观察酪氨酸羟化酶(TH)阳性细胞及TH阳性纤维着色情况.结果正常黑质细胞体形较大,富含黑色素颗粒,可见尼氏体.TH着色结果提示B组与A组之间无显著差异,P>0.05.实验组C与A、B、D组比较均有显著性差异,P<0.01.结论外源性Glu受体拮抗剂KYNA通过阻滞Glu受体一定时间阶段内能减轻6-OHDA诱导的黑质DA能神经元毒性损害.     

Genetic targeting of cerebellar Purkinje cells: history,current status and novel strategies

This review is an account of developments in the field of transgenic and gene targeting approaches with special emphasis on the cerebellar Purkinje cell. A critical discussion of the available genetic tools is provided. As genetic engineering of the mouse is still a rapidly moving field, we felt it appropriate to include some ideas on novel strategies for refined genetic manipulations.     

Developmental dynamics of Purkinje cells and dendritic spines in rat cerebellar cortex

Quantitative morphological changes of the developing Purkinje cells were studied from 6 to 90 postnatal (PN) days in the IVth lobule of vermis in the cerebellum of rats. The soma size (mean diameter) of Purkinje cells increased rapidly between 6 PN (on average 10 μm) and 18 PN (about 17 μm) days; it did not change between 18 and 25 PN days, but increased moderately again between 25 and 48 PN days (22–23 μm) and stabilized on the same value. In contrast, the number of Purkinje cells/100 μm (the “linear density”) decreased rapidly from 6 to 18 PN days. The molecular layer area belonging to 1 Purkinje cell increased rapidly from 6 to 25 PN days (from about 370 to 6,200 μm²) and less rapidly between PN days 30 to 48 (up to 9,300 μm²), followed by a moderate decrease at PN day 90 (about 6,600 μm²). The volume belonging to 1 Purkinje cell dendritic arbor was about 5,500 μm³ at PN day 6,93,000 μm³ at PN day 25, and 100,000 μm³ at PN day 90. The numerical density of dendritic spines in the molecular layer showed a biphasic curve: a rapid increase from PN days 6 to 21 followed by a significant but short decrease at PN day 25, moderate rise from PN days 25 to 48, and a subsequent decline between PN days 48 and 90. The number of spines belonging to 1 Purkinje cell showed two developmental “peaks”: the first peak at 21 PN days was moderate (5.6 × 10⁴ spines/Purkinje cell) while the second maximum at 48 PN days was more significant (1.2 × 10⁵ spines/Purkinje cell), which then declined to 6.3 × 10⁴ spines/Purkinje cell at PN day 90. It is suggested that the temporary overproduction and the following decline in the number of Purkinje dendritic spines during the development of the cerebellar cortex may be the morphological indicator of the dynamics of synaptogenetic and of synaptic stabilization processes. © 1994 Wiley-Liss, Inc.     

Focal axonal swellings in rat cerebellar Purkinje cells during normal development

Focal axonal swellings are characteristic of a wide range of neuropathies. Three neuron-specific monoclonal antibodies have been used to identify focal axonal swellings in the normal developing rat cerebellar cortex. Between 7 and 15 days postnatal, swellings are a common feature of the granular layer and white matter tracts. Using a Purkinje cell-specific antibody, the majority of swellings were shown to occur in Purkinje cell axons. Focal axonal swellings therefore seem to be a normal adjunct of Purkinje cell maturation.     

Serotonergic interactions with rat cerebellar Purkinje cells

Recent immunocytochemical and histofluorescent studies have established the vastness of the serotonergic fibers arising from the raphe complex and projecting to the cerebellar cortex. Ultrastructurally, the indoleaminergic fibers are known to establish synaptic contacts as well as diffuse meandering nonsynaptic terminations near Purkinje cells. The objective of this study was to define the changes elicited by serotonin on spontaneous and chemically- and electrically-evoked excitatory synaptic activities on cerebellar Purkinje cells. Serotonin (10-50 nA) applied for 30-60 sec elicited predominantly a decrease in firing rate followed by rebound excitation (25 cells out of a total of 39 cells). Studies in which serotonin was continuously iontophoresed for prolonged periods, this amine produced an increase in the spontaneous firing rate in 55% of the Purkinje cells tested. A comparison of evoked excitation and spontaneous activity revealed that when serotonin was applied continuously there was a net decrease in evoked excitation to spontaneous activity ratio. Based on the evoked excitation studies, glutamate (5-30 nA) was applied for 30-40 sec in the presence of serotonin (10-30 nA). Seventy-seven percent of the cells displayed a decrease in responsiveness to glutamate while the remaining 23% were potentiated. More definitive explanation of these results are given in the body of the test.     

Activity of cerebellar Purkinje cells during fictitious scratch reflex in the cat

The activity of Purkinje cells (PCs) was recorded in the anterior lobe (the vermis and pars intermedia) and in the paramedian lobule of the cerebellum during the fictitious scratch reflex in thalamic cats immobilized with Flaxedil. In the anterior lobe, the activity of many PCs was rhythmically modulated in relation to the scratch cycle: they generated bursts of impulses separated by periods of silence. Different PCs were active in different phases of the scratch cycle. In many cases the discharge modulation was irregular: the burst duration and the discharge rate in the burst varied considerably in subsequent cycles. The rhythmical activity of PCs was determined by modulation of the frequency of 'simple spikes' reflecting the mossy fiber input. Generation of 'complex spikes' reflecting the climbing fiber input in most PCs was not related with the scratch rhythm. In the paramedian lobule, rhythmical modulation of PCs was practically absent. Rhythmical modulation of PCs in immobilized cats is determined by signals coming from the central spinal mechanism of scratching via the ventral spinocerebellar tract (VSCT) and the spinoreticulocerebellar pathway (SRCP). Results of separate transections of these pathways demonstrated that the VSCT plays the crucial role in modulating the PCs.     

Role of 5-hydroxytryptamine expression in cerebellar Purkinje cells in obstructive sleep apnea syndrome

In the present study, electrical stimulation to the rat insular cortex induced apnea or respiratory disturbance, reduced amplitude of genioglossal electromyogram, and decreased electromyogram integrals. In addition, arterial blood gas analysis showed arterial blood acidosis, reduced pH values, increased alkali reserve negative values, decreased peripheral blood 5-hydroxytryptamine content, and increased 5-hydroxytryptamine expression in cerebellar Purkinje cells. Following lidocaine injection to block the habenular nucleus, abnormalities in breath, genioglossal electromyogram, and blood gas values disappeared, and peripheral blood 5-hydroxytryptamine content returned to levels prior to electric stimulation. However, 5-hydroxytryptamine expression in cerebellar Purkinje cells remained high. The results suggested that 5-hydroxytryptamine expression in Purkinje cells did not correlate with ventilation function involving insular cortex and habenular nucleus.     

Unravelling the Purkinje neuron

Purkinje cells, the first nerve cells to be described, are still one of the most interesting and useful experimental models to investigate different aspects of neural function. This special issue of The Cerebellum is dedicated to this very special neuron and contains a number of articles covering different topics of Purkinje cell research, from developmental neurobiology to neurophysiology and neuropathology. More than 150 years after their discovery, Purkinje cells remain one of the most popular neurons in the neuroscience community.     

Enhanced sensitivity of cerebellar Purkinje cells to iontophoretically-applied serotonin in thiamine deficiency

Electrophysiological aspects of thiamine depletion in the rat induced by dietary deficiency are described. Behavioral changes as well as qualitative and quantitative alterations in the sensitivity of cerebellar Purkinje cells to iontophoretically-applied 5-hydroxytryptamine (5-HT) were observed. Thiamine-deficient rats were characterized essentially by ataxia, piloerection, paresis, apparent weakness, and hypothermia after 4–6 weeks on a thiamine-free diet. Basal Purkinje cell firing frequency was unaffected by thiamine deficiency. The response of Purkinje cells to iontophoretically-applied 5-HT was solely inhibitory in deficient rats. In control rats, however, responses to 5-HT were excitatory, biphasic, or inhibitory. Neurons in the thiamine-deficient animals were more sensitive to the inhibitory effects of 5-HT, as demonstrated by a significant parallel shift to the left of the dose-response curve. Durations of 5-HT effects were similar in both groups. Dose-response relationships for GABA-induced inhibition of Purkinje cell firing from thiamine deficient and control rats did not differ from one another. These data demonstrate a relatively selective effect of thiamine depletion on cerebellar serotonergic neurotransmission assessed electrophysiologically. We believe there is up-regulation of 5-HT receptors on Purkinje cells caused by thiamine deficiency-induced impairment of indoleamine input to the cerebellum from raphe and related nuclei.     

Protein kinase C activity modulates dendritic differentiation of rat Purkinje cells in cerebellar slice cultures

The molecular mechanisms underlying dendritic differentiation in neurons are currently poorly understood. We used slice cultures from rat cerebellum of postnatal day 8 to investigate the effect of protein kinase C (PKC) activity on dendritic development of Purkinje cells. After 12 days in culture under control conditions, Purkinje cells had developed a typical dendritic tree consisting of a few long primary dendrites with shorter side branches. Following treatment with the PKC agonist, phorbol-12-myristate-13-acetate (PMA), the dendritic tree area was strongly reduced to 32% of control and primary dendrites were short with only a few side branches. Delayed addition of PMA after 6 days resulted in a retraction of existing dendrites, whereas discontinuation of PMA treatment after 6 days resulted in a recovery of the dendritic tree to almost control values. In the presence of the PKC inhibitor, 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide (GF109203X), the dendritic tree area was increased to 158% of control with much more ramified branches after 12 days. The overall morphology of the cultures and the survival of Purkinje cells were unaffected by PKC modulators. Our data show that increased activity of PKC inhibits, and reduced activity of PKC promotes dendritic growth. This suggests that PKC activity is a critical regulator of dendritic growth and differentiation in cerebellar Purkinje cells.