Caspase-3反义寡核苷酸抗凋亡作用的研究

目的 研究caspase-3反义寡核苷酸对6-OHDA诱导大鼠中脑多巴胺神经元凋亡的保护作用.方法 向四组SD大鼠的中脑黑质部位注入反义、错义、正义寡核苷酸及NS,然后再注入6-OHDA,取中脑做连续切片,原位杂交及免疫组化检测黑质caspase-3的表达及TH的表达,原位末端标记法(Tunel)检测黑质细胞的凋亡.结果 反义组Tunel阳性细胞数为82±8.6,方差分析显示反义组阳性细胞数显著性低于其他各组(P＜0.05);反义组手术侧TH阳性细胞数为168.6±11.4,与对侧阳性细胞比值为63%±11.3%,显著性高于其余各组(P＜0.05).结论 有效阻断caspase-3的表达可减轻6-OHDA诱导的多巴胺神经元凋亡,caspase-3可作为保护性治疗帕金森病的靶点.     

双靶点注射6-OHDA建立帕金森病大鼠模型并提高成功率

目的建立大鼠帕金森病模型,观测其行为学、中脑黑质多巴胺能神经元及超微结构的变化。方法利用立体定向技术,注射6-OHDA至大鼠中脑黑质SNc和中脑腹侧被盖区VTA,于注射后2、4、6、8周观测阿朴吗啡诱导的大鼠旋转行为学变化;采用免疫组化染色检测TH的表达,了解中脑黑质多巴胺能阳性神经元变化;利用透射电镜了解黑质区超微结构的变化。结果所有大鼠进行阿朴吗啡诱导旋转行为测试,旋转圈数>7r/min,并且>210r/30min,为合格的PD大鼠模型。大鼠模型于第4、6和8周时,大鼠行为学变化较为恒定。第8周时,50只大鼠中出现32只大鼠大鼠旋转次数平均为11.5±1.2/分,造模成功率为64%。PD模型大鼠超微结构观察,黑质神经元数目明显减少,线粒体肿胀,粗面内质网囊性扩张、脱颗粒,髓鞘扩张。免疫组化检测,成功模型光镜下分别计数双侧黑质TH阳性神经元,损毁侧黑质TH阳性神经元占正常侧的3.0%,即毁损侧TH阳性神经元减少97.0%。结论利用立体定向技术,选择黑质和中脑腹侧被盖区等双靶点,可建立6-OHDA大鼠PD模型,具有明确病理变化,提高模型成功率。     

6-羟基多巴定向注射建立帕金森病大鼠模型的实验研究

目的探讨立体定向间隔注射6-羟基多巴(6-OHDA)毁损黑质致密部(SNc)和中脑腹侧被盖(VTA)建立类似于人类帕金森病(PD)中晚期的PD大鼠模型方法。方法近交系Wistar大鼠50只,脑立体定向将6-OHDA注入大鼠右侧VTA及SNc,间隔两周,对阿朴吗啡(Apo)诱发旋转后旋转不明显或无稳定左侧旋转模型再次制模,并观察大鼠行为学改变,免疫组化检测黑质多巴胺(DA)能神经元的数量以及高效液相-荧光法检测黑质纹状体中DA含量的变化。结果(1)50只大鼠中有41只经APO诱导表现为恒定左侧旋转且结果稳定,旋转圈数>210r/30min,视为成功PD大鼠模型,部分大鼠伴有震颤、活动迟缓、嗅探、觅食、竖尾等异常行为改变,并且可持续存在16周;(2)免疫组化结果:PD大鼠模型毁损侧黑质区多巴胺能神经元较对侧及对照组减少大于90%;(3)PD大鼠右侧黑质纹状体中DA含量较左侧及对照组减少90%以上。结论6-OHDA毁损SNc及VTA间隔注射法可有效建立模拟人类PD中晚期的大鼠PD模型。     

6-羟基多巴胺单侧纹状体注射对大鼠双侧多巴胺能神经元的影响

目的探讨帕金森病大鼠模型中6-羟基多巴胺（6-OHDA）单侧纹状体注射对双侧黑质纹状体多巴胺能神经元的影响。方法大鼠随机分成模型组和对照组，模型组自一侧纹状体注射6-OHDA，对照组注射PBS；用免疫组织化学方法分别检测大鼠双侧黑质和纹状体区酪氨酸羟化酶（TH）阳性细胞和纤维的表达；高效液相色谱检测双侧纹状体多巴胺（DA）及其代谢产物3，4-二羟基苯乙酸（DOPAC）和高香草酸（HVA）含量。结果模型组大鼠双侧（毁损侧与其对侧）黑质致密区TH阳性细胞数量均少于对照组（P〈0．01），模型组双侧纹状体区TH阳性纤维密度均低于对照组；模型组大鼠双侧纹状体区DA含量均低于对照组（P〈0．01）；双侧DOPAC和HVA含量也降低。结论6-羟多巴胺单侧纹状体注射制作的帕金森病大鼠模型的对侧黑质纹状体也有损伤。     

早期损毁丘脑底核可阻止6—OHDA对大鼠多巴胺能神经元的损伤作用

目的 :观察不同时期毁损丘脑底核 (STN)对大鼠中脑黑质多巴胺 (DA)神经元 6 -羟基多巴胺 (6 OH DA)损伤的保护作用。方法 :将 6 0只Wistar大鼠随机分为 6组 ,每组 10只。对照组采用 6 -OHDA立体定向注入大鼠右侧前脑内侧束 (MFB)和中脑被盖腹侧区 (VTA) ,制成偏侧帕金森病 (PD)模型。实验组分为第Ⅰ、Ⅱ、Ⅲ、Ⅳ和Ⅴ组 ,分别于 6 OHDA注射前 7d、注射后 1h、2h、3d、7d5个不同时间点 ,局部注射海人藻酸 (KA)破坏STN。 4周后处死大鼠 ,采用免疫组化染色方法 ,定量测量各组大鼠黑质致密区 (SNc)区TH免疫阳性反应神经元数目。实验数据采用方差分析和t检验统计学处理。结果 :第Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ实验组以及对照组的注射侧TH神经元存活数目分别为 71.46± 6 .84、5 7.0 7± 5 .5 4、5 1.0 9± 4.85、12 .6 8± 2 .6 7、4.15± 1.6 0和 3.40± 1.5 4个 /每张切片 ,为对侧的 96 .7%、72 .9%、6 9.8%、17.2 %、5 .6 %及 4.4%。各实验组注射侧TH神经元均比对照组同侧数目多 (P <0 .0 1、0 .0 5 ) ,但V组无显著性差异 (P >0 .0 5 )。各组实验组之间比较 ,均有明显差异 ,其中以Ⅰ组TH神经元存活的数量最多 (P <0 .0 1)。结论 :早期毁损STN ,可减轻 6 -OHDA对SNc区DA能神经元的损伤和细胞数量的缺失 ,晚期毁损对     

重组人促红细胞生成素预处理对黑质多巴胺能神经元凋亡的影响

目的研究重组人促红细胞生成素(rhEPO)对离体帕金森病模型中黑质多巴胺神经元凋亡的影响。方法以6-羟基多巴胺(6-OHDA)为毁损剂建立大鼠离体帕金森病(PD)模型。用6u/mlrhEPO预处理黑质多巴胺神经元,然后用免疫组化方法观察黑质中酪氨酸羟化酶(TH)免疫反应阳性细胞数和半胱天冬酶-3(Caspase-3)免疫反应阳性细胞数的变化,TUNEL法观察黑质中多巴胺神经元的凋亡情况。结果与6-OHDA组(44.2±5.0)相比,rhEPO预处理组TH免疫反应阳性细胞(63.8±6.2,P<0.01)增多;与6-OHDA组(22.3±2.8)相比,rhEPO预处理组多巴胺神经元中Caspase-3表达减少,Caspase-3免疫反应阳性细胞染色较淡,数量减少(13.7±1.8,P<0.01);与6-OHDA组(20.3±3.1)相比,rhEPO预处理组TUNEL阳性细胞染色较淡,数量减少(10.7±1.5,P<0.01)。结论rhEPO预处理可以减轻6-OHDA对离体帕金森病模型中多巴胺神经元的损伤,其机制可能与rhEPO抑制黑质多巴胺神经元凋亡有关。     

同型半胱氨酸对多巴胺神经元的影响及其机制研究

目的研究同型半胱氨酸（homocysteine，Hcy）对帕金森病（PD）模型动物的影响及其机制。方法将63只大鼠随机分成3组，即吡咯烷二硫代氨基甲酸盐（PDTC）组27只、生理盐水对照组27只、假手术组9只。分别在实验前1h腹腔注射PDTC或生理盐水，以后每天1次，连续注射7d，通过脑立体定向注射6-羟多巴胺（6-OHDA）建立大鼠PD模型，2h后同侧脑立体定向注射Hcy或生理盐水，采用TUNEL法、免疫组化技术，选择实验后1d、7d及14d为研究时点，观察黑质多巴胺神经元数量、形态改变，黑质细胞凋亡数，以及黑质细胞NF-κB p65的阳性细胞数的变化。结果（1）局部注射Hcy能明显增加6-OHDA引起的黑质多巴胺神经元变性；（2）局部注射Hcy能明显增加6—OHDA引起的黑质细胞凋亡；（3）局部注射Hcy能明显增加黑质细胞NF—κB p65阳性细胞数；（4）PDTC可抑制Hcy和（或）6-0HDA引起的NF—κB p65活化，减少黑质细胞凋亡，增加多巴胺神经元数目。结论NF—κB p65的激活是Hcy促进6-OHDA引起的黑质多巴胺神经元变性及黑质细胞凋亡机制中的重要因素之一，PDTC可显著抑制NF—κB p65的活化，多巴胺神经元变性和黑质细胞凋亡。     

帕金森病样大鼠模型的稳定性观察

6-OHDA损毁大鼠一侧中脑黑质细胞造成偏侧帕金森病(PD)样大鼠模型,用小动物旋转行为记录仪记录阿朴吗啡诱发大鼠的旋转行为;TH免疫细胞化学染色观察中脑黑质细胞的损毁状况.连续观察10个月其旋转行为无自发性恢复;PD样模型大鼠损毁侧中脑无TH阳性细胞存在.结果表明6-OHDA损毁大鼠一侧中脑黑质细胞可造成稳定、可靠的类似PD病人病理变化的偏侧动物模型.     

损毁丘脑底核阻止6-OHDA对大鼠多巴胺能神经元的损伤作用

目的 观察不同时期毁损丘脑底核对大鼠中脑黑质多巴胺神经元6－羟基多巴胺（6－OHDA）损伤的保护作用。方法 将60只Wistar大鼠随机分为6组,每组10只。对照组采用6－OHDA立体定向注入大鼠右侧前脑内侧束（MFB）和中脑被盖腹侧区（VTA）,制成偏侧帕金森病（PD）模型。实验组分为第Ⅰ、Ⅱ、Ⅲ、Ⅳ和Ⅴ组,分别于6－OHDA注射前7d、注射后1h、2h、3d、7d 5个不同时间点,局部注射海藻氨酸（KA）破坏STN。4周后处死大鼠,采用免疫组化染色方法,定量测量各组大鼠黑质致密区（SNc）区TH免疫阳性反应神经元数目。实验数据采用方差分析和t检验统计学处理。结果 第Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ实验组以及对照组的注射侧TH神经元存活数目分别为71．46±6．84、57．07±5．54、51．09±4．85、12．68±2．67、4．15±1．60和3．40±1．54个每张切片,为对侧的96．7％、72．9％、69．8％、17．2％、5．6％及4．4％。各实验组注射侧TH神经元均比对照组同侧数目多（P ＜ 0．05）,但Ⅴ组无显著性差异（P ＞0．05）。各实验组之间比较,均有明显差异,其中以Ⅰ组TH神经元存活的数量最多（P ＜0．01）。结论 早期毁损丘脑底核（STN）可减轻6－OHDA对黑质致密部多巴胺（DA）能神经元的损伤和细胞数量的缺失;晚期毁损对其无明显     

脑源性神经营养因子对帕金森病大鼠多巴胺能神经元的影响

目的观察脑源性神经营养因子对帕金森病(Parkinson’s disease,PD)大鼠模型黑质多巴胺能神经元的影响。方法选用Wistar种系大白鼠30只,体质量230~250g,随机分3组,通过左侧中脑黑质立体定向注射法,组1为生理盐水对照组(简称对照组)10只,注射相应量(5μL)的生理盐水;组2为注射6-OHDA制作帕金森病模型组(简称6-OHDA组)10只,注射6-OHDA,5μL(2μg/μL);组3为(6-OHDA+BDNF)组,在制成帕金森病模型后再向同侧中脑黑质注射BDNF 5μL(3μg/5μL),连续6d,1次/d。分别观察动物的旋转行为,免疫组化染色方法观察黑质酪氨酸羟化酶(tyrosine hydroxylase,TH)阳性神经元的数量,高效液相法测定纹状体部多巴胺(dopamine,DA)含量的变化。结果单侧黑质内注入6-OHDA制成帕金森病大鼠模型后,6-OHDA组与对照组比较,产生旋转行为,(6-OHDA+BDNF)组在观察旋转行为时,症状明显改善;镜下见TH阳性神经元主要见于对照组的黑质致密部,数量为(42.3±7.56)个/μm2,模型组黑质致密部TH阳性神经元数明显减少为(2.41±1.07)个/μm2,(6-OHDA+BDNF)组黑质致密部TH阳性神经元数为(15.36+3.04)个/μm2;纹状体部多巴胺含量:生理盐水组为(11.4±1.2)μg/g,6-OHDA组(3.6±0.5)μg/g,(6-OHDA+BDNF)组(5.5±0.6)μg/g。结论 BDNF能改善6-OHDA所致的帕金森病大鼠黑质多巴胺能神经元数目的减少;明显抑制6-OHDA引起的纹状体部多巴胺含量降低;并可抑制6-OHDA对黑质多巴胺能神经元的毒性作用。     

Differential contribution of Ih to the integration of excitatory synaptic inputs in substantia nigra pars compacta and ventral tegmental area dopaminergic neurons

The selective vulnerability of substantia nigra pars compacta (SNc) dopaminergic (DA) neurons is an enigmatic trait of Parkinson's disease (PD), especially if compared to the remarkable resistance of closely related DA neurons in the neighboring ventral tegmental area (VTA). Overall evidence indicates that specific electrophysiological, metabolic and molecular factors underlie SNc vulnerability, although many pieces of the puzzle are still missing. In this respect, we recently demonstrated that 1‐methyl‐4‐phenylpyridinium (MPP+), the active metabolite of the parkinsonizing toxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), alters the electrophysiological properties of SNc DA neurons in vitro by inhibiting the hyperpolarization‐activated current (Ih). Here, we present an electrophysiological investigation of the functional role of Ih in the integration of synaptic inputs in identified SNc and VTA DA neurons, comparatively, in acute midbrain slices from TH‐GFP mice. We show that pharmacological suppression of Ih increases the amplitude and decay time of excitatory postsynaptic potentials, leading to temporal summation of multiple excitatory potentials at somatic level. Importantly, these effects are quantitatively more evident in SNc DA neurons. We conclude that Ih regulates the responsiveness to excitatory synaptic transmission in SNc and VTA DA neurons differentially. Finally, we present the hypothesis that Ih loss of function may be linked to PD trigger mechanisms, such as mitochondrial failure and ATP depletion, and act in concert with SNc‐specific synaptic connectivity to promote selective vulnerability.     

Nociceptin/orphanin FQ and opioid receptor-like receptor mRNA expression in dopamine systems

Although nociceptin/orphanin FQ (N/OFQ) influences dopamine (DA) neuronal activity, it is not known whether N/OFQ acts directly on DA neurons, indirectly by means of local circuitry, or both. We used two parallel approaches, dual in situ hybridization (ISH) and neurotoxic lesions of DA neurons by using 6-hydroxydopamine (6-OHDA), to ascertain whether N/OFQ and the N/OFQ receptor (NOP) mRNA are expressed in DA neurons in the ventral tegmental area (VTA) and substantia nigra compacta (SNc). In the VTA and SNc, small populations (approximately 6-10%) of N/OFQ-containing neurons coexpressed mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme for DA synthesis. Similarly, very few (1-2%) TH-positive neurons contained N/OFQ mRNA signal. A majority of NOP-positive neurons (approximately 75%) expressed TH mRNA and roughly half of the TH-containing neurons expressed NOP mRNA. Many N/OFQ neurons (approximately 50-60%) expressed glutamic acid decarboxylase 65 and 67 mRNAs, markers for gamma-aminobutyric acid (GABA) neurons. In the 6-OHDA lesion studies, NOP mRNA levels were nearly 80 and 85% lower in the VTA and SNc, respectively, on the lesioned side. These lesions appear to lead to compensatory changes, with N/OFQ mRNA levels approximately 60% and 300% higher in the VTA and SNc, respectively, after 6-OHDA lesions. Finally, N/OFQ-stimulated [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate levels were decreased in the VTA and SNc but not the prefrontal cortex after 6-OHDA lesions. Accordingly, it appears that N/OFQ mRNA was found largely on nondopaminergic (i.e., GABA) neurons, whereas NOP mRNA was located on DA neurons. N/OFQ is in a position to influence DA neuronal activity by means of the NOP located on DA neurons.     

SK channel function regulates the dopamine phenotype of neurons in the substantia nigra pars compacta

Parkinson's disease (PD) is characterized by loss of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNc). It is widely believed that replacing lost SNc DA neurons is a key to longer-term effective treatment of PD motor symptoms, but generating new SNc DA neurons in PD patients has proven difficult. Following loss of tyrosine hydroxylase-positive (TH+) SNc neurons in the rodent 6-hydroxy-DA (6-OHDA) model of PD, the number of TH+ neurons partially recovers and there is evidence this occurs via phenotype “shift” from TH− to TH+ cells. Understanding how this putative phenotype shift occurs may help increase SNc DAergic neurons in PD patients. In this study we characterize the electrophysiology of SNc TH− and TH+ cells during recovery from 6-OHDA in mice. Three distinct phenotypes were observed: (1) TH− were fast discharging with a short duration action potential (AP), short afterhyperpolarization (AHP) and no small conductance Ca²⁺-activated K⁺ (SK) current; (2) TH+ were slow discharging with a long AP, long AHP and prominent SK current; and (3) cells with features “intermediate” between these TH− and TH+ phenotypes. The same 3 phenotypes were present also in the normal and D2 DA receptor knock-out SNc suggesting they are more closely related to the biology of TH expression than recovery from 6-OHDA. Acute inhibition of SK channel function shifted the electrophysiological phenotype of TH+ neurons toward TH− and chronic (2 weeks) inhibition of SK channel function in normal mice shifted the neurochemical phenotype of SNc from TH+ to TH− (i.e. decreased TH+ and increased TH− cell numbers). Importantly, chronic facilitation of SK channel function shifted the neurochemical phenotype of SNc from TH− to TH+ (i.e. increased TH+ and decreased TH− cell numbers). We conclude that SK channel function bidirectionally regulates the DA phenotype of SNc cells and facilitation of SK channels may be a novel way to increase the number of SNc DAergic neurons in PD patients.     

Somatodendritic ion channel expression in substantia nigra pars compacta dopaminergic neurons across postnatal development

Dopaminergic neurons of the substantia nigra pars compacta (SNc) are involved in the control of movement, sleep, reward, learning, and nervous system disorders and disease. To date, a thorough characterization of the ion channel phenotype of this important neuronal population is lacking. Using immunohistochemistry, we analyzed the somatodendritic expression of voltage‐gated ion channel subunits that are involved in pacemaking activity in SNc dopaminergic neurons in 6‐, 21‐, and 40‐day‐old rats. Our results demonstrate that the same complement of somatodendritic ion channels is present in SNc dopaminergic neurons from P6 to P40. The major developmental changes were an increase in the dendritic range of the immunolabeling for the HCN, T‐type calcium, Kv4.3, delayed rectifier, and SK channels. Our study sheds light on the ion channel subunits that contribute to the somatodendritic delayed rectifier (Kv1.3, Kv2.1, Kv3.2, Kv3.3), A‐type (Kv4.3) and calcium‐activated SK (SK1, SK2, SK3) potassium currents, I_H (mainly HCN2, HCN4), and the L‐ (Cav1.2, Cav1.3) and T‐type (mainly Cav3.1, Cav3.3) calcium currents in SNc dopaminergic neurons. Finally, no robust differences in voltage‐gated ion channel immunolabeling were observed across the population of SNc dopaminergic neurons for each age examined, suggesting that differing levels of individual ion channels are unlikely to distinguish between specific subpopulations of SNc dopaminergic neurons. This is significant in light of previous studies suggesting that age‐ or region‐associated variations in the expression profile of voltage‐gated ion channels in SNc dopaminergic neurons may underlie their vulnerability to dysfunction and disease. © 2014 Wiley Periodicals, Inc.     

Consequences of partial and severe dopaminergic lesion on basal ganglia oscillatory activity and akinesia

Severe chronic dopamine (DA) depletion increases the proportion of neurons in the basal ganglia that fire rhythmic bursts of action potential (LFO units) synchronously with the cortical oscillations. Here we report on how different levels of mesencephalic DA denervation affect substantia nigra pars reticulata (SNpr) neuronal activity in the rat and its relationship to akinesia (stepping test). Chronic nigrostriatal lesion induced with 0 (control group), 4, 6 or 8 microg of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle resulted in a dose-dependent decrease of tyrosine hydroxylase positive (TH+) neurons in the SN and ventral tegmental area (VTA). Although 4 microg of 6-OHDA reduced the number of TH+ neurons in the SN by approximately 60%, both stepping test performance and SNpr neuronal activity remained indistinguishable from control animals. By contrast, animals that received 6 microg of 6-OHDA showed a marked reduction of TH+ cells in the SN ( approximately 75%) and VTA ( approximately 55%), a significant stepping test deficit and an increased proportion of LFO units. These changes were not dramatically enhanced with 8 microg 6-OHDA, a dose that induced an extensive DA lesion (> 95%) in the SN and approximately 70% reduction of DA neurons in the VTA. These results suggest a threshold level of DA denervation for both the appearance of motor deficits and LFO units. Thus, the presence of LFO activity in the SNpr is not related to a complete nigrostriatal DA neuron depletion (ultimate stage parkinsonism); instead, it may reflect a functional disruption of cortico-basal ganglia dynamics associated with clinically relevant stages of the disease.     

Increased neuronal responsiveness to cholecystokinin and dopamine induced by lesioning mesolimbic dopaminergic neurons: an electrophysiological study in the rat

In the rat, cholecystokinin (CCK) and dopamine (DA) coexist in a subpopulation of neurons of the ventral tegmental area (VTA) projecting to the nucleus accumbens. However, in the dorsal hippocampus, dopaminergic projections from the VTA do not contain CCK, the latter neurotransmitter being mainly localized in intrinsic hippocampal neurons. The present experiments were undertaken in order to compare the interactions of CCK and DA and the effects of lesioning VTA dopaminergic neurons in a region where these neurotransmitters coexist and in one where they do not. The effects of microiontophoretic applications of CCK, kainate (KA), glutamate (GLU) and DA were determined in control rats and in rats pretreated with a local injection of 6-hydroxydopamine (6-OHDA) in the VTA. In the nucleus accumbens and in the hippocampus of intact rats, DA exerted a similar depressant effect whether applied during CCK-, KA- or GLU-induced activations. The 6-OHDA lesion enhanced responsiveness of accumbens neurons to KA, GLU and CCK (the responsiveness to this latter peptide being increased by more than 15-fold) and the depressant effect of DA when applied during neuronal activation by KA or GLU but not when the same neurons were activated with CCK. In the dorsal hippocampus, the 6-OHDA lesion enhanced neuronal responsiveness to KA and DA in the CA1, but not in the CA3 region, whereas the responsiveness to CCK remained unchanged in both regions. These results suggest a physiological role for the coexistence of CCK and DA in the nucleus accumbens. The induction of a supersensitivity to DA in the CA1, but not in the CA3, region of the dorsal hippocampus following a VTA lesion is consistent with the regional distribution of the dopaminergic innervation in this structure.     

Generation and survival of midbrain dopaminergic neurons in weaver mice

Generation and survival of midbrain dopaminergic (DA) neurons were investigated using tyrosine hydroxylase (TH) immunocytochemistry combined with tritiated thymidine autoradiography at appropriate anatomical levels throughout the anteroposterior (A/P) axes of the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA). The wild-type (+/+) and homozygous weaver (wv/wv) mice used here were the offspring of pregnant dams injected with the radioactive precursor when the mesencephalic neurons were being produced (gestational days 11-15). Data reveal that, at postnatal day 90, depletion of TH-stained cells in the wv/wv presented an A/P pattern of increasing severity and, therefore, the DA cells located in posterior parts of the SNc or the VTA appear to be more vulnerable than the settled anterior neurons. When the time of neuron origin is inferred for each level of these cell groups, it is found that the neurogenesis span is similar for both experimental groups, although significant deficits in the frequency of wv/wv late-generated neurons were observed in any level considered. On the other hand, it has been found that TH-positive neurons were settled along the extent of the SNc and the VTA following precise and differential neurogenetic gradients. Thus, the acute rostrocaudal increase in the proportion of late-generated neurons detected in both+/+DA-cell groups is disturbed in the weaver homozygotes due to the indicated A/P depletion.     

Cellular localization of tyrosine hydroxylase mRNA and cholecystokinin mRNA-containing cells in the ventral mesencephalon of the common marmoset: effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

In situ hybridization histochemistry was used to localize tyrosine hydroxylase (TH) mRNA and cholecystokinin (CCK) mRNA-expressing cells in the ventral mesencephalon of the common marmoset (Callithrix jacchus) and to examine the effects of the dopaminergic (DA) neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on these two populations of neurons in the pars compacta of the substantia nigra (SNc) and ventral tegmental area (VTA). X-ray film and liquid emulsion autoradiography of brain sections hybridized with an 35S-labelled synthetic 45-mer antisense human TH oligonucleotide probe showed strong hybridization signals and dense populations of TH mRNA expressing cells in the SNc and VTA at all levels, in the control marmoset brain. In the MPTP-treated brain, there was a substantial reduction of TH mRNA in the ventral midbrain. The loss of TH mRNA-expressing cells amounted to 98% in the lateral SNc, 88% in the medial SNc and 33% in the VTA. In situ hybridization of adjacent sections with an 35S-labelled synthetic 45-mer antisense human CCK oligonucleotide probe showed a weak hybridization signal for CCK mRNA in the ventral midbrain of the control brain. Emulsion autoradiography demonstrated CCK mRNA expressing cells in the SNc and VTA at all levels with the number of cells in the VTA similar to that for TH mRNA. However, the number of cells in the SNc expressing CCK mRNA was a fraction (1/4) of that expressing TH mRNA; moreover, the level of expression per cell was substantially less than that for TH mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-terminal tripeptide of IGF-1 (GPE) prevents the loss of TH positive neurons after 6-OHDA induced nigral lesion in rats

The effect of the N-terminal tripeptide of insulin-like growth factor (IGF)-1, glycine-proline-glutamate (GPE), as a neuroprotective agent for nigro-striatal dopaminergic neurons was examined in the present study using a rat model of Parkinson's disease. A unilateral nigro-striatal lesion was induced in rats by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle (MFB). GPE (3 microgram) or its vehicle was administered intracerebroventricularly (i.c.v.) 2 h after the 6-OHDA lesion. Tyrosine-hydroxylase (TH) immunohistochemistry in the substantia nigra compacta (SNc) and the striatum were examined 2 weeks after the lesion. Following 6-OHDA injection, the number of TH immunopositive neurons in the ipsilateral SNc was reduced. The density of TH immunostaining was also reduced in the ipsilateral SNc and the striatum. Treatment with a single dose of GPE (n=9) significantly prevented the loss of TH immunopositive neurons (p<0. 001) and restored the TH immunoreactivity in both the SNc and the striatum compared with the vehicle control group (n=9, p<0.001). The results suggest that GPE showed promise as a potential treatment for Parkinson's disease.