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首页> 外文学位 >Phenotypic and immunohistochemical characterization of conditional knockout mice with a deletion in glutamic acid decarboxylase (GAD) in Gpr88 containing neurons and the role of striatal gad in L-Dopa induced dyskinesia.
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Phenotypic and immunohistochemical characterization of conditional knockout mice with a deletion in glutamic acid decarboxylase (GAD) in Gpr88 containing neurons and the role of striatal gad in L-Dopa induced dyskinesia.

机译：表型和免疫组化特征的条件性基因敲除小鼠，其中含有神经元的Gpr88中的谷氨酸脱羧酶（GAD）缺失以及纹状体在L-多巴引起的运动障碍中的作用。

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摘要

Glutamic Acid Decarboxylase (GAD) is a rate-limiting enzyme responsible for synthesis of the inhibitory neurotransmitter GABA. Dopaminergic denervation in rodents by unilateral injections of 6-OHDA or MPTP causes an increase in Gad67 mRNA in the striatum, which is further exacerbated by administration of L-Dopa (Horvath et al., 2011; Katz et al., 2005 Bacci et al., 2002). Denervation of nigrostriatal neurons is the key pathological hallmark of Parkinson's disease, which results in hypokinetic movement and rigidity. Medium spiny projection neurons of the striatum comprise 95% of the neuronal population and utilize Gad67 (encoded by the Gad1 gene) for the synthesis of basal levels of GABA. The contribution of Gad67 to GABA signaling in medium spiny projection neurons in the striatum has not been thoroughly understood in normal or Parkinsonian states. Mice with a deletion in Gad67 in Gpr88 expressing neurons were generated by crossing mice with a floxed exon 2 of Gad1 with mice expressing Cre recombinase under the control of the Gpr88 promoter. The aim of this study was first, to characterize mice with a deletion in striatal Gad67 by immunohistochmical, electriophysiological and behavioral examination to determine whether Gad67 expression contributes to sensorimotor and learning tasks. And next, to investigate whether a downregulation in striatal Gad67 would decrease dyskinesia and affect the impaired motor symptoms following dopaminergic denervation with a unilateral 6-OHDA lesion and subsequent treatment with L-Dopa. In this study, neuronal Gpr88 expression was indicated by GFP reporter expression, which resulted from Cre-mediated excision of exon 2 of the Gad1 gene. Gpr88 expression was confirmed in the striatum, olfactory tubercle, cortex and brain stem. Furthermore, Gpr88 was confined to striatonigral and striatopallidal MSNs in the striatum. Additionally, Cre-mediated GFP reporter expression indicated that Gpr88 expression occurs throughout various brain regions, including the motor and visual areas of the cortex, amygdala, hippocampus and cerebellum during development. The developmental expression of Gpr88 seems to be a highly regulated process that occurs throughout the brain. In the conditional knockout mouse, deleting striatal Gad67 resulted in an upregualtion of Gad67 in the globus pallidus and downregulation in the substantia nigra. The changes in Gad67 expression indicate the effects of inactivating GABAergic signaling in striatonigral and striatopallidal MSNs in the direct and indirect pathways. Mice with a deletion in striatal Gad67 demonstrated compromised performance in spatial learning in the Morris water maze, suggesting that GABAergic striatal signaling in the direct and indirect pathways accounts for cue-based learning and spatial memory. However, inactivation of GABAergic signaling in striatonigral and striatopallidal MSNs does not account for motor deficits such as bradykinesia, akinesia or hypokinesia in intact mice; instead it perpetuates hyperkinetic motor activity. In the second experiment of this study, dopaminergic denervation by a unilateral 6-OHDA lesion induced bradykinesia and hypokinetic motor behavior, as demonstrated by impaired performance in the rota-rod and pole test. Additionally, L-Dopa administration to 6-OHDA lesioned mice evoked abnormal involuntary movements (AIMs) to the same degree in all dyskinetic mice. A deletion in striatal Gad67 did not decrease symptoms of dyskinesia, nor cause a lessening of motor impairment caused by dopaminergic denervation. Complete inactivation of the indirect pathway is believed to limit the inhibition of unwanted actions and may perpetuate dyskinesia, even when striatonigral MSNs of the direct pathway are inactive.
机译：谷氨酸脱羧酶（GAD）是限速酶，负责合成抑制性神经递质GABA。通过单侧注射6-OHDA或MPTP引起的啮齿动物多巴胺能神经支配引起纹状体中Gad67 mRNA的增加，而通过施用L-Dopa会进一步加剧（Horvath等人，2011； Katz等人，2005 Bacci等人） （2002年）。黑纹状体神经元的去神经支配是帕金森氏病的关键病理标志，导致运动不足和僵硬。纹状体的中型多刺投射神经元占神经元种群的95％，并利用Gad67（由Gad1基因编码）来合成基础水平的GABA。在正常或帕金森病状态下，尚未完全了解Gad67对纹状体中段多刺投射神经元中GABA信号的贡献。在具有Gpr88启动子的控制下，通过将具有Gad1的外显子2浮雕的小鼠与表达Cre重组酶的小鼠杂交，来生成Gpr88表达神经元中Gad67缺失的小鼠。这项研究的目的是首先通过免疫组织化学，电生理和行为检查来鉴定纹状体Gad67缺失的小鼠，以确定Gad67表达是否有助于感觉运动和学习任务。接下来，研究在单侧6-OHDA损伤多巴胺能神经支配后，L-Dopa治疗后纹状体Gad67的下调是否会减少运动障碍并影响运动症状的减弱。在这项研究中，神经元Gpr88表达由GFP报告基因表达指示，这是由Cre介导的Gad1基因外显子2切除所致。在纹状体，嗅结节，皮层和脑干中证实了Gpr88表达。此外，Gpr88仅限于纹状体的纹状体黑质和纹状体的MSNs。此外，Cre介导的GFP报告基因表达表明，Gpr88表达遍布于整个大脑区域，包括发育过程中皮质，杏仁核，海马和小脑的运动和视觉区域。 Gpr88的发育表达似乎是一个高度调节的过程，发生在整个大脑中。在条件基因敲除小鼠中，删除纹状体Gad67会导致苍白球Gad67上调，而黑质则下调。 Gad67表达的变化表明在直接途径和间接途径中，纹状体黑质和纹状体异常MSN中GABA能信号的失活作用。纹状体Gad67缺失的小鼠在莫里斯水迷宫中的空间学习中表现出受损的表现，这表明直接和间接途径中的GABA能性纹状体信号转导了基于提示的学习和空间记忆。然而，在纹状体顶和纹状体顶体MSN中，GABA能信号的失活不能解释运动缺陷，例如完整小鼠的运动迟缓，运动障碍或运动不足。相反，它使运动亢进运动永久存在。在这项研究的第二个实验中，单侧6-OHDA损伤引起的多巴胺能神经支配引起运动迟缓和运动功能低下的运动行为，这在旋转杆和杆试验中表现受损。另外，对6-OHDA损伤的小鼠进行L-Dopa给药在所有运动障碍小鼠中均引起了异常的非自愿运动（AIM）。纹状体Gad67的缺失不会减轻运动障碍的症状，也不会减轻由多巴胺能神经支配引起的运动障碍。间接途径的完全失活被认为限制了对有害作用的抑制，并且即使当直接途径的纹状体MSN失活时，也可能使运动障碍长期存在。

著录项

  • 作者

    Labak, Samantha.;

  • 作者单位

    Boston University.;

  • 授予单位 Boston University.;
  • 学科 Biology Neuroscience.;Agriculture Animal Pathology.;Psychology Behavioral.
  • 学位 M.S.
  • 年度 2014
  • 页码 110 p.
  • 总页数 110
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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