Plasticity of bridge collaterals in Parkinonian state and treatment

 描述（由申请人提供）：在帕金森病（PD）中，多巴胺能神经元变性导致的症状包括运动缓慢、僵硬、震颤和步态异常。多巴胺（DA）前体左旋多巴（L-DOPA）显著减轻运动症状，但在长期治疗后可引起副作用，例如L-DOPA诱导的运动障碍（LID），从而限制其治疗用途。PD中运动缺陷和LID中过度运动的关键介质是纹状体中棘神经元（MSNs），其形成纹状体的唯一输出并在正常条件下接收DA输入。PD时DA输入不足，慢性左旋多巴后DA输入过多，导致MSN功能异常，分别导致运动迟缓和LID。经典的基底神经节（BG）模型认为MSNs可分为两条独立的通路：i）D1-MSNs，表达多巴胺D1受体（D1 Rs），形成“直接通路”，仅靶向BG的输出核团; ii）D2-MSN，其表达D2受体（D2 R）并形成“间接途径”，经由外部苍白球（GPe）间接靶向BG的输出核。然而，随后的解剖学研究发现，来自D1-MSNs的轴突形成也投射到GPe的侧枝。因此，这些“桥接侧支”允许D1-MSN影响直接和间接途径的靶标。我们最近发现，这些桥接侧枝的末端密度是高度可塑性的，并随着DA信号的改变而改变。D-1 MSN桥接侧支被慢性D2 R阻断减少，但被D2 R过表达几乎加倍。重要的是，桥接侧支密度的变化显著改变了D1-MSN对运动输出的影响。虽然D1-MSNs的光遗传学刺激通常增加运动行为，但通过D2 R过表达增加桥接侧枝引起相同的刺激以抑制运动行为。通过慢性D2 R阻滞使桥接侧支末梢密度正常化，将这种运动抑制逆转回运动激活。这里提出的实验将测试这一假设，即桥接侧枝也被PD和LID中的异常DA信号改变。我们将使用在Drd 1a启动子控制下表达绿色荧光蛋白（GFP）的转基因小鼠，这使得D1-MSN的轴突投射可以通过针对GFP的免疫组织化学来可视化。因此，D1- MSN桥接侧支的末端密度可以通过GPe中的GFP染色强度来确定。通过6-羟基多巴胺的DA去神经支配将用于诱导偏侧帕金森症。肢体使用偏倚将用于评估运动不能，LID将由长期左旋多巴治疗诱导。我们建议，DA耗竭将逐步减少桥接侧支末梢密度，这将平行的渐进能力的D1 R激动剂逆转运动不能。此外，我们认为，长期左旋多巴治疗，这会导致LID的严重程度增加，将与进一步改变的终端密度的桥接侧支。PD中异常桥接侧支变化的识别将证明未来开发逆转这些变化的治疗是合理的。

 DESCRIPTION (provided by applicant): In Parkinson's disease (PD), degeneration of dopaminergic neurons leads to symptoms including slowness of movement, rigidity, tremor, and gait abnormality. The dopamine (DA) precursor levodopa (L-DOPA) dramatically alleviates motor symptoms, but can cause side effects such as L-DOPA-induced dyskinesia (LID) after prolonged treatment, thereby limiting its therapeutic use. A critical mediator of motor deficit in PD and excessive movement in LID is the striatal medium spiny neurons (MSNs), which form the sole output of the striatum and receive DA input under normal conditions. The lack of DA input in PD, and the excessive, non-physiological DA input after chronic L-DOPA leads to abnormal MSN function and contribute to slowness of movement and LID respectively. The classical model of basal ganglia (BG) proposes that MSNs can be divided into two segregated pathways: i) D1-MSNs, which express dopamine D1 receptors (D1Rs) and form the "direct pathway", targeting only the output nuclei of BG; ii) D2-MSNs, which express D2 receptors (D2Rs) and form the "indirect pathway", targeting the output nuclei of BG indirectly via the external globus pallidus (GPe). However, subsequent anatomical studies found that axons from D1-MSNs form collaterals that also project to the GPe. These "bridging collaterals" thus allow D1-MSNs to influence the targets of both direct and indirect pathway. We recently found that the terminal density of these bridging collaterals is highly plastic and changes in response to alterations in DA signaling. D-1MSN bridging collateral is reduced by chronic D2R blockade but is almost doubled by D2R overexpression. Importantly, changes in bridging collateral density dramatically alter D1-MSN's influence on motor output. While optogenetic stimulation of D1-MSNs normally increases locomotor behavior, increasing bridging collaterals by D2R overexpression caused the same stimulation to inhibit locomotor behavior. This motor inhibition was reversed back to motor activation by normalizing bridging collateral terminal density via chronic D2R blockade. Experiments proposed here will test the hypothesis that bridging collaterals are also altered by abnormal DA signaling in PD and LID. We will use transgenic mice that express green fluorescent protein (GFP) under the control of the Drd1a promoter, which allows the axonal projections of D1-MSNs to be visualized by immunohistochemistry against GFP. The terminal density of D1- MSN bridging collateral can therefore be determined by GFP staining intensity in the GPe. DA denervation by 6-hydroxydopamine will be used to induce hemi-parkinsonism. Limb-use bias will be used to assess akinesia, and LID will be induced by chronic L-DOPA treatment. We propose that DA depletion will progressively reduce bridging collateral terminal density, and that it would parallel the progressive ability of a D1R agonist to reverse akinesia. Furthermore, we propose that chronic L-DOPA treatment, which causes increases in LID severity, will be associated with further alterations in the terminal density of bridging collaterals. The identification of abnormal bridging collateral changes in PD will justify future development of therapy to reverse these changes.