Experience-Dependent Enhancement of Neuronal Integration

描述(由申请人提供)： 该实验的目的是提高受损或变性的大脑神经元回路的修复效率，重点关注成年小鼠大脑皮质或皮质-丘脑神经元的在体恢复。未成熟的神经元和神经前体将被移植到皮质中，经历一种明确和可控的选择性神经元的靶向性变性(神经变性)。这种凋亡性神经元变性将由靶向分子的光激活在生物物理上诱导，在体内仅对所需的神经元亚群造成特定的变性。在这个项目中，新神经元的存活和分化将受到感觉经验的调节，这将在成年小鼠的躯体感觉皮质产生活动依赖的可塑性变化。Macklis教授的团队已经证明，在选定的神经元有针对性地退化后，未成熟的神经母细胞可以被移植到大脑皮层中来取代神经元。这些新整合的细胞可以正确地分化为正确类型的神经元，重新建立适当的远距离投射，发展成熟的表型，并与受体电路建立适当的连接。Macklis实验室还首次表明，可以在成年小鼠的新皮质中诱导神经发生，从而可以从原位操纵的内源性前体中招募新的神经元，而不需要移植。据推测，靶向性神经细胞死亡产生了负责神经元定向迁移的发育信号的局部重新表达， 移植的神经母细胞或移植的或内源性的前体细胞的分化和连通性。与这些结果相补充的是，越来越多的文献证据表明，在完整的成年小鼠大脑中观察到的海马体和嗅球的内源性神经元增殖和分化(神经发生)可以由经验调节。在拟议的研究项目中，将评估是否可以通过实验操作小鼠躯体感觉皮质中的神经母细胞来改善其回路活动的分化、整合和在回路内存活的有效性。这项拟议的研究将评估通过感觉刺激和感觉剥夺诱导成人躯体感觉皮质活动依赖的可塑性变化的效果。将动物安置在全新的“触觉丰富”区域(丰富的环境)或通过强烈的触觉胡须的强迫运动(触须的机械刺激)，将产生强烈的、持久的感觉刺激。感觉剥夺将基于长时间的触觉胡须修剪或触觉切除(限制感觉输入到皮质)。在经历实验诱导的神经变性和一系列刺激依赖性激活的成人皮质中，移植的神经母细胞对存活、分化和整合的影响将通过在Macklis实验室建立的一系列成熟的免疫细胞化学、神经解剖学和遗传示踪方法来研究，并使用荧光和共聚焦显微镜进行分析，以评估细胞整合、突触电路形成和跨突触功能激活。

DESCRIPTION (provided by applicant): The objective of the proposed experiments is to increase the repair efficiency of damaged or degenerated neuronal circuits in the brain, focusing the interest on restoration of corticocortical or cortico-thalamic neurons of adult mice in vivo. Immature neurons and neural precursors will be transplanted in the cortex undergoing a defined and controllable targeted degeneration of selective neurons (neurodegeneration). This apoptotic neuronal degeneration will be biophysically induced by photoactivation of targeting molecules, causing specific degeneration only to desired subpopulations of neurons in vivo. In this project, survival and differentiation of new neurons will be modulated by sensory experience, which produces activity-dependent plastic changes in the somatosensory cortex of adult mice. The group of Prof. Macklis has shown that neurons can be replaced by immature neuroblasts transplanted into the cerebral cortex after targeted degeneration of selected neurons. These newly incorporated cells can differentiate appropriately into precisely the right kind of neuron, re-establish appropriate long-distance projections, develop a mature phenotype, and make appropriate connections with recipient circuitry. The Macklis lab also showed, for the first time, that neurogenesis can be induced in the adult mouse neocortex, and new neurons can thereby be recruited from endogenous precursors manipulated in situ, without transplantation. It is postulated that targeted neuronal cell death produces local reexpression of developmental signals responsible for directed neuronal migration, differentiation, and connectivity of transplanted neuroblasts, or either transplanted or endogenous precursors. Complementary to these results, there is growing evidence in the literature suggesting that endogenous proliferation and differentiation of neurons (neurogenesis), which in the intact adult mouse brain is observed to the hippocampus and olfactory bulb, can be regulated by experience. In the proposed research project, it will be evaluated whether the effectiveness of differentiation, integration, and survival within circuitry by transplanted neuroblasts in the somatosensory cortex of mice can be improved by experimental manipulations of their circuit activity. The proposed research will evaluate the effects of induction of activity-dependent plastic changes in the adult somatosensory cortex, achieved by sensory stimulation and sensory deprivation. Intense long-lasting sensory stimulation will be induced by housing animals in completely new "tactile-rich" area (enriched environment) or by intense forced movement of tactile whiskers (mechanical stimulation of vibrissae). Sensory deprivation will base on long-lasting tactile whisker trimming or vibrissectomy (restriction of sensory input to the cortex). The effects on survival, differentiation, and integration of transplanted neuroblasts in the adult cortex undergoing experimentally-induced neurodegeneration and a range of stimulation-dependent activation will be studied with a range of established immunocytochemical, neuroanatomical, and genetic tracer methods well established in the Macklis lab, analyzed using fluorescence and confocal microscopy to evaluate cellular integration, synaptic circuit formation, and trans-synaptic functional activation.