Synaptic Regulation of Neurogenesis in the Dentate Gyrus

描述（申请人提供）：成年期，新的神经元不断添加到齿状回的颗粒细胞（GC）层。在2周内，超过50%的这些颗粒细胞死亡，大多数存活的GC并入齿状回回路。虽然齿状核神经发生的功能意义尚不清楚，但减少GC神经发生的治疗会损害联想记忆，而联想学习任务会增加新GC的存活率。因此，GC存活的调节对于联想学习显得很重要。我们的初步研究表明，低频刺激的穿通通路，主要的神经元投射到齿状回，赋予完整的生存不成熟的GC神经元8天的年龄。此外，随后激活相同的穿通路径纤维1周后诱导未成熟神经元中的立即早期基因（IEG）Zif 268。我们的主要假设是传入活动促进GC存活，最有可能是通过GABA α受体激活。这反过来又启动了未成熟GC向功能性成熟GC的快速发育，成熟GC显示NMDA受体和突触可塑性，如IEG Zif 268的活性依赖性表达所反映的。为了验证这些假设，我们将使用在穿支通路和齿状回中永久植入电极的成年大鼠。通过施用溴脱氧尿苷（BrdU）（一种仅掺入分裂细胞中的胸苷类似物）结合无偏体视学和荧光免疫细胞化学来定量BrdU阳性细胞、IEG蛋白表达和GC发育的标志物（祖细胞、未成熟的和成熟的）来评估穿通通路刺激对GC神经发生的影响。在特定目标1的研究中，我们将确定有丝分裂后的“关键期”，在此期间，贯穿通路刺激可以赋予新生成的GC神经元存活。我们还将探讨由传入活动提供的因素，赋予GC生存，并确定是否局部或全身给药的GABA α，AMPA，或NMDA受体拮抗剂阻断GC的生存介导的贯穿路径刺激。在具体目标2中，我们将确定赋予8天龄GC的存活是否也加速其功能发育。这将通过确定NR 2B NMDAR亚基和Zif 268（一种涉及突触可塑性的IEG蛋白）在传入诱导的GC存活后与相同年龄的未刺激GC相比可以表达的最早时间点来评估。我们还将探讨传入活动提供的Zif 268的表达是至关重要的可能因素，并确定是否GABAa，AMPA，或NMDA受体拮抗剂阻断Zif 268在幸存的GC的表达。在具体目标3中，我们将确定增加或减少8天大的生存能力GC的数量是否会促进或损害空间或情景记忆任务的表现。这些研究将有助于理解干细胞存活，这对治疗神经系统疾病至关重要，并将促进PI的发展目标，即获得主流资金，以维持少数民族机构可行的研究/教学实验室环境。 公共卫生相关性：了解成年齿状回的神经发生将提供有关未成熟颗粒细胞的功能发育是如何通过体内突触活动调节的信息。神经元存活所必需的因素和从成体干细胞产生的神经元的适当靶向仍然未知，限制了移植细胞用作治疗各种疾病的能力-例如阿尔茨海默病，帕金森病，缺血和脑创伤-涉及神经元的损失。由于调节神经元干细胞存活对于实现干细胞治疗的目标至关重要，这些研究将提供关于神经元干细胞如何存活并整合到现存神经回路中的关键信息，并解决干细胞治疗中的主要警告。

DESCRIPTION (provided by applicant): New neurons are continually added to the granule cell (GC) layer of the dentate gyrus during adulthood. Within 2 weeks, over 50% of these granule cells die, with most surviving GCs incorporating into the dentate circuit. Although the functional significance of dentate neurogenesis is unclear, treatments that reduce GC neurogenesis impair associative memory, and associative learning tasks increase the survival of new GCs. Thus the regulation of GC survival appears important for associative learning. Our preliminary studies show low-frequency stimulation of the perforant pathway, the principal glutamatergic projection to the dentate gyrus, confers complete survival of immature GC neurons 8 days of age. Furthermore, subsequent activation of the same perforant path fibers 1 week later induces the immediate-early gene (IEG) Zif268 in immature neurons. Our primary hypothesis is afferent activity promotes GC survival, most likely via GABAa receptor activation. This, in turn, initiates rapid development of immature GCs into functional, mature GCs that display NMDA receptors and synaptic plasticity as reflected in the activity-dependent expression of the IEG Zif268. To test these hypotheses, we will use adult rats with permanently implanted electrodes in the perforant pathway and dentate gyrus. The effects of perforant path stimulation on GC neurogenesis is assessed by administering bromodeoxyuridine (BrdU), a thymidine analog incorporated only in dividing cells, in conjunction with unbiased stereology and fluorescent immuno-cytochemistry to quantify BrdU-positive cells, IEG protein expression and markers of GC development (progenitor, immature and mature). In the studies of Specific Aim 1, we will determine the `critical period' after mitosis during which perforant path stimulation can confer survival of newly- generated GC neurons. We also will explore the factor(s) that are provided by afferent activity that confer GC survival, and determine whether local or systemic administration of GABAa, AMPA, or NMDA receptor antagonists block GC survival mediated by perforant path stimulation. In Specific Aim 2, we will determine if conferring survival of 8 day old GCs also accelerates their functional development This will be assessed by determining the earliest time point when NR2B NMDAR subunits and Zif268, an IEG protein implicated in synaptic plasticity, can be expressed following afferent-induced GC survival when compared with unstimulated GCs of the same age. We also will explore possible factors provided by afferent activity that are crucial for Zif268 expression, and determine if GABAa, AMPA, or NMDA receptor antagonists block Zif268 expression in surviving GCs. In Specific Aim 3, we will determine if increasing or depleting the population of 8-day old, survival-competent GCs facilitates or impairs performance in spatial or episodic memory tasks. These studies will contribute to understanding stem cell survival, crucial for treating neurological disorders, and will facilitate the PI's development goal of obtaining mainstream funding to maintain a viable research/teaching laboratory environment at a minority institution. Public Health Relevance: An understanding of neurogenesis in the adult dentate gyrus will provide information regarding how the functional development of immature granule cells is regulated by synaptic activity in vivo. The factors necessary for neuronal survival and appropriate targeting of neurons generated from adult stem cells remain unknown, limiting the ability of transplanted cells to be used as a treatment for various diseases- such as Alzheimer's disease, Parkinson's disease, ischemia, and brain trauma - that involve a loss of neurons. As regulating neuronal stem cell survival is essential to achieve the goal of treatment with stem cells, these studies will provide crucial information on how neuronal stem cells survive and incorporate into extant neural circuits, and address a major caveat in stem cell therapy.