Synaptic Regulation of Neurogenesis in the Dentate Gyrus

描述（申请人提供）：成年期齿状回颗粒细胞（GC）层不断增加新的神经元。在2周内，超过50%的颗粒细胞死亡，大多数存活的gc并入齿状回路。虽然齿状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和联想学习任务，从而增加新GC的存活率。因此，GC存活的调节对联想学习很重要。我们的初步研究表明，低频刺激穿孔通路（主要的谷氨酸能投射到齿状回）可以使未成熟的GC神经元在8天龄时完全存活。此外，1周后，同样的穿孔路径纤维的后续激活诱导了未成熟神经元的即时早期基因（IEG） Zif268。我们的主要假设是传入活动促进GC存活，最可能是通过GABAa受体激活。这反过来又启动了未成熟的GCs快速发育为功能成熟的GCs，这些GCs显示NMDA受体和突触可塑性，这反映在IEG Zif268的活性依赖性表达中。为了验证这些假设，我们将使用在穿孔通路和齿状回永久植入电极的成年大鼠。通过给予溴脱氧尿嘧啶（BrdU）（一种仅存在于分裂细胞中的胸腺嘧啶类似物），结合无偏体视学和荧光免疫细胞化学来量化BrdU阳性细胞、IEG蛋白表达和GC发育标志物（祖细胞、未成熟细胞和成熟细胞），评估穿通路径刺激对GC神经发生的影响。在Specific Aim 1的研究中，我们将确定有丝分裂后的“关键时期”，在此期间穿孔路径刺激可以使新生成的GC神经元存活。我们还将探索由传入活动提供的赋予GC存活的因素，并确定局部或全身给药GABAa、AMPA或NMDA受体拮抗剂是否会阻断由穿孔路径刺激介导的GC存活。在Specific Aim 2中，我们将确定给予8日龄GCs存活是否也会加速其功能发育。这将通过确定NR2B NMDAR亚基和Zif268（一种与突触可塑性有关的IEG蛋白）在事件诱导的GC存活后表达的最早时间点来评估，并与未刺激的相同年龄的GCs进行比较。我们还将探索对Zif268表达至关重要的传入活性提供的可能因素，并确定GABAa、AMPA或NMDA受体拮抗剂是否会阻断存活的GCs中Zif268的表达。在具体目标3中，我们将确定增加或减少8天大、具有生存能力的GCs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，在少数民族机构维持一个可行的研究/教学实验室环境。

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.