Live imaging of second messengers in developing retina

长期的目标是描绘的细胞机制，其中自发相关的神经活动是在发育中的哺乳动物视网膜产生。在大脑发育的早期，在感觉体验成为可能之前，电和化学活动都是在整个未成熟的神经系统中自发产生的。越来越多的证据表明，这种早期活动对于神经回路的适当发展至关重要。发展对人类神经系统正常发育的组织原则的详细了解，可能使我们有可能了解神经性出生缺陷的起源。此外，它将提供关键的见解，设计策略，使神经系统重新布线正常功能的神经回路，以应对发育异常， 弱视（lazy eye）视网膜中自发活动的细胞基础主要通过电生理学和钙成像来研究。自发性视网膜活动的特征在于以分钟量级的周期发生的去极化。事实上，我们可以在分离的视网膜神经元中重现这种缓慢的周期性，这表明这种周期性背后的起搏器可能是细胞自主的。在这里，我们建议测试的假设，这种缓慢的周期性是由第二信使，cAMP的振荡。这项建议有两个目标。首先，我们建议在视网膜神经元中使用两个指标-一个 对第二信使cAMP水平敏感，对蛋白激酶A活性敏感。其次，我们将使用这些指标，以确定是否自发振荡cAMP的基础上观察到的周期性在完整的视网膜以及自发活动的网络形成的解离的视网膜神经元。

The long-term objective is to delineate the cellular mechanisms by which spontaneous correlated neural activity is generated in the developing mammalian retina. Very early in brain development, before sensory experience is possible, both electrical and chemical activity is generated spontaneously throughout the immature nervous system. There is growing evidence that this early activity is critical for the appropriate development of neural circuits. Developing a detailed understanding of the organizing principles that govern the normal development of the human nervous system may make it possible to understand the origin of neurological birth defects. In addition, it will provide critical insights into devising strategies that allow the nervous system to rewire normal functioning neural circuits in response to developmental abnormalities such as amblyopia (lazy eye). The cellular basis of spontaneous activity in the retina has been studied primarily by electrophysiology and calcium imaging. Spontaneous retinal activity is characterized by depolarizations that occur with a period on the order of minutes. Indeed, we can reproduce this slow periodicity in dissociated retina neurons, indicating that the pacemaker underlying this periodicity may be cell autonomous. Here we propose to test the hypothesis that this slow periodicity is set by oscillations in the second messenger, cAMP. The goals of this proposal are two fold. First, we propose to implement in retinal neurons the use of two indicators - one sensitive to levels of the second-messenger cAMP levels, and the second sensitive to activity of protein kinase-A. Second, we will then use these indicators to determine whether spontaneous oscillations in cAMP underlie the periodicity observed in both the intact retina as well spontaneously active networks formed by dissociated retinal neurons.