Live imaging of second messengers in developing retina

长期目标是描述在发育中的哺乳动物视网膜中自发相关神经活动产生的细胞机制。在大脑发育的非常早期，在感觉体验成为可能之前，电和化学活动在整个未成熟的神经系统中自发地产生。越来越多的证据表明，这种早期活动对神经回路的适当发育至关重要。对支配人类神经系统正常发展的组织原则有了详细的了解，可能会使理解神经出生缺陷的起源成为可能。此外，它还将为设计允许神经系统重新连接正常功能的神经回路以应对发育异常的策略提供关键的见解 弱视(懒眼)。视网膜自发活动的细胞基础主要是通过电生理学和钙成像来研究的。自发性视网膜活动的特征是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复制这种缓慢的周期性，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，我们建议检验这样一个假设，即这种慢周期是由第二信使cAMP中的振荡设置的。这项提案的目标有两个。首先，我们建议在视网膜神经元中使用两种指示器--一种 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来确定cAMP的自发振荡是否构成了在完整视网膜以及由分离的视网膜神经元形成的自发活动网络中观察到的周期性的基础。

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.