Optical methods for imaging and manipulating dendritic spines in vivo

树突棘覆盖大多数哺乳动物神经元的树突，并接收几乎 大脑皮层的所有兴奋性连接。尽管它们在这些回路中的作用是 因此，脊椎的功能可能是至关重要的，但人们对它的了解仍然很少。 脊椎是化学隔间，这可以提供生化 隔离是实现特定于输入的突触可塑性所必需的。但最近 实验表明，此外，脊椎还可以将 电压。这可能会对兴奋性突触电位产生重大影响，改变 当它们被注入树突时。事实上，通过调节脊椎颈部 维度、树突棘可以快速控制突触强度。在那里的时候 体外数据是否支持这一假说，目前还没有直接的 活体脊柱电压的测量。 我们的目标是制造工具来确定脊柱是否真的具有电功能 在活体内。我们提出了两种光学工具来成像和光学操作 活体中的小鼠视皮层中的脊椎。在第一个目标中，我们将建立、校准和 测试两个新的遗传编码电压指示器(GEVI)，它们将是 设计用于最佳双光子截面和树枝晶靶向 脊椎。在第二个目标中，我们将试点同时使用双光子 体内单个脊椎的成像和光遗传学，我们还将合成 并在体内测试Rubi Cage-TTX的双光子光释放。 我们的研究将开发工具，使系统研究 树突棘和其他神经元纳米隔间的功能。测试 脊椎的电功能也有助于更好地理解 许多精神发育迟滞综合征的病理生理学，其特征是 异常长的脊椎。

Dendritic spines cover dendrites of most mammalian neurons and receive almost all excitatory connections in the cortex. Although their role in these circuits is therefore likely to be crucial, the function of spines is still poorly understood. Spines are chemical compartments, and this could provide the biochemical isolation necessary to implement input-specific synaptic plasticity. But recent experiments have suggested that, in addition, spines could compartmentalize voltage. This could have a major impact on excitatory synaptic potentials, altering them as they are injected into the dendrites. In fact, by regulating the spine neck dimensions, dendritic spines could rapidly control synaptic strength. While there is in vitro data supporting this hypothesis, there is currently no direct measurements of spine voltages in vivo. Our goal is to build tools to determine if spines indeed have an electrical function in vivo. We propose two types of optical tools to image and optically manipulate spines in mouse visual cortex in vivo. In the first aim we will build, calibrate and test two novel Genetically Encoded Voltage Indicators (GEVIs), which will be designed for optimal two-photon cross section and for targeting to dendritic spines. In the second aim, we will pilot the use of simultaneous two-photon imaging and optogenetics of individual spines in vivo and we will also synthesize and test a RuBi caged-TTX for two-photon photorelease in vivo. Our research will develop tools that could enable the systematic study of the function of dendritic spines and other neuronal nanocompartments. Testing the electrical function of spines could also help to better understand the pathophysiology of many mental retardation syndromes, characterized by abnormally long spines.