Use of dynamic photostimulation to investigate synaptic integration in vitro

描述(申请人提供)：这个项目的主要目的是建立和测试一种新的方法，该方法将扩大体外切片准备的使用，以研究突触整合的时空方面以及正在进行的网络活动对神经元计算的影响。急性脑片是研究神经系统功能的有用的体外模型。然而，一个重大的缺陷是缺乏背景神经元活动。在活体中，神经元维持一个恒定的自发活动水平，这转化为每个神经元的突触输入。突触输入已知会影响神经元的计算特性。目前，突触整合的体外研究依赖于通过在胞体或树突上注入电流来模拟突触活动。这项技术仅限于一个或两个注射部位，不能解决仍然知之甚少的分布式突触输入的树突整合。在活体中，突触输入不断到达数千个树突部位。为了在更真实的水平上研究突触整合，有必要在测量突触后神经元反应特性的同时，在空间和时间上控制大量突触输入。在这里，我们提出了一种新的实验技术，它将允许我们用精确控制的时空刺激模式来刺激体外培养的皮质锥体细胞的树突树。我们的具体目标是：目标1)采用数字光处理(DLP)技术(由数千个单独控制的微型镜组成的矩阵)作为动态光刺激(DPS)设备。刺激通过兴奋性神经递质谷氨酸的光解作用发生。用于谷氨酸去除的紫外光将由DLP控制，允许无伪影产生2维静态或动态刺激模式。目的2)用动态光刺激(DPS)检测同步“背景”输入对新皮质神经元增益的影响。我们将使用DPS在皮质锥体细胞的树状结构树上生成精确定义的时空输入模式，同时测量神经元响应。急性切片是许多与人类健康相关的研究的首选方法，例如药物滥用的药理学研究或增强记忆的药物的评估。我们的新方法带来的改进将显著扩大急性切片准备中可以解决的问题的范围。使用活的脑组织切片来研究神经系统是许多与人类健康相关的研究的首选方法，例如药物滥用的药理学研究或增强记忆的药物的评估。我们在这里建议开发的新方法带来的改进将显著扩大活体脑片制备中可以解决的问题的范围。

DESCRIPTION (provided by applicant): The main purpose of this project is to establish and test a new method that will expand the use of the vitro slice preparations to study spatio-temporal aspects of synaptic integration and the effects of ongoing network activity on neuronal computation. Acute slices are a useful in vitro model for the investigation of nervous system function. However, a significant pitfall is the lack of background neuronal activity. In vivo, neurons maintain a constant level of spontaneous activity which translates into synaptic input for each individual neuron. Synaptic input is known to affect the computational properties of neurons. Currently, in vitro investigation of synaptic integration relies on simulating synaptic activity through current injection at the soma or dendrite. This technique is limited to only one or two injection sites and can not address the still poorly understood dendritic integration of distributed synaptic inputs. In vivo, synaptic inputs constantly arrive at thousands of dendritic sites. In order to investigate synaptic integration at a more realistic level, it is necessary to control large numbers of synaptic inputs in space and time while measuring the response properties of the postsynaptic neuron. Here we propose to develop a new experimental technique, which will allow us to stimulate the dendritic trees of cortical pyramidal cells in vitro with precisely controlled spatio-temporal stimulus patterns. Our specific aims are: Aim 1) Adapt Digital Light Processing (DLP) technology (a matrix of several thousand individually controlled miniature mirrors) to be used as a dynamic photo stimulation (DPS) device. Stimulation occurs through photolytic uncaging of the excitatory neurotransmitter glutamate. UV light for glutamate uncaging will be controlled with DLP, allowing the artifact-free generation of 2-dimensional static or dynamic stimulus patterns. Aim 2) Use dynamic photo stimulation (DPS) to test the influence of synchronous "background" input on gain in neocortical neurons. We will use DPS to generate precisely defined spatio-temporal input patterns on the dendritic tree of cortical pyramidal cells while measuring neuronal responses. Acute slices are the method of choice for many studies related to human health, such as pharmacological studies of drug abuse or the evaluation of memory enhancing drugs. The improvements brought about by our new method will significantly expand the range of questions that can be addressed in acute slice preparations. The use of living slices of brain tissue to study the nervous system is the method of choice for many studies related to human health, such as pharmacological studies of drug abuse or the evaluation of memory enhancing drugs. The improvements brought about by the new method we propose to develop here will significantly expand the range of questions that can be addressed in living brain slice preparations.