Probing presynaptic receptor function with two-photon uncaging, Ca2+ imaging and photobleaching

在神经元之间传递信息的突触受到内在和外在因素相互作用的高度多样化的调节。这既有助于丰富大脑回路的信息处理，也有助于它们的稳定性，然而，在癫痫等疾病状态下，大脑回路可能会崩溃。有几种方法可用来间接研究化学受体如何与单个神经递质释放神经过程中的钙信号相互作用。然而，它们不能很容易地相互结合：一些只能与活的神经元一起使用，而另一些则需要组织冷冻或化学处理。我们建议通过结合一种新的方法，通过激光激发分子来操纵化学活性物质的局部浓度，从而实现对单个突触功能的更详细的了解。这种方法依赖于用两个光子非常快速地相继撞击单个电子，从而引起原子状态的高度局域化和短暂变化。这种局部操作对于研究突触是必要的，因为它们通常在微米和毫秒的空间和时间尺度上发挥作用。我们将把这种方法应用到几个与正常大脑功能和癫痫相关的热点问题上。具体地说，我们将研究神经递质受体的空间分布和介导钙进入神经突起的不同蛋白质是如何相互作用的。我们还将询问癫痫中发生的一些变化是否由这些受体的局部表达变化引起的。这项工作将阐明神经元信号如何构成认知过程的基础，以及突触功能异常如何导致癫痫发作。

Synapses that transmit information among neurons are modulated in a highly diverse manner by an interplay of intrinsic and extrinsic factors. This contributes both to the richness of information processing by brain circuits and to their stability, which can however break down in disease states such as epilepsy. Several methods are available to study rather indirectly how chemical receptors interact with calcium signals within individual neurotransmitter-releasing nerve processes. However, they cannot be readily integrated with one another: some can only be used with living neurons, while others require the tissue to be frozen or chemically treated. We propose to achieve a much more detailed insight into the function of individual synapses by incorporating a novel method to manipulate the local concentration of chemically active substances with laser-evoked excitation of molecules. This method relies on hitting individual electrons with two photons in very rapid succession, thus evoking a highly localised and brief change in the state of the atoms. Such localised manipulations are necessary to study synapses, because they normally function on a space- and time-scale of microns and milliseconds. We will apply this method to several topical questions that are relevant to normal brain function and to epilepsy. Specifically, we will investigate how the spatial distribution of neurotransmitter receptors and different proteins mediating calcium movements into nerve processes interact with one another. We will also ask whether some changes that occur in epilepsy are caused by alterations in the local expression of these receptors. The work will shed light on how neuronal signalling underlies cognitive processes and how abnormal synaptic function contributes to seizures.