Mechanism of Synaptically-Driven Gene Expression

值得注意的是，经验可以以一种快速的方式改变神经元的功能，这种方式可以作为长期记忆持续一生。现在已经确定，特定的突触活动模式可以引起突触强度的改变，这些改变（称为LTP和LTD）被认为是记忆储存和大脑发育活动依赖性微调的基础。LTP和LTD都被证明具有需要合成新蛋白质的晚期阶段。因此，突触驱动的基因转录可能是形成长期记忆的关键事件。人们普遍 一致认为，这一过程需要突触后钙流入。然而，钙内流是如何耦合到转录事件的细节仍然知之甚少。钙信号在神经元中触发转录的空间和时间要求是什么？试图解决这个问题几乎完全涉及浴应用谷氨酸或高钾解离的神经元培养。鉴于这些刺激的非生理性质，出现相互矛盾的结果并不奇怪，一些研究人员声称需要在细胞核中进行钙瞬变，而另一些研究人员则报告说，仅限于树突的钙瞬变就足够了。我们将使用一种更接近完整大脑的制剂来解决这个问题。在这里，我们建议在脑切片中刺激撞击神经元树突的突触，同时使用多光子显微镜以高分辨率测量整个神经元中的Ca浓度，并同时测量两个体细胞膜 潜力和转录因子活性。后者将涉及使用CREB/CBP FRET系统的动态测量和使用高分辨率原位杂交（CATFISH）和磷酸化状态特异性抗体的免疫组织化学的事后分析。该分析将在具有不同树突形态和放电特性的两种类型的CNS神经元中进行。神经元中钙离子触发转录事件的关键核靶点是什么？许多注意力都集中在转录因子CREB上，而排除了其他潜在的重要靶点。细胞培养实验已经表明，转录因子SRF（血清反应因子）被神经元中的Ca信号强烈激活。此外，许多（如果不是全部）活性依赖性IEGs含有SRF及其相关因子的结合位点。因此，我们假设SRF是支持启动新基因转录的突触驱动的Ca信号的关键靶点。我们建议使用新的空间和 时间基因消融技术，以解决SRF的必要性，用于激活一组神经元即刻早期基因和已知具有转录依赖性晚期相（包括海马LTP和小脑LTD）的几种形式的活性依赖性可塑性。此外，一种新的小分子SRF激活剂将用于在突触刺激不存在的情况下急性诱导SRF依赖性转录。后一种方法将用于解决SRF依赖性转录对于突触功效和IEG转录的长期变化的充分性。

It is remarkable that experience can modify neuronal function in a manner that is rapid and which can last for an entire lifetime as long-term memory. It is now well established that particular patterns of synaptic activity can give rise to alterations in synaptic strength, and these alterations (called LTP and LTD) are believed to underlie both memory storage and the activity-dependent fine-tuning of brain development. Both LTP and LTD have been shown to have late phases that require the synthesis of new proteins. Thus, synaptically-driven gene transcription is likely to be a key event in laying down long-term memories. There is general agreement that this process requires postsynaptic Ca influx. However, the details of how Ca influx is coupled to transcriptional events remain poorly understood. What are the spatial and temporal requirements for Ca signals to trigger transcription in neurons? Attempts to address this question have almost exclusively involved bath application of glutamate or high K to dissociated neuronal cultures. Given the nonphysiological nature of these stimuli, it is not surprising that conflicting results have emerged, with some investigators claiming a requirement for a Ca transient in the nucleus while others have reported that a Ca transient restricted to dendrites is sufficient. We will address this issue using a preparation that more closely resembles the intact brain. Here, we propose to stimulate glutamatergic synapses impinging upon dendrites of neurons in brain slices while measuring Ca concentration throughout the neuron at high resolution using multiphoton microscopy and simultaneously measuring both somatic membrane potential and transcription factor activity. The latter will involve both dynamic measurements using a CREB/CBP FRET system and posthoc analyses using high resolution in situ hybridization (CATFISH) and immunohistochemistry with phosphorylation-state specific antibodies. This analysis will be performed in two types of CNS neurons with different dendritic morphologies and firing properties. What are the critical nuclear targets for Ca triggered transcriptional events in neurons? Much attention has been directed towards the transcription factor CREB, to the exclusion of other potentially important targets. Cell culture experiments have indicated that the transcription factor SRF (Serum Response Factor) is robustly activated by Ca signaling in neurons. Moreover, many if not all activity-dependent IEGs contain binding sites for SRF and its associated factors. Thus we hypothesize that SRF is a key target of synaptically-driven Ca signals that supports initiation of new gene transcription. We propose to use novel spatial and temporal gene ablation techniques to address the necessity of SRF for the activation of a panel of neuronal immediate-early genes and in several forms of activity-dependent plasticity known to have transcription-dependent late phases (including hippocampal LTP and cerebellar LTD). Moreover, a novel small molecule SRF activator will be used to acutely induce SRF-dependent transcription in the absence of synaptic stimulation. The latter approach will be used to address the sufficiency of SRF-dependent transcription for long-term changes in synaptic efficacy and IEG transcription.