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，这种方式可以像长期记忆一样持续整个一生。现已证实，突触活动的特定模式可以引起突触强度的变化，这些变化(称为LTP和LTD)被认为是记忆存储和大脑发育依赖活动微调的基础。LTP和LTD都被证明有需要合成新蛋白质的晚期。因此，突触驱动的基因转录很可能是奠定长期记忆的关键事件。有一般的 一致认为这一过程需要突触后钙内流。然而，关于钙内流如何与转录事件相关联的细节仍然知之甚少。钙信号触发神经元转录的空间和时间要求是什么？解决这个问题的尝试几乎完全涉及到在分离的神经元培养中应用谷氨酸或高钾。考虑到这些刺激的非生理学性质，出现相互矛盾的结果也就不足为奇了，一些研究人员声称细胞核中需要钙瞬变，而另一些人则报告说，仅限于树突的钙瞬变就足够了。我们将使用一种更接近于完整大脑的制剂来解决这个问题。在这里，我们建议刺激谷氨酸能突触撞击脑片中神经元的树突，同时使用多光子显微镜以高分辨率测量整个神经元的钙浓度，并同时测量两个体细胞膜 潜能和转录因子活性。后者将包括使用CREB/CBP FRET系统的动态测量和使用高分辨率原位杂交(CATFISH)的POTHOC分析，以及使用磷酸化状态特异性抗体的免疫组织化学。这项分析将在具有不同树突形态和放电特性的两种类型的中枢神经系统神经元中进行。钙离子触发神经元转录事件的关键核靶点是什么？人们对转录因子CREB有很大的关注，而忽略了其他潜在的重要靶点。细胞培养实验表明，转录因子SRF(血清反应因子)在神经元中被钙信号强烈激活。此外，许多(如果不是全部)依赖活性的IEG含有SRF及其相关因子的结合位点。因此，我们假设SRF是突触驱动的钙信号的关键靶点，支持新基因转录的启动。我们建议使用新颖的空间和 时间基因消融技术，以解决SRF的必要性，以激活一组神经元即刻-早期基因，并在几种形式的活动依赖可塑性已知具有转录依赖的晚期(包括海马LTP和小脑LTD)。此外，一种新的小分子SRF激活剂将被用来在没有突触刺激的情况下强烈地诱导SRF依赖的转录。后一种方法将被用来解决SRF依赖的转录对于突触有效性和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.