Tracing neuronal circuits in transgenic animals by transneuronal control of transcription (TRACT).
Tracing neuronal circuits in transgenic animals by transneuronal control of transcription (TRACT).
复制标题
通过转录（Tract）的跨神经元控制在转基因动物中追踪神经元回路。
作者：
Huang TH;Niesman P;Arasu D;Lee D;De La Cruz AL;Callejas A;Hong EJ;Lois C
理解大脑回路中发生的计算需要识别这些回路中的神经元是如何相互连接的。我们描述了一种称为TRACT（TRAnsneuronal Control of Transcription）的技术，该技术基于配体诱导的膜内蛋白水解，以揭示来自感兴趣的基因标记神经元的单突触连接。在这种策略中，表达人工配体的神经元（“供体”神经元）结合并激活其突触伴侣（“受体”神经元）上的基因工程人工受体。当配体-受体在突触处结合时，受体在其跨膜结构域中被切割并释放激活突触伴侣中的转录的蛋白质片段。在果蝇中使用TRACT，我们已经证实了嗅觉受体神经元与其突触后靶点之间的连接，并发现了昼夜节律回路中神经元之间潜在的新连接。我们的研究结果表明，TRACT方法可以用来研究大脑中的神经元回路的连接。理解大脑如何工作的主要障碍之一是，我们对大脑的神经细胞或神经元如何相互连接知之甚少。这些连接构成了大脑的线路图。目前揭示这种连接的方法都有局限性。最流行的方法--连续电子显微镜--可以非常详细地揭示大脑一个小区域中的连接，但它不能显示相距甚远的神经元之间的连接。Huang等人现在已经创建了一个遗传系统来可视化这些连接。为了让神经元进行交流，一个神经元必须产生一种称为配体的信号。然后，这种配体可以结合并激活其伴侣神经元。Huang等人修改了神经元的DNA，这样每当这些细胞产生特定的配体时，它们也会产生红色荧光蛋白。类似的修饰确保了每当配体激活一个配对神经元时，激活的神经元就会产生一个绿色荧光蛋白。在显微镜下观察红色和绿色神经元使Huang等人能够看到哪些细胞与其他细胞进行交流。虽然这些实验是在果蝇身上进行的，但同样的方法也适用于其他实验室动物，包括鱼类、小鼠和大鼠。一旦我们知道了大脑的线路图，下一步就是研究各种连接的作用。例如，为了理解计算机是如何工作的，我们可以改变其电路组件之间的连接，并观察这如何影响计算机的输出。通过这种新方法，我们可以改变大脑中神经元之间的交流方式，然后观察对行为的影响。这将为人类大脑的运作提供深入的见解，并为精神分裂症和自闭症等疾病的问题提供线索。
Understanding the computations that take place in brain circuits requires identifying how neurons in those circuits are connected to one another. We describe a technique called TRACT (TRAnsneuronal Control of Transcription) based on ligand-induced intramembrane proteolysis to reveal monosynaptic connections arising from genetically labeled neurons of interest. In this strategy, neurons expressing an artificial ligand (‘donor’ neurons) bind to and activate a genetically-engineered artificial receptor on their synaptic partners (‘receiver’ neurons). Upon ligand-receptor binding at synapses the receptor is cleaved in its transmembrane domain and releases a protein fragment that activates transcription in the synaptic partners. Using TRACT in Drosophila we have confirmed the connectivity between olfactory receptor neurons and their postsynaptic targets, and have discovered potential new connections between neurons in the circadian circuit. Our results demonstrate that the TRACT method can be used to investigate the connectivity of neuronal circuits in the brain. One of the main obstacles to understanding how the brain works is that we know relatively little about how its nerve cells or neurons are connected to one another. These connections make up the brain’s wiring diagram. Current methods for revealing this wiring all have limitations. The most popular method – serial electron microscopy – can reveal the connections in a small region of the brain in great detail, but it cannot show connections between neurons that are far apart. Huang et al. have now created a genetic system for visualizing these connections. For neurons to communicate, one neuron must produce a signal called a ligand. This ligand can then bind to and activate its partner neuron. Huang et al. modified the DNA of neurons so that every time those cells produced a specific ligand, they also produced a red fluorescent protein. Similar modifications ensured that every time the ligand activated a partner neuron, the activated neuron produced a green fluorescent protein. Viewing the red and green neurons under a microscope enabled Huang et al. to see which cells were communicating with which others. While these experiments took place in fruit flies, the same approach should also work in other laboratory animals, including fish, mice and rats. Once we know the wiring diagram of the brain, the next step is to investigate the role of the various connections. To understand how a computer works, for example, we might change the connections between its circuit components and look at how this affects the computer’s output. With this new method, we can change how neurons communicate with one another in the brain, and then look at the effects on behavior. This should provide insights into the workings of the human brain, and clues to what goes wrong in disorders like schizophrenia and autism.