DEVELOPMENT OF SYNAPSES BETWEEN IDENTIFIED NEURONS

神经系统的功能依赖于突触回路的准确建立。这项工作的长期目标是了解特定突触连接形成的机制，以及这些连接在发育过程中调整的方式。这些问题正在使用蟑螂美洲大熊猫颈部系统中明确定义的感觉神经元和中间神经元群体来解决。这项提议的具体目标描述如下：在从第一期幼虫过渡到第二期幼虫的过程中，感官输入增加了20倍，周期仅为8天。我们的第一个目标是确定新的感觉神经元的出生、轴突生长和突触形成的时间，这是后面实验的基本前提。第二个目的是验证这样的假设，即积聚的蛋白质形成位置信息系统的一部分，该系统决定了感觉神经元的轴突解剖和突触连接。使用细胞内染料注射、电子显微镜和电生理学方法，评估反义寡核苷酸和错误表达Enraed对发育中的感觉神经元轴突分支和突触连接的影响。第三个目的是测试Hedgehog信号通路的拮抗剂cAMP依赖的蛋白激酶(PKA)在决定轴突树枝形成和突触连接中的作用。PKA在药物作用下可被激活或被抑制。第四个目标是研究感觉神经元与神经元间突触的强度是如何随着动物的生长和新的输入进入神经系统而调整的。实验方法包括细胞内记录、定量分析、细胞内染料注射和形态计量分析。许多控制神经回路形成的基本机制在进化过程中似乎是保守的，因此这项研究的结果应该与人类神经系统发育和再生过程中突触的形成有关。

The functioning of the nervous system is dependent on the accurate establishment of synaptic circuits. The long-term goal of this work is to understand the mechanisms by which specific synaptic connections are formed, and the ways that these connections are adjusted during development. These questions are being addressed using a well-defined population of sensory neurons and interneurons in the cercal system of cockroach Periplaneta americana. The specific aims of this proposal are described below: There is a 20-fold increase in sensory input during the transition from first to second stage larva, a period of only 8 days. Our first aim is to determine the times of birth, axonal ingrowth and synapse formation of the new sensory neurons, an essential prerequisite for the later experiments. The second aim is to test the hypothesis that the Engrailed protein forms part of a system of positional information which determines the axon anatomy and synaptic connectivity of sensory neurons. The effects of antisense oligonucleotides and misexpression of Enrailed on the axonal arborizations and synaptic connections of developing sensory neurons will be assessed, using intracellular dye injection, electron microscopy and electrophysiology. The third aim is to test the role of cyclic AMP-dependent protein kinase (PKA), an antagonist of the Hedgehog signaling pathway, in determining axon arborization and synaptic connectivity. PKA will be activated or inhibited pharmacologically. The fourth aim is to investigate how the strengths of sensory neuron to interneuron synapses are adjusted as the animal grows and new inputs enter the nervous system. The experimental approaches include intracellular recording, quantal analysis, intracellular dye injection, and morphometric analysis. Many of the basic mechanisms which control the formation of neural circuitry appear to have been conserved during evolution, so the results derived from this study should be relevant to synapse formation during the development and regeneration of the human nervous system.