Direct nicotinic excitation of layer 5 neocortical pyramidal neurons

描述(申请人提供)：新皮质在许多高级功能中起着中心作用，如解释感觉信息、理解语言和控制随意运动。这些突起中的许多是由基底前脑复合体(主要是基底核)神经元上升的胆碱能驱动形成的。这一通路的功能障碍会导致涉及新皮质的许多行为出现缺陷，并与抑郁症、帕金森氏症和阿尔茨海默病等临床疾病有关。我们的长期目标是了解基底核ACh如此深刻地影响皮质功能的细胞和网络机制。ACh作用于M受体和烟碱型ACh受体(mAChRs和nAChRs)，它们广泛表达于新皮质。新皮质中的锥体神经元表达这两种受体，但锥体神经元中nAChRs的生理功能尚不清楚。在这个方案中，我们描述了nAChRs在锥体神经元上的功能。我们使用光遗传学、病毒工具、转基因小鼠、免疫细胞化学、细胞电生理学、双光子显微镜和电子显微镜等技术组合，直接评估由基底核轴突释放的ACh如何影响运动皮质中的锥体神经元。我们在基底核的胆碱能神经元和新皮质的胆碱能神经元表达通道视紫红质-2。在初步实验中，胆碱能轴突的激活通过nAChRs去极化并促进第5层锥体神经元的放电，使我们假设ACh促进了信息通过新皮质网络从上升的兴奋性输入(例如来自丘脑)传递到靶结构(例如脊髓中的运动神经回路)。在这 我们将确定ACh对锥体神经元的影响是否支持这一假说。我们将研究nAChRs影响放电和涉及的nAChR受体亚单位的机制(特定目标1)，确定激活的nAChRs位于锥体神经元树突树内的位置(特定目标2)，并确定这些突触后nAChRs是否介导了其他层和新皮质区域的ACh的影响(特定目标3)。我们的结果将揭示ACh调节锥体神经元兴奋性的新机制。据我所知，我们的研究还将提供第一个证据，证明ACH具有特定于蛋鸡的效果。由此产生的假说有可能改变我们对来自基底核的胆碱能通路改变新皮质网络功能的方式的理解，因此可能对帕金森病和阿尔茨海默病等衰弱疾病具有重要意义。

DESCRIPTION (provided by applicant): The neocortex plays a central role in many higher-order functions such as the interpretation of sensory information, comprehension of language and control of voluntary movements. Many of these processes are shaped by ascending cholinergic drive from neurons in the basal forebrain complex, principally nucleus basalis. Dysfunction of this pathway leads to deficits in many behaviors that involve neocortex and has been tied to clinical conditions such as depression, Parkinson's disease and Alzheimer's disease. Our long-term aim is to understand the cellular and network mechanisms by which ACh from nucleus basalis so profoundly influences cortical function. ACh acts at muscarinic and nicotinic ACh receptors (mAChRs and nAChRs), which are widely expressed in neocortex. Pyramidal neurons in neocortex express both of these receptor types, but the physiological functions of nAChRs in pyramidal neurons are unknown. In this proposal we describe the functions of nAChRs on pyramidal neurons. We directly assess how ACh, released by axons from nucleus basalis, affects pyramidal neurons in motor cortex, using a combination of techniques, including optogenetics, viral tools, genetically-modified mice, immuno-cytochemistry, cellular electrophysiology, two-photon microscopy and electron microscopy. We express channelrhodopsin-2 in cholinergic neurons in nucleus basalis and their axons in neocortex. In preliminary experiments, activation of cholinergic axons depolarized and promoted spiking of layer 5 pyramidal neurons via nAChRs, leading us to hypothesize that ACh facilitates the transfer of information through neocortical networks from ascending excitatory inputs, e.g. from thalamus, to target structures, e.g. motor circuits in the spinal cord. In this proposal we will determine whether the effects of ACh on pyramidal neurons support this hypothesis. We will investigate the mechanisms by which nAChRs affect spiking and the nAChR receptor subunits involved (specific aim 1), determine where in the activated nAChRs are located within the dendritic trees of pyramidal neurons (specific aim 2), and determine whether these postsynaptic nAChRs mediated the effects of ACh in pyramidal neurons in other layers and neocortical areas (specific aim3). Our results will reveal a new mechanism by which ACh modulates the excitability of pyramidal neurons. Our studies will also provide the first evidence, to my knowledge, that ACh has layer-specific effects. The resulting hypothesis has the potential to transform our understanding of the manner in which the cholinergic pathway from nucleus basalis changes network function in neocortex and may therefore have important implications for debilitating conditions such as Parkinson's disease and Alzheimer's disease.