MCA Pilot PUI: Neural Signaling and Mechanisms Underlying Sensory Integration and Plasticity

长期以来，研究人员一直在研究单个神经元和神经回路是如何接收和处理信号的。然而，关于神经回路如何调和驱动相反行为反应的竞争信号，人们知之甚少；因此，研究神经回路如何整合和/或调和相反的反应回路可能会导致发现新的和令人兴奋的学习过程。为了观察神经回路是如何完成这种信号整合的，本项目利用了一种由主要研究者先前描述的在线虫中的学习实验。在这个学习协议中，两个刺激分别驱动一个先天的，相反的运动反应（即向前和向后运动）是配对的。这种配对不会增强对单一刺激的后期反应，而是导致一种新的反应结果，即停止运动；因此，这些刺激所激活的先天反应会对条件反应产生质的影响。这种习得的反应在几分钟内稳定下来，可能涉及到信号整合可能发生的中间神经元下游神经元的调节（例如，运动神经元）。该奖项支持主要研究者和两个教师合作伙伴之间的合作，以追求多种调查途径。通过这些合作，主要研究者将获得并扩展她的技能集，包括最先进的遗传工具和先进的显微镜技术，这些技术将允许观察单个神经元的激活，无论是在活动动物的刺激配对期间还是在静止动物的刺激配对之后，单个突触或预计涉及的突触子集的靶向激活。并对突变株进行行为分析，以探讨神经元支持神经胶质细胞在形成学习反应中的可能作用。这个项目将为一些本科生提供有意义的研究机会，在主要研究者的实验室和基于课程的本科生经验（CURE）实验课程中。通过在主要研究者的家庭机构建立这些技术，该项目将扩大科学企业的可用工具。该项目结合现代遗传工具和成像方法，可视化神经元活动的动态，揭示秀丽隐杆线虫神经回路信号整合和学习的细胞机制。为了识别在对立的运动反应神经回路配对期间和之后激活的神经元，来自NeuroPAL独特神经元标记的荧光将与GcAMP（一种基因编码的钙指示剂）荧光同时被捕获。表达针对特定神经元的光敏离子通道的秀丽隐杆线虫菌株将在成对信号呈现期间用于激活/灭活靶向电路。为了针对参与电路集成的单个突触或一组突触，该项目将光遗传学与图案点照明和超分辨率/FLIM共聚焦显微镜相结合。最后，利用靶向突变，神经胶质细胞在促进这种刺激配对后的学习中的潜在作用将被探索。这个项目有可能揭示单个神经元、电路和/或胶质细胞如何参与整合同时对立的反应信号，以及这种经历如何改变生物体的行为。该奖项为首席研究员提供了学习这些遗传和显微镜方法的时间，并将这些技能纳入其实验室的研究项目和实验课程，为本科生提供了有意义的研究经验，并有机会与科学领域少数族裔女性首席研究员一起工作。主要研究者所在机构的教员将受益于这些现代技术的引进所扩大的当地工具。这些方法也将通过下一届本科神经科学学院会议的研讨会传播给其他本科院校的神经科学学院。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Researchers have long investigated how signals are received and processed by individual neurons and within neural circuits. However, little is known with regards to how neural circuits reconcile competing signals that drive opposing behavioral responses; therefore, investigating how neural circuits integrate and/or reconcile opposing response circuits could lead to the discovery of new and exciting learning processes. To observe how neural circuits accomplish this signal integration, this project utilizes a learning assay in a nematode, previously described by the primary investigator. For this learning protocol, two stimuli that each drive an innate, opposing locomotor response (i.e., forward vs backward locomotion) are paired. This pairing does not augment the later response to a single stimulus, but rather results in a novel response outcome, a cessation of locomotion; thus, the innate responses activated by these stimuli qualitatively impact the conditioned response. This learned response stabilizes over minutes and may involve modulation from neurons that are downstream from the interneurons where signal integration likely occurs (e.g., motor neurons). This award supports collaborations between the primary investigator and two faculty partners to pursue multiple avenues of investigation. Through these collaborations, the primary investigator will gain access and expand her skill set to include state of the art genetic tools as well as advanced microscopy techniques that will allow for observing activation of single neurons either during stimulus pairing in behaving animals or following stimulus pairing in immobilized animals, targeted activation of single synapses or subset of synapses predicted to be involved, and behavioral analysis of mutant strains to investigate the possible role of the neuron-supporting glial cells in forming the learned response. This project will provide meaningful research opportunities to a number of undergraduates in the primary investigator’s laboratory and in a course-based undergraduate experience (CURE) lab course. By establishing these techniques at the primary investigator’s home institution, this project will expand the available tools for scientific enterprise. This project combines modern genetic tools and imaging approaches to visualize the dynamics in neuron activity and uncover cellular mechanisms involved in neural circuit signal integration and learning in C. elegans. To identify neurons activated during and after pairing of opposing locomotor response neural circuits, fluorescence from the NeuroPAL unique neuron marker will be captured simultaneously with GcAMP (a genetically-encoded calcium indicator) fluorescence. Strains of C. elegans that express light-sensitive ion channels genetically targeted to specific neurons will be employed to activate/deactivate targeted circuits during paired signal presentation. To target a single synapse or set of synapses involved in circuit integration, this project will combine optogenetics with patterned point illumination and super resolution/FLIM confocal microscopy. Finally, using targeted mutations the potential role of glial cells in facilitating learning following this stimulus pairing will be explored. This project has the potential to reveal a new understanding for how individual neurons, circuits, and/or glia participate in integrating simultaneous opposing response signals and how this experience alters an organisms’ behavior. This award affords the principal investigator supported time to learn these genetic and microscopy approaches and incorporate these skills into her laboratory’s research program and lab courses, providing undergraduates with a meaningful research experience and the opportunity to work with a female primary investigator who is an underrepresented minority in science. Faculty at the primary investigator’s home institution will benefit from the expanded local tools available from the introduction of these modern techniques. These approaches will also be disseminated to other Neuroscience faculty at undergraduate institutions through a workshop at the next Faculty for Undergraduate Neuroscience Conference.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.