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如何接收和处理信号。然而，关于神经回路如何协调驱动相反行为反应的相互竞争的信号，人们知之甚少；因此，研究神经回路如何整合和/或协调相反的反应电路可能会导致发现新的、令人兴奋的学习过程。为了观察神经电路如何完成这种信号整合，这个项目利用了主要研究人员之前描述的线虫的学习测试。在这种学习方案中，两个刺激配对，每个刺激都驱动先天的、相反的运动反应(即向前运动与向后运动)。这种配对不会增强对单一刺激的后一种反应，而是导致一种新的反应结果，即运动停止；因此，由这些刺激激活的固有反应定性地影响条件反应。这种习得的反应在几分钟内稳定下来，可能涉及中间神经元下游的神经元(例如运动神经元)的调制，这些神经元可能发生信号整合。该奖项支持首席研究员和两个教师合作伙伴之间的合作，以寻求多种调查途径。通过这些合作，主要研究人员将获得并扩展她的技能集，包括最先进的遗传工具以及先进的显微技术，这些技术将允许观察单个神经元在行为动物的刺激配对期间或在被固定的动物的刺激配对之后的激活，对单个突触或预计涉及的突触子集的定向激活，以及对突变株的行为分析，以调查支持神经元的神经胶质细胞在形成学习反应中的可能作用。这个项目将为一些在初级调查员实验室和以课程为基础的本科经验(CURE)实验室课程的本科生提供有意义的研究机会。通过在主要研究人员的家乡机构建立这些技术，该项目将为科学事业扩展可用的工具。该项目结合了现代遗传工具和成像方法，以可视化神经元活动的动力学，并揭示线虫神经回路、信号整合和学习的细胞机制。为了识别在相反的运动反应神经回路配对过程中和配对后激活的神经元，来自NeuroPAL唯一神经元标记的荧光将与GCaMP(一种遗传编码的钙指示剂)荧光同时捕获。表达针对特定神经元的遗传目标的光敏离子通道的线虫菌株将在配对信号呈现期间被用来激活/去激活目标电路。为了针对电路集成中涉及的单个突触或一组突触，该项目将结合光遗传学与图案化的点照明和超分辨率/薄膜共聚焦显微镜。最后，利用定向突变，将探索神经胶质细胞在促进这种刺激配对后学习方面的潜在作用。这个项目有可能揭示一个新的理解，即单个神经元、电路和/或胶质细胞如何参与整合同时的相反反应信号，以及这种经历如何改变生物体的行为。这一奖项为首席研究员提供了学习这些遗传学和显微镜方法的时间，并将这些技能纳入她的实验室的研究计划和实验室课程，为本科生提供了有意义的研究经验，并有机会与科学界代表性较低的女性首席研究员合作。初级调查员所在机构的教员将受益于这些现代技术的引入所提供的扩大的当地工具。这些方法也将通过下一届本科生神经科学会议学院的研讨会传播给本科机构的其他神经科学教师。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.