NeuroNex Technology Hub: Advancing neuronal and genetic approaches to animal behavior research

神经科学是现代生物医学研究中最突出的领域之一，但它对其他领域的影响，特别是与环境和有机生物学相关的领域，一直有限。这是因为越来越多的神经科学研究集中在少数“模型”动物物种上，比如老鼠和果蝇，它们已经开发出强大的遗传工具来操纵它们的基因组。这种关注与早期的神经科学研究形成鲜明对比，在早期，更广泛的生物多样性采样导致了重要的发现。突出的例子包括发现鱿鱼的神经元是如何被激活的，以及海蛞蝓是如何建立长期记忆的。该提案的主要目标是通过开发和应用现代神经遗传学工具来观察和操纵任何动物物种的神经元活动，从而增加动物物种的多样性，从而促进神经科学研究。为了证明这一原理，为蜜蜂和美国蚱蜢开发了最先进的工具。除了对基础神经科学产生直接影响外，该项目还对应用研究产生影响，因为这两种选择的物种分别是重要的害虫和传粉者。新工具和研究成果将通过nsf资助的增加遗传研究物种多样性的倡议以及国内和国际研讨会传播给研究界。传播方面的努力包括在华盛顿大学开设年度暑期强化课程，为那些对将现代神经科学和遗传工具纳入其研究项目感兴趣的生物生物学家提供讲座和实践经验。该项目提供的其他教育和培训机会包括圣路易斯地区的公共神经科学推广工作、博士后培训，以及在假设驱动的神经科学研究中指导研究生、本科生和高中生。现代神经科学研究中物种多样性的缺乏限制了一般神经生物学原理在有机体、生态和进化问题中的适用性和解释。因此，本提案的主要目标是通过易于采用通用的遗传和转基因工具来监测和操纵任何动物物种的神经元活动，从而增加系统和行为神经科学研究中的动物物种多样性，特别强调昆虫。建议的方法由两个步骤组成。首先，使用Cas9/ crispr依赖性基因组编辑将非必需基因white替换为DNA盒，该DNA盒包含眼睛特异性红色荧光蛋白（RFP），两侧是两个定向phic31整合酶attP位点，从而能够快速筛选白色眼睛颜色和RFP表达作为成功种系转化的标记。其次，使用高效的phiC31-Integrase反应将RFP盒替换为选择的转基因。作为原理证明，在蜜蜂Apis mellifera和美国蚱蜢Schistocerca americana的特定神经元群体中产生了表达Ca2+报告基因GCaMP6的转基因系，并对其可行性进行了测试。通过利用现代遗传工具加强对这两种具有重要经济意义的昆虫物种的神经科学研究，这两种昆虫物种也是基础有机生物学研究的重要模型，该项目可能会在农业、神经行为学、动物行为学、害虫生态学和行为神经科学等多个研究领域产生广泛影响。这个神经技术中心奖是大脑计划和国家科学基金会理解大脑活动的一部分。

Neuroscience is one of the most prominent areas of modern biomedical research, but its impact on other fields, particularly those related to environmental and organismal biology, has been limited. This is because an increasing proportion of neuroscience research has been concentrated on just a few "model" animal species, such as the mouse and fruit fly, for which powerful genetic tools have been developed to manipulate their genomes. This focus stands in stark contrast to the early days of neuroscience research, in which a broader sampling of biodiversity led to important discoveries. Prominent examples include the discovery of how neurons become activated in the squid and how long-term memory is established in a marine slug. The primary goal of this proposal is to increase the diversity of animal species that can be used to advance neuroscience research by developing and applying modern neurogenetic tools for observing and manipulating neuronal activity in any animal species. As a proof-of-principle, state-of-the-art tools are developed for the honey bee and the American grasshopper. In addition to having immediate impact on basic neuroscience, the project has impact on applied research, as these two selected species are important pests and pollinators, respectively. The new tools and research findings from the proposed work is disseminated to the research community via NSF-funded initiatives to increase species diversity in genetic studies, and national and international workshops. The dissemination efforts include the development of an annual intensive summer course at Washington University that entails both lectures and hands-on experiences for organismal biologists who are interested in incorporating modern neuroscience and genetic tools into their research programs. Additional educational and training opportunities served by the project include public neuroscience outreach efforts in the St. Louis region, training of postdoctoral fellows, as well as mentoring of graduate, undergraduate, and high school students in hypothesis-driven neuroscience research.The lack of species diversity in modern neuroscience research restricts the applicability and interpretations of general neurobiological principles in the context of organismal, ecological, and evolutionary questions. Therefore, the primary goal of this proposal is to increase animal species diversity in systems and behavioral neuroscience research by enabling easy adoption of universal genetic and transgenic tools for monitoring and manipulating neuronal activity in any animal species, with a specific emphasis on insects. The proposed approach is comprised of two steps. First, Cas9/CRISPR-dependent genome editing is used to replace the non-essential gene white with a DNA cassette that includes an eye-specific red fluorescent protein (RFP) flanked by two directional phiC31-integrase attP sites, enabling rapid screening of both white eye-color and RFP expression as markers of successful germline transformation. Second, the efficient phiC31-Integrase reaction is used to replace the RFP cassette with a transgene of choice. As a proof-of-principle, transgenic lines that express the Ca2+ reporter GCaMP6 in defined neuronal populations in the honey bee Apis mellifera and the American grasshopper Schistocerca americana are generated and tested for feasibility. By enabling the use of modern genetic tools to enhance neuroscience research in these two economically important insect species, which also serve as important models for basic organismal biological research, the proposed project is likely to have broad impact relevant to diverse research fields, including agriculture, neuroethology, animal behavior, pest ecology, and behavioral neuroscience. This NeuroTechnology Hub award is part of the BRAIN Initiative and NSF's Understanding the Brain activities.