Talmage Lab - New Space Activation

我们今年的第一个目标是在国立卫生研究院(NIH)的贝塞斯达校区建立一个功能实验室。 分目标1：建立物理空间。翻修工程原计划于2019年8月/9月完工。这一日期被修改为10月下旬、12月中旬、2020年1月底，最后是2020年3月中下旬。3月份，当机组人员完成翻修时，我们开始打开购买的物品，当时由于新冠肺炎疫情，我们的维修工作降到了最低限度。在最低限度的维护期间完成了翻修，大部分空间接近正常使用。最后的设备安装和培训计划在2020年8月/9月进行。 到目前为止，美国国立卫生研究院的实验室还没有进行过任何科学研究。 次级目标2：招聘关键实验室人员。我招募了以下人员： 1名专职科学家、2名博士后研究员和1名博士后。 次级目标3：装备和供应实验室。我们基本上已经做到了这一点。截至2020年8月初，实验室的所有区域都已搭建完毕，除了我们的切片电生理装置(计划于8月中旬安装)、我们的成像设施(在过程的最后阶段订购显微镜，以及我们的共焦空间等待另一名研究人员搬出设备)。 次目标4：建立我们的鼠群。我们将三个品系的小鼠从石溪大学运往美国国立卫生研究院，由NIMH转基因核心进行再衍生。到目前为止，三条线路中的两条已经成功重新衍生，第三条预定在未来两周内重新衍生。来自1号线的第一批动物现在正在繁殖，我们已经确定了纯合子后代，用于建立我们的繁殖群体。来自第二个品系的动物现在已经进行了基因分型，杂合子被转移到我们新生的殖民地。 我们的第二个目标是开始旨在定义Nrg1相互作用体的实验--在神经元中与Nrg1蛋白物理相互作用的蛋白质星系。为此，我构建了表达结构，以允许在亲和纯化之后对相互作用子进行邻近标记。这些构建物已经用HEK293细胞进行了验证。我已经与NINDS蛋白质组学核心设施主任李燕博士建立了合作关系，以识别相互作用的蛋白质。我们还对野生型和突变斜坡体的海马区进行了初步的RNAseq分析，以检查干扰依赖于伽马分泌酶的Nrg1信号的转录后果。 我们的第三个目标是促进和保持与角色实验室(现在是NINDS中的电路、突触和分子信号实验室)、Ari Kaufman博士(计算机科学系)、Parsey博士和De Lorenzo博士(精神病学系)(都在石溪大学)以及与耶鲁大学精神病学系的Marina Picciotto博士的持续合作。与Role博士和Kaufman博士合作，产生了两份正在修订的手稿(开发使用人工智能方法预测衰老期间胆碱能终端场损失轨迹的独特图像处理算法)，(B)Parsey和De Lorenzo博士致力于建立一项关于正常和病理性衰老期间胆碱能系统的跨物种研究，以及(C)Picciotto博士正在修订一份手稿，第二份将于未来几周提交。后两项研究确定了参与不同类型记忆的胆碱能神经元的特定群体，并首次证明胆碱能神经元直接参与记忆印记。

Our first objective for the current year was to establish a functional laboratory at the Bethesda campus of the National Institutes of Health (NIH). Sub-objective 1: set up the physical space. Renovations were originally scheduled to be completed by August / September 2019. This date was modified to late October, mid-December, end of January 2020 and finally mid-to-late March of 2020. We began to unpack purchases in March while the crew was finishing renovations when we went to minimal maintenance due to the COVID-19 pandemic. Renovations were completed during minimal maintenance and most of the space is nearing functional. Final equipment installation and training is scheduled for August / September 2020. No science has been done in the lab at NIH yet. Sub-objective 2: recruit key lab personnel. I have recruited: A staff scientist, 2 post-doctoral fellows and a post-bac. Sub-objective 3: Equip and supply the lab. We have largely accomplished this. As of the beginning of August 2020, all areas of the lab are set up with the exception of our slice electrophysiology rig (installation scheduled for mid-August), our imaging facilities (ordering of microscopes in final phases of the process, and space for our confocal awaits the move out of equipment of another investigator). Sub-objective 4: Establish our mouse colony. We shipped three lines of mice from Stony Brook University to the NIH for re-derivation by the NIMH transgenic core. To date, two of the three lines have been successfully rederived, the third is scheduled for rederivation in the next two weeks. The first animals from line 1 are now breeding and we have identified homozygous offspring to use for establishing our breeding colony. Animals from the second line have now been genotyped and heterozygotes transferred to our nascent colony. Our second objective was to begin experiments aimed at defining the Nrg1 interactome--the galaxy of proteins that physically interact with the Nrg1 protein in neurons. To this end I have built expression constructs to allow proximity tagging of interactors followed by affinity purification. The constructs have been validated using HEK293 cells. I have established a collaboration with Dr. Yan Li, Director of the NINDS proteomics core facility, for identifying interacting proteins. We have also performed an initial set of RNAseq analyses on the hippocampi of wild type and mutant littermates to examine the transcriptional consequences of disrupting gamma secretase dependent Nrg1 signaling. Our third objective was to foster and sustain ongoing collaborations with the Role lab (now Laboratory of Circuits, Synapses and Molecular Signaling in the NINDS), with Dr. Ari Kaufman (Department of Computer Sciences) and Drs. Parsey and De Lorenzo (Department of Psychiatry) (all at Stony Brook University) and with Dr. Marina Picciotto at Yale University Department of Psychiatry. The collaboration with Dr. Role and with (a) Dr. Kaufman yielded two manuscripts that under revision (developing unique image process algorithms that use AI approaches to predict trajectories of cholinergic terminal field loss during aging), (b) Drs Parsey and De Lorenzo the establishment of a cross species study of the cholinergic system during normal and pathological aging, and (c) Dr. Picciotto a manuscript under revision and a second that will be submitted in the coming weeks. These latter two studies identify specific populations of cholinergic neurons that participate in distinct types of memory and demonstrate for the first time that cholinergic neurons participate directly in memory engrams.