Talmage Lab - New Space Activation

我们今年的第一个目标是在美国国立卫生研究院（NIH）的贝塞斯达校区建立一个功能实验室。 次级目标1：建立物理空间。翻新工程原定于2019年8月/9月完成。该日期被修改为10月下旬，12月中旬，2020年1月底，最后是2020年3月中下旬。我们于3月开始拆包采购，当时船员正在完成装修，由于COVID-19疫情，我们进行了最低限度的维护。翻修是在最低限度的维护和大部分的空间是接近功能完成。最终设备安装和培训计划于2020年8月/9月进行。 在NIH的实验室里还没有科学研究。 次级目标2：招募关键实验室人员。我招募了： 一名科学家，两名博士后研究员和一名博士后。 次级目标3：为实验室提供设备和用品。我们在很大程度上做到了这一点。截至2020年8月初，实验室的所有区域都已设置完毕，除了我们的切片电生理设备（安装计划于8月中旬），我们的成像设施（在过程的最后阶段订购显微镜，以及我们的共聚焦空间等待另一位研究者的设备搬出）。 子目标4：建立我们的小鼠群体。我们从斯托尼布鲁克大学运送了三个品系的小鼠到NIH进行NIMH转基因核心的再衍生。到目前为止，三条线路中的两条已经成功地重新推导，第三条线路计划在未来两周内重新推导。来自1号系的第一批动物现在正在繁殖，我们已经确定了纯合后代用于建立我们的繁殖群体。来自第二线的动物现在已经进行了基因分型，杂合子转移到我们的新生群体。 我们的第二个目标是开始旨在定义Nrg 1相互作用组的实验--在神经元中与Nrg 1蛋白发生物理相互作用的蛋白质的星系。为此，我构建了表达构建体，以允许相互作用物的邻近标记，然后进行亲和纯化。已使用HEK 293细胞验证了构建体。我与NINDS蛋白质组学核心设施主任Yan Li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型同窝仔的海马进行了初始的一组RNAseq分析，以检查破坏γ分泌酶依赖性Nrg 1信号传导的转录后果。 我们的第三个目标是促进和维持与角色实验室（现在的NINDS电路，突触和分子信号实验室），Ari考夫曼博士（计算机科学系）和Parsey博士和De Lorenzo博士（精神病学系）（都在斯托尼布鲁克大学）以及耶鲁大学精神病学系的Marina Picciotto博士的持续合作。与Role博士和（a）考夫曼博士的合作产生了两份正在修订的手稿（开发独特的图像处理算法，使用AI方法预测衰老期间胆碱能终末场损失的轨迹），（B）Parsey和De Lorenzo博士建立了正常和病理衰老期间胆碱能系统的跨物种研究，以及（c）皮乔托博士一份正在修订的手稿，第二份将在未来几周内提交。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痕迹。

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.