CRCNS: Multiple Time Scale Memory Consolidation in Neural Networks

动物拟定用途的详细描述，包括种属， 使用的菌株、年龄、性别和数量; 分离的原代培养物将从以下任一种新生小鼠的皮质制备： 性别（小家鼠，出生后第0-1天）。这些实验将使用 从与Floxopatch交配的VRES 1-IRES 2-Cre菌株获得的幼仔（Lou等人，（2016年） 菌株，使锥体细胞将表达通道视紫红质CheRiff和电压 指标QuasR 2。最多可培养5个培养物，可用于5个实验。 使用动物的理由、种属的选择和使用的数量; 描述尽量减少不适、痛苦、疼痛和伤害的程序; 安乐死的方法及其选择的理由。 将使用转基因小鼠（C类），因为实验依赖于 用于光学刺激/记录的CheRiff和QuasR 2的表达。没有 替代物种。 所有的实验都是终端。新生小鼠将在颈部注射前在冰中冷却。 脱位并切除脑以制备培养物。所有程序都在 根据纽约大学动物福利委员会的指导方针。 以下动物的估计数量确保我们有持续的供应， 每天进行实验。根据我们过去3年的经验， 年，每只出生后第0-1天（P0-P1）小鼠将产生至少5个培养物 准备工作一个有活力的培养物将具有高密度的神经元， Floxopatch构建体，其因小鼠而异。如果一种文化不是 所有来自该小鼠的培养物也是不可用的。成功率 大约50%。因为培养物只能从P0-1天大的小鼠制备， 因为不可能确定表达是否足够，直到 实验2-3周后，我们将需要每周制作2组培养物。因此，在本发明中， 我们将需要每周保持2对繁殖对或每月保持8对繁殖对， 有源源不断的文化资源。繁殖对将由野生型 小鼠（用于病毒注射）或VAP 11-IRES 2-Cre-Gt（ROSA）26 Sor（Risopatch）小鼠。 繁殖对将每六个月更换一次。 实验动物总数：32只小鼠/年（=16只小鼠（8对育种）x 2/年 更换育种对）。 为了维持3个品系，VEGF 1-IRES 2-Cre各2只雄性和2只雌性， Gt（ROSA）26 Sor和野生型小鼠将保持在分开的笼中（4+4+4=12只小鼠 共计）。每6个月（每年两次），将对每种类型的小鼠进行繁殖。从他们 将来自每个品系的4只雄性和4只雌性后代断奶并分开饲养 笼中的小鼠（4+ 4 + 4 = 12），并对4对原始繁殖对（12只小鼠）实施安乐死。 总体而言，将使用24只小鼠/年来维持品系。 小鼠总数/年：56只（=24只小鼠/年以维持品系+32只小鼠/年用于实验）。 3年内的小鼠总数= 168只小鼠。 参考 Lou等人，（2016）转基因靶向全光学电生理学 Cre-Dependent Optopatch Mouse，J. Neurosci. 36：11059-73 1 53 来自CRCNS以前支持的结果 私家侦探和联合私家侦探以前没有得到过CRCNS的支持 协调计划 劳动力的分工P.I.（A. Reyes）和他在纽约大学的研究小组将进行实验和分析 在本提案的目标1和2中详细说明。Co-P.I（S. Fusi）和他在哥伦比亚大学的团队 将负责理论和计算模型的发展， 实验数据 纽约大学和哥伦比亚大学之间的数据共享：分析数据、模型代码和手稿 将通过Google Drive在各小组之间传播。所有福斯和雷耶斯的成员 团体可通过电子邮件请求访问。较大的原始数据将存储在本地服务器上， 通过FTP。 纽约大学和哥伦比亚大学之间的协调因为纽约大学和哥伦比亚大学很容易进入- 我们将每月轮流在纽约大学和哥伦比亚大学之间举行联合实验室会议。研究生 将鼓励学生和博士后研究员提交联合海报介绍或讲座， 如Cosyne和神经科学学会会议。 16 54

Detailed description of the proposed use of the animals, including species, strains, ages, sex, and number to be used; Dissociated, primary cultures will be prepared from the cortex of new born mice of either sex (mus musculus, Postnatal day 0-1). These experiments will be performed using pups obtained from Vglut1-IRES2-Cre strains mated with Floxopatch (Lou et al., 2016) strains so that pyramidal cells will express channel rhodopsin CheRiff and voltage indicator QuasR2. Up to 5 cultures can be grown, which can be used for 5 experiments. Justification for the use of animals, choice of species, and numbers to be used; Description of procedures for minimizing discomfort, distress, pain, and injury; and Method of euthanasia and the reasons for its selection. Transgenic mice (category C) will be used because the experiments rely on the expression of CheRiff and QuasR2 for optical stimulation/recording. There are no alternative species. All experiments are terminal. Newborn mice will be cooled in ice prior to cervical dislocation and excision of the brain for culture preparation. All procedures are in accordance with guideleines of the NYU Animal Welfare Committee. The estimated number of animals below ensure that we have a continuous supply of cultures to perform experiments on a daily basis. Based on our experience in the past 3 years, each postnatal day 0-1 (P0-P1) mouse will generate at least 5 culture preparations. A viable culture will have a have a high density of neurons that express the Floxopatch construct, which varies from mouse to mouse. Thus, if one culture is not viable, all cultures derived from that mouse are also unusable. The success rate is approximately 50%. Because cultures can be made only form P0-1 day old mice and because it is not possible to determine whether expression is sufficient until the day of the experiment 2-3 weeks later, we will need to make 2 sets of cultures per week. Thus, we will need to maintain 2 breeding pairs per week or 8 breeding pairs per month to have a continuous supply of cultures. The breeding pairs will either consist of wildtype mice (for viral injection) or Vglut1-IRES2-Cre - Gt(ROSA)26Sor (floxopatch) mice. Breeding pairs will be replaced every six months. Total animals for experiments: 32 mice/year (=16 mice (8 breeding pairs) x 2/year replacement of breeding pairs). To maintain the 3 lines, 2 males and 2 females each of Vglut1-IRES2-Cre, Gt(ROSA)26Sor, and wildtype mice will be kept in separate cages (4+4+4=12 mice total). Every 6 months (twice per year), the mice of each type will be bred. From their offsprings, 4 males and 4 females from each line will be weaned and kept in separated cages as above (4+ 4 + 4 = 12) and the 4 original breeding pairs (12 mice) euthanized. Overall, 24 mice/year will be used to maintain the lines. Total mice/year: 56 (=24 mice/year to maintain lines + 32 mice/year for experiment). Total mice over 3 years = 168 mice. Reference Lou et al., (2016) Genetically Targeted All-Optical Electrophysiology with a Transgenic Cre-Dependent Optopatch Mouse, J. Neurosci. 36:11059-73 1 53 Results from previous support from CRCNS The P.I. and co-P.I. have not received previous CRCNS support Coordination Plan Division of labor The P.I. (A. Reyes) and his group at NYU will perform the experiments and analyses detailed in Aims 1 and 2 of this proposal. The co-P.I (S. Fusi) and his group at Columbia University will be responsible for the development of the theory and computational models that will incorporate the experimental data. Data Sharing between NYU and Columbia groups: Analyzed data, model codes, and manuscripts in progress will be disseminated between the groups via Google drive. All members of the Fusi and Reyes groups will have access upon email request. Larger, raw data will be stored on local servers and accessible via FTP. Coordination between NYU and Columbia University Because NYU and Columbia are easily acces- sible by subway, we will have monthly joint lab meetings, alternating between NYU and Columbia. Graduate students and postdoctoral fellows will be encouraged to submit joint poster presentations or talks to annual meetings such as Cosyne and Society for Neuroscience Meetings. 16 54