Imaging dendritic spines across the ovarian cycle in the awake, intact mouse

 描述(申请人提供)：人们普遍假设，记忆存储在大脑中，作为神经元之间的接线图和突触连接强度的持久变化。大多数突触的接受结构是树突棘，这是一个微米级的突起，从神经元的树突发出，感受由紧密相对的突触前终末释放的神经递质。一些证据表明，新皮质中树突棘的密度和周转率受到学习的影响，并与学习速度呈正相关。自20世纪90年代初以来，人们已经证实，在雌性大鼠、小鼠和非人灵长类动物的卵巢周期中，树突棘密度的变化约为35%，动情前期和动情间期的树突密度较高，动情期的树突密度较低。同样，手术切除卵巢会导致脊柱密度下降约40%，这可以通过17-β-雌二醇(E2)治疗迅速逆转。这些发现提出了一个重要的问题：如果记忆主要编码在带刺的突触中，如果棘突密度在卵巢周期中波动约40%，那么面对这种波动，雌性哺乳动物如何保持长期记忆？为什么记忆力不会随着每个卵巢周期而退化？到目前为止，对与卵巢周期相关的脊柱的测量依赖于应用于死后组织的传统解剖学方法。这些方法排除了跨越时间的动物内和树突内的比较。在这里，我们建议使用体内双光子显微镜和现有的转基因小鼠品系(包括用于稀疏标记第5层锥体细胞的Thy1M-EGFP)来产生新皮质中已识别的带刺树突的延时图像，从而解决两个关键问题。目标1：当卵巢切除后失去脊椎时，雌激素水平升高后，新的脊椎是否会在相同的树突位置重新生长？试点研究将使用传统的组织学技术来确定在I-III层中可以看到最大和最可靠的脊椎丢失/恢复的新皮质区域。 用于指导颅窗的放置，以进行延时成像、过骨切除或假手术以及随后的E2或赋形剂治疗。目标2：在发情前期/发情间期，当雌激素水平再次上升时，当脊椎在发情期间丢失时，它们是否倾向于在相同的树突位置重新生长？与目标1一样，最初的组织学实验将指导颅窗的放置。然后，每天的监测将与雌二醇酶联免疫吸附试验和卵巢周期状态的阴道拭子检测一起开始。树突棘的延时成像以及通过外源性处理(目标1)或自然周期(目标2)操纵雌激素水平，将使我们能够确定在低雌激素条件下丢失的树突棘是否在高雌激素水平恢复时在相同的树突位置重新生长。几个周期的成像(目标2)将使我们能够确定发情期脊柱丢失是否倾向于发生在特定的脊柱位置或脊柱类型的子集，这些位置或类型定义了高周转池。最后，我们将使用Thy1M-GFP小鼠在丘脑后内侧注射编码红色荧光团的病毒作为第一次尝试，以确定再生的脊椎是否重新接触它们在新皮质第一层的原始丘脑皮质轴突。这项工作将具有重要的临床意义 用于卵巢切除或更年期后的激素替代治疗。

 DESCRIPTION (provided by applicant): It is widely hypothesized that memory is stored in the brain as enduring changes in the wiring diagram and strength of synaptic connections between neurons. The receptive structure for most of these synapses is the dendritic spine, a micron-scale protrusion emitted from the neuron's dendrite which senses neurotransmitter released by the closely-opposed presynaptic terminal. Several lines of evidence suggest that dendritic spine density and turnover in the neocortex is modified by learning and is positively correlated with learning rate. Since the early 1990s, it has been established that dendritic spine density varies by about 35% over the course of the ovarian cycle in female rats, mice and nonhuman primates, being higher in proestrus and diestrus and lower in estrus. Similarly, surgical ovariectomy leads to an ~40% loss of spine density which can be rapidly reversed by 17-beta-estradiol (E2) treatment. These findings have suggested an important question: If memory is largely encoded in spiny synapses and if spine density fluctuates by ~40% over the ovarian cycle, then how does long-term memory persist in female mammals in the face of this fluctuation? Why doesn't memory degrade with each ovarian cycle? To date, measurements of spines in relationship to the ovarian cycle have relied upon traditional anatomical methods applied to postmortem tissue. These methods preclude within-animal and within-dendrite comparisons across time. Here, we propose to use in vivo two-photon microscopy together with existing transgenic mouse lines (including Thy1M-EGFP for sparse labeling of layer 5 pyramidal cells) to produce time-lapse images of identified spiny dendrites in the neocortex and thereby address two crucial questions. Aim 1: When spines are lost following ovariectomy, do new spine regrow in those same dendritic locations when estrogen levels rise following E2 treatment? Pilot studies will use traditional histological techniques to determine the regions of the neocortex in which the largest and most reliable loss/recovery of spines can be seen in layers I - III. This information will then be used to guide the placement of cranial windows for time-lapse imaging spanning overiectomy or sham surgery and subsequent E2 or vehicle treatment. Aim 2: When spines are lost during estrus do they tend to regrow in that same dendritic location when estrogen levels rise again in proestrus/diestrus? As in Aim 1, initial histological experiments will guide the placement of cranial windows. Then, daily monitoring will commence together with E2 ELISA and vaginal swab assays of ovarian cycle status. Time-lapse imaging of dendritic spines together with manipulation of estrogen levels either though exogenous manipulation (Aim 1) or natural cycles (Aim 2) will allow us to determine whether dendritic spines lost during low-estrogen conditions are regrown at the same dendritic location when high estrogen levels return. Imaging over several cycles (Aim 2) will allow us to determine if spine loss in estrus tends to occur in a particular subset of spine locations or spine types that define a high turnover pool. Finally, we will use Thy1M-GFP mice injected with virus encoding red fluorophore in posteromedial thalamus as a first attempt to determine whether regrown spines are re-contacting their original thalamo-cortical axons in neocortical layer 1. This work shall have important clinical implications for hormone replacement therapy after ovariectomy or menopause.