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

 描述（由申请人提供）：人们普遍假设，记忆是作为神经元之间突触连接的接线图和强度的持久变化存储在大脑中的。大多数突触的接受结构是树突棘，这是一种从神经元树突发出的微米级突起，它可以感知由紧密相对的突触前末梢释放的神经递质。几条证据表明，树突棘密度和营业额在新皮层的学习修改，并与学习率呈正相关。自20世纪90年代初以来，已经确定在雌性大鼠、小鼠和非人灵长类动物的卵巢周期过程中，树突棘密度变化约35%，在发情前期和发情间期较高，发情期较低。同样，卵巢切除术会导致脊柱密度损失约40%，但可以通过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：如果记忆主要是在棘状突触中编码的，如果棘密度在卵巢周期中波动约40%，那么面对这种波动，雌性哺乳动物的长期记忆是如何保持的？为什么记忆不会随着每个卵巢周期而退化？到目前为止，测量脊柱与卵巢周期的关系依赖于传统的解剖学方法应用于死后组织。这些方法排除了跨时间的动物内和树突内比较。在这里，我们建议使用体内双光子显微镜与现有的转基因小鼠品系（包括Thy 1 M-EGFP用于稀疏标记第5层锥体细胞），以产生新皮层中已识别的棘状树突的延时图像，从而解决两个关键问题。目标1：当卵巢切除术后脊柱丢失时，当雌激素水平在E2治疗后升高时，新的脊柱会在相同的树突位置重新生长吗？初步研究将使用传统的组织学技术来确定新皮层的区域，在这些区域中，可以在层I-III中看到最大和最可靠的棘丢失/恢复。这些信息将 用于指导颅骨窗的放置，以进行跨覆盖切除术或假手术以及随后的E2或载体治疗的延时成像。目标二：当在发情期失去棘时，当雌激素水平在发情前期/间情期再次升高时，它们是否倾向于在同一树突位置重新生长？与目标1一样，最初的组织学实验将指导颅窗的放置。然后，每日监测将与E2 ELISA和阴道拭子检测卵巢周期状态一起开始。树突棘的延时成像以及通过外源性操作（Aim 1）或自然周期（Aim 2）来操纵雌激素水平，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返回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定义高周转池的脊柱位置或脊柱类型的特定子集中。最后，我们将使用Thy 1 M-GFP小鼠注射病毒编码的红色荧光团在后内侧丘脑作为第一次尝试，以确定是否重新生长的脊椎重新接触其原始的丘脑皮质轴突在新皮层层1。这项工作将具有重要的临床意义 用于卵巢切除术或绝经后的激素替代治疗。

 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.