Cell type-specific calcium imaging during hippocampus-dependent memory

描述（由申请人提供）：来自人类和动物的证据表明，海马体在长期情景记忆的形成中起着关键作用。海马体由解剖学上分离的亚区组成，这些亚区具有多种神经元类型，可以编码经历的不同方面。尽管海马体神经元活动依赖性可塑性的分子机制已被深入研究，但我们对特定神经元群体在记忆编码、巩固和回忆过程中所表现出的活动模式的理解有限。因此，迫切需要开发新的成像方法，以便在海马回路被认为支持的行为的背景下询问海马回路的活动。由于神经元尖峰与细胞内大的钙传导有关，钙指标可以用作神经元激活的代理。在拟议的研究中，我们将使用光纤荧光内镜（FFE）来长期监测海马依赖任务小鼠中特定海马神经元的激活。基因编码的钙指示物可在CA1锥体细胞、齿状颗粒细胞和CA3锥体细胞中选择性表达Cre重组酶的转基因小鼠中有条件地表达。我们在完全清醒、不受限制的小鼠中进行的初步原理验证实验表明，这种方法对于单个神经元活动的大规模成像是可行的。在我们的第一个目标中，我们将优化FFE所需的参数，以实现不同神经元群体的稳定、长期成像。在我们的第二个目标中，我们将使用这种方法来定义编码情境恐惧条件反射和痕迹恐惧条件反射的神经元集合，这是两种依赖海马体的联想记忆变体，假设它们参与不同的回路。延时实验可以比较在训练、巩固和回忆过程中观察到的激活模式。通过对这些记忆阶段中的每一个进行遗传或药理干预，我们将把选定神经元的活动与认知联系起来。因此，拟议的研究将通过引入FFE作为研究记忆机制的一种新颖的体内方法来推进该领域，并将使用这种技术开始解决以前无法解决的记忆细胞基础问题。

DESCRIPTION (provided by applicant): Evidence from humans and animals indicate a critical role for the hippocampus in the formation of long- term episodic memories. The hippocampus consists of anatomically segregated subregions with a diversity of neuronal types hypothesized to encode different aspects of an experience. Although the molecular mechanisms of activity-dependent plasticity in hippocampal neurons have been intensely scrutinized, we have limited understanding of the activity patterns shown by select neuronal populations during memory encoding, consolidation and recall. Therefore, there is a strong demand to develop new imaging approaches that can interrogate the activity of hippocampal circuits in the context of the behavior that they are thought to support. Because neuronal spikes are associated with large intracellular calcium conductances, calcium indicators can be used as a proxy for neuronal activation. In the proposed research, we will implement the use of fiber optic fluorescence endomicroscopy (FFE) to chronically monitor the activation of defined hippocampal neurons in mice subjected to hippocampus-dependent tasks. A genetically-encoded calcium indicator will be conditionally expressed in transgenic mice selectively expressing Cre recombinase in CA1 pyramidal cells, or in dentate granule cells and CA3 pyramidal cells. Our initial proof-of-principl experiments in fully awake, unrestricted mice advocate the feasibility of this approach for large-population imaging of single-neuron activity. In our first aim, we will optimize the parameters of FFE required to achieve stable, long-term imaging of the different neuronal populations. In our second aim, we will use this method to define the neuronal ensembles that encode contextual- and trace-fear conditioning, two hippocampus-dependent variants of associative memory hypothesized to engage different circuits. Time-lapse experiments will allow comparisons of the activation patterns observed during training, consolidation and recall. Through genetic or pharmacological interventions against each one of these memory phases, we will link the activity of select neurons to cognition. Thus, the proposed research will advance the field by introducing FFE as a novel in vivo approach to study memory mechanisms, and will use this technique to begin to address previously inaccessible questions on the cellular basis of memory.