Cao Vania Y, Ye Yizhou, Mastwal Surjeet S, Lovinger David M, Costa Rui M, Wang Kuan H
Unit on Neural Circuits and Adaptive Behaviors, Genes Cognition and Psychosis Program, National Institute of Mental Health, USA.
J Vis Exp. 2013 Jan 5(71):50148. doi: 10.3791/50148.
The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc). The transcription of Arc is rapidly and highly induced by intensified neuronal activity and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes.
大脑响应经验而发生变化的能力对于健康的脑功能至关重要，而这一过程中的异常会导致多种脑部疾病。为了更好地理解脑回路对动物经验作出反应的机制，需要具备在活体动物中长时间监测特定神经元群体中依赖经验的分子变化的能力。虽然已知经验和相关神经活动会触发神经元中的基因表达变化，但大多数检测此类变化的方法不允许在多天内对同一神经元进行重复观察，或者没有足够的分辨率来观察单个神经元。在此，我们描述了一种方法，该方法将体内双光子显微镜与基因编码的荧光报告基因相结合，以追踪日常经验过程中单个皮质神经元中依赖经验的基因表达变化。一个已被充分证实的依赖经验的基因是活性调节细胞骨架相关蛋白（Arc）。Arc的转录会被增强的神经元活动迅速且高度诱导，其蛋白质产物调节谷氨酸受体的内吞作用和长期突触可塑性。Arc的表达已被广泛用作绘制参与特定行为的神经回路的分子标记。在大多数此类研究中，通过固定脑切片中的原位杂交或免疫组织化学检测Arc的表达。尽管这些方法揭示了行为经验后Arc的表达定位于一部分兴奋性神经元，但未研究Arc表达的细胞模式如何随数天内多次重复或独特的经验而变化。体内双光子显微镜提供了一种强大的方法来检查活体大脑中依赖经验的细胞变化。为了能够通过双光子显微镜检查活神经元中的Arc表达，我们之前构建了一个敲入小鼠品系，其中绿色荧光蛋白（GFP）报告基因置于内源性Arc启动子的控制之下。本方案描述了在活体动物中追踪神经元群体中依赖经验的Arc-GFP表达模式的手术准备和成像程序。在该方法中，首先在Arc-GFP小鼠的感兴趣皮质区域植入慢性颅骨视窗。然后在几天的时间里，在期望的行为范式后，通过双光子显微镜对这些动物进行反复成像。该方法可能普遍适用于携带其他依赖经验的分子变化荧光报告基因的动物。
