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In vivo two-photon imaging of experience-dependent molecular changes in cortical neurons. | LitMetric

In vivo two-photon imaging of experience-dependent molecular changes in cortical neurons.

J Vis Exp

Unit on Neural Circuits and Adaptive Behaviors, Genes Cognition and Psychosis Program, National Institute of Mental Health, USA.

Published: January 2013

AI Article Synopsis

  • The brain's adaptability to experiences is crucial for its health, and disruptions in this process can lead to various disorders.
  • Current methods to monitor molecular changes in neurons during these experiences are limited, lacking the ability to observe the same neurons repeatedly over time.
  • This research introduces a technique combining in vivo two-photon microscopy and a fluorescent reporter, enabling tracking of the experience-dependent gene Arc in individual living neurons across multiple days of activity.

Article Abstract

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.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3582675PMC
http://dx.doi.org/10.3791/50148DOI Listing

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