The authors would like to thank L. Belluscio for surgery filming equipment, D. Kwon for filming assistance, K. Liu for video editing assistance, and K. MacLeod for all background music. K.W. acknowledges the generous support of the NIMH Division of Intramural Research Programs and the Genes, Cognition and Psychosis Program. This work was supported by the NIMH Intramural Research Program (V.C., Y.Y., S.M. K.W.) and the NIAAA Division of Intramural Clinical and Biological Research Program (V.C., R.M.C., D.M.L.).

The experimental procedures described below were approved by the National Institute of Mental Health Animal Care and Use Committee and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
1. Pre-operative Preparation
<ol>
 <li>Clean all tools in a hot bead sterilizer before aseptic surgery, clean the surgery site with 70% ethanol, and put down clean drop cloths. Wear sterile gloves. Anesthetize the animal with freshly prepare...

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No conflicts of interest declared.

The in vivo imaging method described here enables repeated examination of Arc gene expression changes in the same sets of neurons over multiple days in the live animal. It is an efficient and versatile method to obtain information about neural plasticity-related molecular dynamics in individual neurons in response to various behavioral experiences. Standard histochemical methods such as in situ hybridization and immunostaining can achieve single-cell resolution3, but lack the ability to tra...

<table border="1">
<tbody>
 <tr>
 <td>Name 			of the Equipment</td>
 <td>Company</td>
 <td>Catalogue 			number</td>
 <td>Comments 			(optional)</td>
 </tr>
 <tr>
 <td>FV1000 			multi-photon laser scanning microscope</td>
 <td>Olympus</td>
 <td>FV1000MPE</td>
 <td>Imaging</td>
 </tr>
 <tr>
 <td>Dissection 			microscope</td>
 <td>Omano</td>
 <td>555V107</td>
 <td>Surgery</td>
 </tr>
 <tr>
 <td>Stereotaxis 			surgery stage for mice</td>
 <td>Harvard 			Apparatus</td>
 <td>726335</td>
 <td>Surgery</td>
 </tr>
 <tr>
 <td>20X 			or 25X water immersion objective</td>
 <td>Olympus</td>
 <td>XLPL25XWMP</td>
 <td>Imaging</td>
 </tr>
 <tr>
 <td>Microscope 			stage with head-fixation frame</td>
 <td>Custom 			made</td>
 <td>N/A</td>
 <td>Imaging</td>
 </tr>
 <tr>
 <td>Fine 			forceps</td>
 <td>Fine 			Science Tools</td>
 <td>11251-20</td>
 <td>Surgery</td>
 </tr>
 <tr>
 <td>Dental 			drill burr</td>
 <td>Fine 			Science Tools</td>
 <td>19007-05</td>
 <td>Surgery</td>
 </tr>
 <tr>
 <td>CCD 			camera</td>
 <td>QImaging</td>
 <td>QICAM 			12-bit</td>
 <td>Imaging</td>
 </tr>
 </tbody>
</table>

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

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 disorders1,2. 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 neurons1,2, 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 neurons3,4. 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)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. 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 brain8,9. 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 promoter10. 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 changes4.

This protocol describes a method to track experience-dependent molecular changes in individual cortical neurons in live animals. A chronic cranial window is first created over a cortical region of interest in a mouse carrying a fluorescent reporter of gene expression. Two-photon microscopy can then be coupled with various behavioral paradigms to observe behaviorally induced molecular changes in individual neurons and track such changes in the same sets of neurons over multiple days (Figure 1).
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Watch this Scientific Journal Video about In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons at JoVE.com

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

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 ...

in-vivo-two-photon-imaging-experience-dependent-molecular-changes

Research

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Neuroscience

21.5K visualizações.  National Institute of Mental Health. Experiência dependentes de alterações moleculares nos neurônios são essenciais para a capacidade do cérebro de se adaptar em resposta aos desafios comportamentais. Um In vivo Dois fótons método de imagem é descrito aqui que permite o acompanhamento de tais alterações moleculares em individuais neurônios corticais através de repórteres codificados geneticamente.