В Vivo Двухфотонные изображений зависящие от опыта молекулярные изменения в корковых нейронов
Summary
Опыт зависит от молекулярных изменений в нейронах имеют важное значение для способности мозга к адаптации в ответ на поведенческие проблемы.<em> В естественных условиях</em> Двухфотонного изображений метод описан здесь, что позволяет отслеживать такие молекулярные изменения в отдельные нейроны коры через генетически закодирована журналистам.
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 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.
Protocol
Representative Results
Discussion
В методе естественных изображений описанный здесь позволяет повторную экспертизу д'Арк изменения экспрессии генов в той же набор нейронов в течение нескольких дней в живых животных. Это эффективный и универсальный метод получения информации о нейронной пластичности связ…
Declarações
The authors have nothing to disclose.
Acknowledgements
Авторы хотели бы поблагодарить Л. Belluscio для хирургии съемках оборудования, Д. Квон для съемок помощи, K. Liu для редактирования видео помощь, и К. Маклеод для всех фоновой музыки. KW признает щедрой поддержке Отдела NIMH очной исследовательских программ и гены, познание и психоз программы. Эта работа была поддержана NIMH Внутренние программы исследований (VC, YY, SMKW) и Отдела NIAAA очной клинической и биологической исследовательской программы (VC, RMC, DML).
Materials
| Name of the Equipment | Company | Catalogue number | Comments (optional) |
| FV1000 multi-photon laser scanning microscope | Olympus | FV1000MPE | Imaging |
| Dissection microscope | Omano | 555V107 | Surgery |
| Stereotaxis surgery stage for mice | Harvard Apparatus | 726335 | Surgery |
| 20X or 25X water immersion objective | Olympus | XLPL25XWMP | Imaging |
| Microscope stage with head-fixation frame | Custom made | N/A | Imaging |
| Fine forceps | Fine Science Tools | 11251-20 | Surgery |
| Dental drill burr | Fine Science Tools | 19007-05 | Surgery |
| CCD camera | QImaging | QICAM 12-bit | Imaging |
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