This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.
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Method Article
A Craniotomy Surgery Procedure for Chronic Brain Imaging
In This Article
Summary
Abstract
Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an extremely useful method, because it allows the study of the live intact brain. With appropriate preparations, this technique allows the observation of the same cortical area chronically, from minutes to months. In this video, we show a preparation for chronic in vivo imaging of the brain using two-photon microscopy. This technique was initially pioneered by Dr. Karel Svoboda, who is now a Howard Hughes Medical Institute Investigator at Janelia Farm. Preparations like the one shown here can be used for imaging of neocortical structure (e.g., dendritic and axonal dynamics), to record neuronal activity using calcium-sensitive dyes, to image cortical blood flow dynamics, or for intrinsic optical imaging studies. Deep imaging of the neocortex is possible with optimal cranial window surgeries. Operating under the most sterile conditions possible to avoid infections, together with using extreme care to do not damage the dura mater during the surgery, will result in successful and long-lasting glass-covered cranial windows.
Protocol
- Anesthetize mice with isoflurane (4% for induction, 1.5-2% for surgery) using IACUC approved procedures. It is important that tail and/or toe pinches are used in order to ensure the animal is fully sedated.
- Using a rodent trimmer, shave the hair from the back of the neck up to the eyes.
- Place the mouse in a stereotaxic frame, over a surgery water re-circulating blanket. Firmly secure the head with ear bars.
- Apply eye ointment, in order to prevent the animal's eye from drying out.
- Administer, subcutaneously, Dexamethasone (0.2 mg/Kg) and Carprofen (5 mg/Kg) to prevent swelling of the brain and/or an inflammatory response, respectively.
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Discussion
As we have shown in the video and in the supplementary figures, the cranial window preparation, combined with the use of two-photon microscopy, is a very powerful tool to study in vivo the structure and function of the neocortex. The technique requires rigorous training to become familiar with the relevant anatomy and the fine surgical procedures and skills that this preparation requires. Only pristine surgeries can be used for chronic imaging. If the dura is manipulated excessively or punctured, the preparation should .......
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Materials
| Name | Company | Catalog Number | Comments | |
|---|---|---|---|---|
| Carprofen (Rimadyl) | Drug | Pfizer Pharma GmbH | ||
| Isoflurane (Aerrane) | Surgery | Baxter Internationl Inc. | ||
| Dexamethasone | Drug | Baxter Internationl Inc. | ||
| Ortho-Jet Powder | Reagent | LANG | To be mixed with the acrylic | |
| Jet-Acrylic Liquid | Reagent | LANG | To be mixed with Ortho-Jet Powder | |
| Round Glass Cover Slip | Tool | Electron Microscopy Sciences | 72195-05 | 5 mm diameter |
| Gelfoam | Surgery | Pharmacia Corporation (Pfizer) | ||
| Titanium bars are custom-made |
References
- Svoboda, K., Denk, W., Kleinfeld, D., Tank, D. W. In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature. 85, 161-165 (1997).
- Lendvai, B., Stern, E. A., Chen, B., Svoboda, K. Experience-dependent plasticity of dendritic spines in the developing....
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