Abstract
Cortico-cortical interactions play crucial roles in various brain functions. Here, we present a detailed surgical procedure for cortical voltage-sensitive dye (VSD) imaging that allows monitoring of spatiotemporal dynamics in cortical activity in living mice. Cortical neurons in the upper layers (layer 1-3) are stained with a VSD, and an image sensor with a fast sampling rate (500 Hz) detects fluorescent changes in corrective activity. The procedure includes fixing a mouse brain to a stereotaxic apparatus, craniotomy on a large cortical area, VSD staining, and wide-field imaging of cortical activity. The entire procedure can be completed in 5 h (from the administration of anesthesia to the start of cortical VSD imaging).
Keywords: Voltage-sensitive dye imaging, Craniotomy, Cortical activity, Wide-field imaging
Materials and Reagents
Equipment
Software
Procedure
All animal experiments were performed in accordance with institutional guidelines and were approved by the Animal Experiment Committee from the RIKEN BSI.
Notes
To prevent phototoxicity of the dye to neurons, the intensity of the excitation light and exposure time should be minimized. We could obtain stable imaging for 2 h when we use 4 sec exposure time every 40 sec. Proper washing of the unbinding dye is helpful in improving the single-to-noise ratio. However, excessive washing decreases stable imaging time.
Recipes
Acknowledgments
This protocol was adapted from Manita et al. (2015). The authors acknowledge support from a Grant-in-Aid for Young Scientists (A) from the JSPS (Japan Society for the Promotion of Science), the Uehara Memorial Foundation, and the Japan Prize Foundation to M. M.; from a Grant-in-Aid for Challenging Exploratory Research from the JSPS to T. S.
References
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