Abstract:
:One of the challenges for modern neuroscience is to understand the rules of concerted neuronal function in vivo. This question can be addressed using noninvasive high-resolution imaging techniques like two-photon microscopy. This protocol describes a versatile approach for in vivo two-photon calcium imaging of neural networks, stained with membrane-permeant fluorescent-indicator dyes. It is based on a targeted pressure ejection of the dye into the tissue of interest and can be used for a large spectrum of indicator dyes, including Oregon Green 488 BAPTA-1 acetoxymethyl ester and Fura-2 acetoxymethyl ester. Through the use of dye mixtures and multicolor imaging, this technique allows the visualization of distinct neurons and glial cells up to 500 microm below the brain surface. It is suitable for staining the brain tissue of various different species (e.g., mouse, rat, cat and zebrafish) at all developmental stages. When combined with brain microendoscopy, it allows the monitoring of intracellular calcium signals in awake, behaving animals. The total time required to carry out the protocol, including dissection and cell staining, is approximately 2 h. Thereafter, imaging experiments might be performed for at least 6 h.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Garaschuk O,Milos RI,Konnerth Adoi
10.1038/nprot.2006.58subject
Has Abstractpub_date
2006-01-01 00:00:00pages
380-6issue
1eissn
1754-2189issn
1750-2799pii
nprot.2006.58journal_volume
1pub_type
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