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Targeted axon-attached recording with fluorescent patch-clamp pipettes in brain slices
Takuya Sasaki1, Norio Matsuki, Yuji Ikegaya
1Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan. t.sasaki.0224@gmail.com
Nature Protocols
|June 2, 2012
Summary
This study presents a new protocol for recording from narrow central nervous system axons. The method uses advanced microscopy and specialized pipettes for successful cell-attached recordings of axonal action potentials.
Area of Science:
- Neuroscience
- Cellular Physiology
- Neurobiology
Background:
- Understanding central nervous system (CNS) physiology necessitates direct experimental access to intact axons.
- Investigating the function of narrow axon fibers (<1 μm) presents significant technical challenges.
Purpose of the Study:
- To describe a reliable protocol for performing cell-attached recordings from narrow CNS axon fibers.
- To enable visual targeting and recording from fluorescently labeled axons in brain slice preparations.
Main Methods:
- Utilizing fluorophore-coated glass pipettes for precise axon labeling.
- Employing Nipkow disk confocal microscopy for online optical control and targeting.
- Performing cell-attached recordings in acute and cultured brain slice preparations.
Main Results:
- Achieved a success rate of approximately 50% for cell-attached recordings from narrow axons.
- Successfully recorded axonal action potentials as extracellular unit-like, sharp negative currents.
- Demonstrated stable recordings lasting at least 30 minutes.
Conclusions:
- The described protocol provides a viable method for studying the physiology of narrow CNS axons.
- This technique facilitates the investigation of axonal function in both acute and cultured brain slice models.
- The protocol is efficient, with labeling and recording completed within 1-2 hours.
