Optical recordings of Ca2+ signaling activities from identified inner ear cells in cochlear slices and hemicochleae.
One of the major obstacles hindering the progress of studies on mammalian cochlear physiology is the inaccessibility of inner ear cells located in a complex structure of the bony labyrinth. We describe here a technique to record cellular fluorescent signals from any identified inner ear cells in cochlear slices and hemicochleae. Cochlear slices were obtained from postnatal rats (P0-P7) before the cochlea completely ossify, and hemicochleae were cut from older animals (P7-adult). Individual inner ear cells were visually identified using infrared differential interference contrast or oblique illumination optics. Techniques were developed for either bulk-loading cells or loading selected single cells with Ca(2+) indicator dyes, and for maintaining functional viability of cochlear slices/hemicochleae for recordings. Robust and reliable responses of ligand-gated receptors were recorded from individual inner ear cells (e.g. hair cells, spiral ganglion neurons etc.) for at least 24 h after slices/hemicochleae were cut by an oscillating tissue slicer. The technique described here allowed direct observations of [Ca(2+)](i) activities from multiple cells simultaneously in situ, thus providing a feasible way to study the intercellular communication or networking activities from identified cells in the inner ear.
Section on Neurobiology, Leslie & Susan Gonda Department of Cell and Molecular Biology, 2100 West Third Street, House Ear Institute, Los Angeles, CA 90057, USA. xlin@hei.org
Xi
X
Lin
Paul
P
Webster
Qingxia
Q
Li
Shanping
S
Chen
Yannan
Y
Ouyang
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D004210:Dissection / Q000379:methods; D001330:Electronic Data Processing / Q000295:instrumentation / Q000379:methods*; D004594:Electrophysiology / Q000295:instrumentation / Q000379:methods*; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D005260:Female; D005456:Fluorescent Dyes; D006199:Hair Cells, Auditory, Inner / Q000254:growth & development / Q000502:physiology* / Q000648:ultrastructure; D008297:Male; D051379:Mice; D008855:Microscopy, Electron, Scanning; D009924:Organ Culture Techniques / Q000295:instrumentation / Q000379:methods*; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley
D018690:Excitatory Amino Acid Agonists; D005456:Fluorescent Dyes; D000255:Adenosine Triphosphate; D011188:Potassium