A procedure to label inner ear afferent nerve endings for calcium imaging.
Characterization of synaptic transmission between the inner ear sensory cells and primary neuron dendrites has been hampered by the limited access to the postsynaptic terminals. Because direct physiological recording of postsynaptic currents are difficult to achieve, no information regarding the synaptic and dendritic events are available. This is due to the small size of the postsynaptic afferent nerve endings that do not allow a clear identification, and thus compromise direct electrophysiological recordings of the buttons. To study the physiology of afferent nerve endings, we have developed a two-photon imaging technique in cochlear and vestibular slice preparations from neonatal rats and turtles. This technique is based on a retrograde labeling of afferent nerve endings with high-affinity calcium-sensitive dyes. Dye filling was achieved by 6 h application of the dextran-amine conjugate of calcium green-1. Calcium changes were measured in afferent nerve endings in line scan and time lap mode. To address recording in a near-physiological situation, iontophoretic application of K+ was performed in the area of the stereocilia whereas glutamate was applied at the basal pole of sensory hair cells. Both types of application cause a reversible and sustained increase of Ca2+ in the button of afferent nerve fibers. Typical recordings are presented and potential interests for pharmacological studies of inner ear sensory cell synapses are discussed.
Boyer
Samuel
S
U 583 INSERM-Physiopathologie et Thérapie des Déficits Sensoriels et Mateurs, Université Montpellier 1 and 2-Hôpital Saint Eloi, 34090 Montpellier Cedex 5, France.
Ruel
Jérôme
J
Puel
Jean-Luc
JL
Chabbert
Christian
C
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D003051:Cochlea / Q000166:cytology / Q000294:innervation* / Q000502:physiology; D004396:Coloring Agents; D004594:Electrophysiology / Q000379:methods; D005456:Fluorescent Dyes; D018698:Glutamic Acid / Q000494:pharmacology; D006198:Hair Cells, Auditory / Q000187:drug effects / Q000502:physiology; D009930:Organic Chemicals; D011188:Potassium / Q000494:pharmacology; D017729:Presynaptic Terminals / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D051381:Rats; D017208:Rats, Wistar; D011984:Sensory Receptor Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D013136:Spiral Ganglion / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D013194:Staining and Labeling / Q000379:methods*; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology; D014426:Turtles; D014725:Vestibular Nerve / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D014722:Vestibule, Labyrinth / Q000166:cytology / Q000294:innervation* / Q000502:physiology; D000159:Vestibulocochlear Nerve / Q000166:cytology / Q000187:drug effects / Q000502:physiology
D004396:Coloring Agents; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C071336:calcium green; D018698:Glutamic Acid; D011188:Potassium; D002118:Calcium