This work was supported by a Deafness Research Foundation Research Grant to EY and NIDCD DC006476 to EG and by NIDCD DC005211 to the Center for Hearing and Balance, Johns Hopkins University. Artwork copyright Tim Phelps, Johns Hopkins University.
LG wrote the initial manuscript; EY and LG filmed the dissection and the recording. All authors provided exemplar figures and contributed to writing the manuscript.

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No conflicts of interest declared.

The critical step in this procedure is the dissection. If the tissue is stretched or damaged during the dissection, afferent fibers will not survive. Tissue from younger rats is more elastic and forgiving. We find that postnatal days 10 to 11 are easiest to dissect and experiments have higher success rates. A significant degree of cochlear maturation occurs postnatally, with rats beginning to hear from around postnatal day 12 14. Therefore, at the age where the dissection is easiest, synapses may not be fully mature 4.

The dissection described here for rats is essentially the same for mice, the main difference being the smaller size of the mouse cochlea. This technique allows the properties of ribbon synapses to be examined in transgenically modified mice 15. Further modifications to this technique include: adding a fluorescent dye to the intracellular solution to label fibers 3; paired recordings with the presynaptic inner hair cell and postsynaptic afferent bouton, allowing the transfer function between pre and post synaptic cells to be determined 2 and loose-seal extracellular recordings at afferent boutons to avoid loss of cellular integrity. The extracellular recording configuration is easier to attain than the whole-cell configuration and experiments are generally longer lasting.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Air table</td>
<td>&nbsp;</td>
<td>TMC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Gibraltar Stage with xy-table</td>
<td>&nbsp;</td>
<td>Burleigh</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Axioscope FS2 upright microscope DIC optics Green filter</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Newvicon camera with controller</td>
<td>&nbsp;</td>
<td>Dage</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Monitor</td>
<td>&nbsp;</td>
<td>Dage</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Multiclamp 700B (or similar)</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Digidata 1322A (or similar)</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
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<td>Manipulator MP285</td>
<td>&nbsp;</td>
<td>Sutter </td>
<td>&nbsp;</td>
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<td>6-channel valve application system for local perfusion (used with hand made perfusion pipettes) </td>
<td>&nbsp;</td>
<td>Warner</td>
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<td>&nbsp;</td>
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<td>PC with acquisition software (PClamp)</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
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			Above is the equipment in our electrophysiological setups used for recording from afferent terminals.		</div>

postsynaptic recordings at afferent dendrites contacting cochlear inner hair cells: monitoring multivesicular release at a ribbon synapse

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse 1-4. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential 5. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes 1, 4, 6-11. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood.
The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 &mu;m in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly 2. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.

Watch this Scientific Journal Video about Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse at JoVE.com

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian cochlea.

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the ...

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