Proceedings of The Physiological Society

Physiology 2014 (London, UK) (2014) Proc Physiol Soc 31, PCB073

Poster Communications

Distribution of synaptic calcium signals in adult inner hair cells of the mouse cochlea

A. M. Garcia de Diego2, J. Ashmore1,2

1. Neuroscience, Physiology & Pharmacology, UCL, London, United Kingdom. 2. UCL Ear Institute, UCL, London, United Kingdom.

The output coding synapse of the mammalian cochlea is a ribbon synapse located around the base of the inner hair cell (IHC). Each IHC makes contact with 10-15 afferent dendrites. It has been known for many years that individual fibres modulate their firing to sound intensity over only a limited range and thus , to cover the full range of intensities, the fibre population is functionally segmented (Frank et al, 2009). Whether the response differences are pre- or post-synaptically determined is not known although, from immunohistochemistry, an inverse relationship exists between ribbon size and postsynaptic receptor area (Liberman et al, 2011) To examine the presynaptic heterogeneities of synaptic release we have detected calcium signals in IHCs of adult mice (CBA/J, P25-P120) as a measure of synapse activity. The temporal bone was removed after rapid killing of the mouse by cervical dislocation and then stuck down onto the recording chamber in normal extracellular perilymph. A small whole in the apical surface of the cochlea was opened to allow access for patch recording and for imaging using a multi photon confocal scanning microscope with a 63x 1.0 WI objective and operating with an excitation of 935 nm. In this preparation cells can be identified in their normal position and orientation in the organ of Corti. A calcium fluorophore OGB5N (200 μM in 0.5 mM EGTA) was introduced into IHCs by patch pipette recording. Cells were depolarized for 100 ms to -20mV from a holding potential of -70 mV and images acquired at up to 80 fps. Up to 4 discrete separable sites where calcium signals were detected were identified in each sectional plane taken in the basal half of an IHC. In cat, afferent fibres segregate depending on their sensitivities on a radial neural-abneural axis of the IHC (Liberman, 1982), a distinction which is has not been established in mouse. To address this issue we have collected calcium responses at multiple different z-planes, spaced by 1 μm and collected over at least a 12 min recording period to build up a 3D map of the calcium entry ‘hotspots'. Each spot was less than 1 μm in diameter, but spread over several sections as a result of the microscope optics. We found that the strongest signals were obtained at the basal pole with a slight emphasis towards the neural side. The peak hotspot signal intensity became progressively less pronounced when determined at more apical regions of the IHC. The results suggest that the functional distribution IHC neurotransmission properties must take a basal-apical axis of the cell into account as well as any radial distribution of synapse heterogeneity.

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