[Super-resolution optical microscopy of the organ of Corti. Investigations on the fine structure of the inner hair cell afferent synapse by the 4Pi and STED techniques]. / Höchstauflösende optische Mikroskopie am Corti-Organ. Feinstrukturaufklärung an der afferenten Synapse innerer Haarzellen mittels 4Pi- und STED-Technik.

BACKGROUND: Inner hair cells encode sound into action potentials in the auditory nerve. Spiral ganglion neurons form the afferent innervation of inner hair cells via the hair cell synapse. The structure and function of this ribbon-type synapse is considered to have a major impact on the sound encoding process itself. In this study we have used conventional confocal microscopy as well as super-resolution techniques to investigate the synaptic organization in the inner hair cells of mice. MATERIAL AND METHODS: Functionally relevant proteins of the afferent inner hair cell synapse were selectively marked using immunohistochemical methods and investigated with conventional confocal and super-resolution 4Pi- and stimulated emission depletion (STED) techniques. RESULTS: Synapse and innervation density was mapped over the entire tonotopic axis. We found inner hair cells in the region of best hearing to have about twice the number of afferent fibres compared to the apex or base of the cochlea. For the first time 4Pi and STED microscopic techniques were employed to resolve the fine structure of these synapses beyond the resolution of conventional light microscopy. With 4Pi a resolution of approximately 100 nm in the z-axis direction is feasible. In practice STED delivers an effective resolution between 150 and 30 nm, depending on the power of the lasers employed. Synapses at different tonotopic positions of the cochlea exhibit no relevant structural differences at this level of resolution. The 4Pi and STED microscopic techniques are capable of showing the structure of afferent synapses in the organ of Corti with unsurpassed resolution. These images contribute to our understanding of sound-encoding mechanisms in the inner ear.

Synaptic organization in cochlear inner hair cells deficient for the CaV1.3 (alpha1D) subunit of L-type Ca2+ channels.

Cochlear inner hair cells (IHCs) release neurotransmitter onto afferent auditory nerve fibers in response to sound stimulation. Normal development and function of inner hair cells require the expression of alpha subunit 1.3 forming L-type voltage-gated Ca(2+) channel (Ca(V)1.3). Here, we used immunohistochemistry and reverse transcription-polymerase chain reaction to study the synaptic organization and expression of large conductance Ca(2+)-activated potassium channels in IHCs of mice lacking the Ca(V)1.3 Ca(2+) channel (Ca(V)1.3(-/-)). Despite the near complete block of evoked afferent synaptic transmission, hair cell ribbon synapses were formed and remained preserved for at least 4 weeks after birth. Moreover, these "silent" afferent synapses held major components of the synaptic machinery such as Bassoon, Piccolo, and CSP. Hence, the block of exocytosis might be solely attributed to the lack of Ca(2+) influx through Ca(V)1.3 channels. Later on, Ca(V)1.3 deficient IHCs subsequently lost their afferent synapses. This was probably due to a secondary degeneration of the postsynaptic spiral ganglion neurons. In line with a prolonged efferent synaptic transmission onto Ca(V)1.3 deficient IHCs, which normally ceases around onset of hearing, we found juxtaposed immunoreactive spots of efferent presynaptic synaptophysin and postsynaptic (IHCs) small conductance Ca(2+)-activated potassium channels (SK channels) up to six weeks after birth. Finally, we show a substantial reduction of mRNA for the alpha subunit of the large conductance Ca(2+)-activated potassium channel (BK) in the apical cochlea, suggesting a reduced transcription of its gene in Ca(V)1.3 deficient IHCs. Ca(V)1.3 deficient IHCs lacked the apical spot-like immunoreactivity of clustered BK channels, which normally contribute to the temporal precision of hair cell afferent synaptic transmission. In summary, these data indicate that the Ca(V)1.3 channels are crucially involved in regulation of the expression of BK and SK channels. Ca(V)1.3 channels seem not to be essential for ribbon synapse formation, but are required for the maintenance of ribbon synapses and spiral ganglion neurons.