Investigating the Calcium Signaling at Ribbon Synapses
Untersuchung von Kalzium-Signalen an Bändersynapsesn
by Thomas Frank
Date of Examination:2010-04-30
Date of issue:2010-05-11
Advisor:Dr. Tobias Moser
Referee:Prof. Dr. Detlev Schild
Persistent Address:
http://dx.doi.org/10.53846/goediss-3319
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Abstract
English
Using whole-cell patch-clamp and fast confocal Ca2+ imaging we show that localized Ca2+ influx sites exist at ribbon synapses in mature inner hair cells of the mouse. Spot detection measurements at these fluorescent hot-spots allowed their precise kinetic characterization, revealing rapid build-up and cessation within few milliseconds. We find that these Ca2+ microdomains represent Ca2+ influx through presynaptic clusters of CaV1.3 channels and that Ca2+-induced Ca2+ release does not play a role in shaping these responses. They were ob-served under a range of different Ca2+ buffering conditions including the presence of endogenous buffers. A combined model of Ca2+ reaction-diffusion and imaging predicted the experimentally observed responses reasonably well, under a set of different conditions. The amplitude and the voltage-dependence of these Ca2+ microdomains showed pronounced variability across individual synapses within single cells. A number of control experiments indicate that this variability is largely of biological origin and only to a minor degree introduced by the experimental observations. Different manipulations, including interference with Ca2+ buffering, Ca2+ channel gating and Ca2+ homeostasis left the variability of Ca2+ microdomain amplitudes unchanged. However, we found substantial variability in the antigen-content between presynaptic Ca2+ channel clusters as well as a positive correlation between the size of the active zone as indicated by a size estimate of the synaptic ribbon and the amplitude of its associated Ca2+ microdomain signal. We propose that individual active zones within a given hair cell differ in the number and gating of Ca2+ channels, resulting in different amplitudes and voltage-dependencies of synaptic Ca2+ influx. These specializations provide two levels of regulation to d ifferentially adjust synaptic transfer characteristics at individual synapses. Im-plementation of our results into a model of the inner hair cell to auditory nerve fiber synapse further suggests a strong impact of presynaptic Ca2+ channel number on the firing characteris-tics of the postsynaptic auditory nerve fibers and thus sound coding. We apply this technique to investigate presynaptic function at different tonotopic locations, revealing that unlike in other species mice do not adjust the characteristics but rather the number of IHC ribbon synapses in a frequency-dependent manner.
Keywords: calcium microdomain; imaging; ribbon synapse; hair cell; auditory system; modeling; coding
Other Languages
Mittels "whole-cell patch-clamp" und schneller, konfokaler Ca2+-Bildgebung werden lokalisierte Ca2+ Signale an einzelnen Bändersynapsen reifer innerer Haarzellen der Maus beschrieben. Des weiteren wird die Abhängigkeit dieser Signale von einer Reihe unterschiedlicher Parameter, inklusive Ca2+-Pufferung, Ca2+-induzierte Ca2+-Freisetzung, und Lage in der Cochlea untersucht. Sowohl die Amplitude als auch die Spannungsabhängigkeit der Ca2+-Signale zeigt ausgeprägte intrazelluläre Heterogeneität über unterschiedliche Synapsen hinweg. In Bezug auf die Amplitudenvariation erscheint eine unterschiedliche Anzahl von Ca2+-Kanälen als wahrscheinliche Erklärung. Zuletzt werden mögliche Auswirkungen der unterschiedlichen präsynaptischen Ca2+-Kanal-Anzahl auf die Antworteigenschaften postsynaptischer Spiral-Ganglion-Neurone theoretisch untersucht sowie diskutiert.
Schlagwörter: Kalzium-Mikrodomäne; Mikroskopie; Bändersynapse; Haarzelle; auditorisches System; Modellierung; Kodierung