Alexander Egner, Tobias Moser, and Leica Microsystems CMS GmbH

The goal of this project is to establish fluorescence measurements of nanometer-scaled neuronal [Ca2+] signals using stimulation emission depletion (STED) microscopy in combination with patch-clamp and modelling.

Neurons are highly differentiated cells using a variety of subcellular functional domains such as dendritic spines or presynaptic active zones to perform their complex tasks. The function of these domains relies on tightly regulated and highly localized signalling, often involving Ca2+ as the key signal. Ultrastructure and physiological observations have long indicated that neuronal Ca2+ signalling occurs on the micrometer or even nanometer scale (reviewed in Neher, 1998b; Augustine et al., 2003; Moser et al., 2006). It became clear that these Ca2+ micro- or nanodomains are the key signalling units regulating neuronal function and its plasticity. Although some success has been reported in imaging such local Ca2+signals by fluorescence microscopy (Llinas et al., 1992; DiGregorio et al., 1999; Becherer et al., 2003; Zenisek et al., 2003), their structure is largely beyond the reach of conventional light microscopy. Hence, direct investigation of local neuronal Ca2+ signals, such as that governing transmitter release at the active zone of synapses, remained impossible. The development and technical implementation of STED microscopy has enabled light microscopy on the nanometer scale (Klar et al., 2000; Hell, 2003; Kastrup et al., 2005). However, these approaches hitherto solely served morphological studies on fixed samples.