Moser Group

We are interested in the mechanisms that enable the impressive temporal precision and long-lasting reliability of synaptic sound encoding. Moreover, we would like to understand how the "dynamic range problem": i.e. perception of sound pressures over six orders of magnitude with encoding over only 1-2 orders of magnitude at each individual synapses is solved by the cochlea. We approach these questions by a systematic analysis of the molecular anatomy and physiology of the ribbon synapse in normal and molecularly manipulated hair cells in collaborations with the groups of Andreas Neef, Ellen Reisinger,Nicola Strenzke and Carolin Wichmann and partners outside the Department of Otolaryngology. Confocal, STED, PALM and 4Pi microscopy are applied to immunofluorescently labeled hair cells for quantification of protein/structure localization, as well as shape and size of structural elements of the synapse such as the ribbon, scaffolds and channel clusters. This work builds on a long-term collaboration with the Dept. of Nanobiophotonics of Stefan Hell at the MPI for biophysical Chemistry (link). Our findings are compared to electron microscopy and tomography studies performed in collaboration with Dietmar Riedel at the MPI for biophysical Chemistry (link) and Carolin Wichman. Functionally we perform patch-clamp measurements of presynaptic currents and membrance capacitance changes (e.g. Moser and Beutner, 2000), the latter reflecting exocytic and endocytic changes in cell surface. Uncaging of caged Ca²⁺ is used to study the intrinsic Ca²⁺ dependence of exocytosis (e.g. Beutner et al., 2001). Combining these approaches to synapse function with morphological studies we have gained insights into the role of the synaptic ribbon, the scaffold protein Bassoon (Khimich et al., 2005; Buran et al., 2010, Frank et al., 2010) and the multi-C2 domain protein Otoferlin (Roux et al., 2006; Pangrsic et al., 2010; Reisinger et al., 2011). Presynaptic patch-clamp is combined with confocal imaging of synaptic Ca²⁺ signaling and membrane turnover using pHluorins. Imaging enables investigation of single synapses as do patch-clamp recordings of postsynaptic boutons, pioneered by Elisabeth Glowatzki (Glowatzki and Fuchs, 2002). Imaging of synaptic Ca²⁺ signals have revealed an intriguing heterogeneity among the synapses of a given inner hair cell (Frank et al., 2009; Meyer et al., 2009).

Neef, Bernstein-Group

The Bernstein-Group at the InnerEarLab works on the interface of experimental biophysics/neuroscience and computation neuroscience. It emerged out of a fruitful collaboration of many years between the InnerEarLab and the group of Theoretical Neurophysics at the Max-Planck-Institute of Dynamics and Self-Organization. The focus lies on establishing quantitative models of signal transduction and sound encoding at the inner ear ribbon synapse. Some peculiar aspects of this system make it very interesting for both, synaptic physiology and computational neuroscience: For one, the molecular machinery of Ca²⁺-secrecion coupling is different from central synapses as key molecular players like complexin and, synaptotagmin are missing from the hair cell; consequently "standard" synaptic models might fail to adequately describe the hair cell synapse. Furthermore, each of the dozen or so presynaptic sites within a hair cell represent the sole input to exactly one auditory nerve cell and each of those nerve cells seems to encode different aspects of the incoming sound. On the biophysical side, the Bernstein-group deals with the advancement of methods and experimental paradigms to measure fundamental parameters of the presynaptic signaling cascade (Neef et al., 2007 a,b). On the side of computational neuroscience we establish biophysically motivated models of signal transduction, comprising the hair cell presynapse, aspects of postsynaptic action potential generation down to synaptic transmission at the next stage, in the cochlear nucleus (Strenzke et al., 2009; Buran et al., 2010). Modeling allows us to link the results of ultrastructural experiments, in vitro and in vivo synaptic physiology. Those models not only guide the experimental design of in-vivo experiments of the "Auditory Systems Physiology" group, but they are the stepping stones to the answer of the question that is our central interest: "Which aspects of the biophysical signaling cascade ultimately shape sound encoding?"

Reisinger, Junior Research Group "Molecular Biology of Cochlear Neurotransmission"

Our group is interested in the molecular mechanisms of hearing and deafness. Specifically, we focus on the mechanism and pathomechanism of synaptic transmission in auditory hair cells. The protein otoferlin is known to play a crucial role for a late step of vesicle exocytosis (Roux et al., Cell 2006). To date, it is largely unknown how otoferlin triggers exocytosis or regulates vesicle replenishment. Our group studies the impact of pathogenic missense mutations in otoferlin on the protein function (Pangrsic et al., Nat Neurosci 2010). Further, we aim to understand the role of the individual protein domains in otoferlin by solving the structure of these and by testing the biochemical properties, like binding to Ca²⁺, phospholipid membranes or other proteins. For X-ray crystallography, we collaborate with Ralf Ficner’s group at the Dept. for Molecular Structural Biology (link). Our collaboration partner Henning Urlaub at the Max-Planck-Institute for Biophysical Chemistry (link) identifies protein interaction partners of otoferlin by mass spectroscopy. In addition to unraveling the biochemical and structural properties of otoferlin, we are interested in rescuing the deafness phenotype by genetic manipulation of auditory hair cells. For this, we use virus transduction to transform auditory hair cells of deaf mouse mutants in vivo and in vitro and analyze rescue of function by auditory brainstem response and patch-clamp recordings.

Canis Group, "In vivo Imaging of Cochlear Function"

Our group is interested in the physiology and pathology of cochlear microcirculation. We combine widefield intravitalmicroscopy and two-photon microscopy to study cochlear microcirculation and vascular architecture with an analysis of cochlear and vestibular function under different pathophysiological and therapeutic conditions. Normal hearing function is dependent on ion and fluid homeostasis which is maintained by cochlear microcirculation. Disturbances of microcirculation are a well-established concept for the onset of a number of inner ear disorders like noise-induced hearing loss as well as sudden sensory-neural hearing loss. Difficulties in analyzing cochlear microcirculation in humans result from the complexity and hidden localization within the temporal bone and limit the options for clinical investigation. To bridge this gap, we have established different animal models to provide further insight into vascular physiology and pathophysiology. 1-photon widefield microscopy enables us to quantify parameters of cochlear microcirculation such as capillary diameter and capillary blood flow velocity. Using 2-photon microscopy in collaboration with the Moser group we will study cellular function e.g. by calcium imaging up to a depth of about one millimeter. Additionally, we conduct clinical studies on vascular-related hearing disorders to comprehend our field from bench to bedside.

Strenzke, Junior Research Group "Auditory Systems Physiology"

Our group employs a system physiology approach to characterize mechanisms and functional consequences of sensorineural hearing loss. In vivo electrophysiological recordings of responses from the cochlea, auditory nerve and brainstem, behavioral tests and morphological studies are used to explore sound encoding in the rodent auditory system under normal and diseased conditions. Our main interest concerns partial defects of the inner hair cell ribbon synapse in which summation potentials from the auditory nerve and brainstem are partly preserved. Here, single fiber recordings from the auditory nerve during acoustic stimulation are a powerful tool to explore synaptic function. Startle responses and behavioral experiments are used to further assay the systems and behavioral consequences of hair cell synapse dysfunction. The correlation of our results with in vitro studies and non-invasive auditory tests aims to improve our understanding of auditory physiology and to seek new diagnostic and therapeutic strategies in hearing-impaired subjects.

Wichmann, Junior Research Group "Molecular architecture of synapses"

We combine immunohistochemistry, high resolution light microscopy and electron microscopy to study the molecular architecture of synapses and how structure relates to function. In general, synapses comprise several morphological and functional distinguishable compartments as the presynaptic cytomatrix at the active zone (CAZ), a mesh of proteins, essential for synaptic vesicle docking and release and the postsynaptic density (PSD) containing a matrix of cell-adhesion molecules, receptors and cytoskeletal elements.

The sensory inner hair cell (IHC) ribbon synapses are of particular interest for us. They exhibit an elaborated ellipsoid ribbon surrounded by a halo of synaptic vesicles. This characteristic structure allows ribbon synapses to support sustained exocytosis over long time periods. Using electron microscopic techniques as electron tomography and high-pressure freezing/freeze-substitution (HPF/FS) we study morphological aspects of wild-type and mutant synapses qualitatively and quantitatively. Electron tomography enables the visualization of fine molecular structures due to its high z-resolution. HPF/FS is a special preparation method avoiding chemical fixation of the tissue. Under high pressure and low temperature samples become immobilized within milliseconds and preserved in a near native state. The combination of electron tomography and HPF/FS allows us to determine synaptic vesicle pools, docking or tethering of synaptic vesicles in high spatio-temporal resolution under different conditions (resting or stimulated). Further, we want to construct a coherent picture of the localization of synaptic proteins at IHC ribbon synapses using immunofluorescence and immunogold labeling methods in combination with high resolution light microscopy and electron tomography or serial 3D reconstructions.

In addition, we have a longstanding interest in the ultrastructure of presynapses and their postsynaptic counterparts in general.