Our group works on encoding and transmission of auditory information in normal and prosthetic hearing.
Molecular anatomy, physiology and pathology of sound encoding
Towards normal hearing we aim to decipher the molecular and cellular mechanisms of sound coding at the hair cell ribbon synapse and signal transmission at the endbulb of Held synapse in the cochlear nucleus. We are interested in the mechanisms that enable the impressive temporal precision and long-lasting reliability of synaptic transmission in the early auditory pathway. Moreover, we aim 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 in each individual neuron is solved by the cochlea. We approach these questions by a systematic analysis of the molecular anatomy and physiology of hair cell ribbon synapses and endbulb synapses 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 and STED 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). These findings are compared to electron microscopy and tomography studies performed by 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 molecular physiology of the hair cell Ca2+ current (e.g. Brandt et al., 2003; 2005; Cui et al., 2007; Neef et al., 2009; Meyer et al., 2009; Schrauwen et al., 2012), 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; Pangršič et al., 2010; Reisinger et al., 2011). We have indicated important differences of hair cell synapses to other synapses regarding their molecular composition (recent reviews in Pangršič et al., 2012, Rutherford and Pangršič, 2012). For example they express Ribeye [Khimich et al., 2005], Otoferlin [Roux et al., 2006] and seem to lack Complexins [Strenzke et al., 2009], Synaptotagmins [Reisinger et al., 2011] and neuronal SNAREs [Nouvian 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 (Pangršič et al., 2010; Rutherford et al., 2012). 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). Active zones differ in the strength and voltage-dependence of their Ca2+ influx. The strength appeared to correlate with the size of the synaptic ribbons. We argue that the heterogeneity of presynaptic Ca2+ signaling is a key mechanism contributing to the wide dynamic range of sound coding. We hypothesize that different active zones drive spiral ganglion neurons with diverse spontaneous rates, sound thresholds, and dynamic range. Studies of synaptic transmission at the endbulb of Held synapse involve patch-clamp, modeling and electron and immunofluorescence microscopy.
Our understanding of normal coding and transmission forms the basis for designing strategies for the restoration of hearing in animal models of human deafness. Using viral gene transfer we aim to restore hearing in mouse mutants lacking essential synaptic proteins such as otoferlin. Challenges are the large size of the cDNA, the efficiency of transfection protocols and adjusting appropriate levels of expression.
Nowadays, hearing can be partially restored to the deaf by CIs, which bypass the cochlear dysfunction via direct electric stimulation of spiral ganglion neurons (SGNs). CIs enable open speech comprehension in most users, but the quality of hearing is low. This results from low frequency and intensity resolution of coding due to the wide spread of electrical current from each electrode contact.
We aim to overcome this fundamental problem by establishing many independent coding channels via spatially confined optical stimulation of channelrhodopsin (ChR)-expressing SGNs by tens of microscale light emitters along the tonotopic axis of the cochlea (cochlear optogenetics). This innovation promises a dramatic increase in the frequency- and intensity-resolution of CIs and a ground-breaking advance of hearing restoration. Cochlear optogenetics will also be of enormous use in auditory research. We already have proof of principle in rodents: we achieved stable virus-mediated expression of ChR in SGNs and recorded light-evoked auditory activity. In the future we aim to develop cochlear optogenetics in larger animals by
- development of viral transfer of suitable ChR variants into SGNs and of
- multichannel optical stimulation using CIs with arrays of microscale light emitting diodes and waveguides,
- characterizing neuronal responses along the auditory pathway using physiological and behavioural methods, and
- comparing optogenetic to acoustic and electric stimulation.
Together, this work will establish cochlear optogenetics as a research tool, validate its potential for improved hearing restoration and prepare translation into the clinic.
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?"
My research interest targets towards a better understanding of hearing, specifically of how sensory cells in the inner ear transmit information to the auditory nerve. With my group, I analyze proteins involved in synaptic transmission from auditory hair cells with molecular biology, protein biochemistry, fluorescence immunohistochemistry, viral transduction and cellular electrophysiology. The protein otoferlin is known to play a crucial role for a late step of vesicle exocytosis (Roux et al., Cell 2006) and is required for fast vesicle replenishment (Pangrsic et al., 2010; reviewed in Pangrsic, Reisinger and Moser 2012). To date, it is largely unknown how otoferlin triggers exocytosis or regulates vesicle replenishment. With my group I study the impact of pathogenic missense mutations in otoferlin on protein function, localization, and on exocytosis from hair cells (e.g., Strenzke et al., 2016). Together with my CRC collaboration partner Henning Urlaub at the Max-Planck-Institute for Biophysical Chemistry (link), we aim at identifying protein interaction partners of otoferlin by mass spectrometry. In addition to unraveling the biochemical and structural properties of otoferlin, we are interested in restoring hearing by genetic manipulation of auditory hair cells. For this, we transduce auditory hair cells of deaf mouse mutants with viruses (e.g. Reisinger et al., 2011) in vivo and in vitro and analyze rescue of function by patch-clamp recordings and auditory brainstem response recordings.
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.
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.
Our group is interested in the molecular and cellular mechanisms governing the fast encoding of sensory information in the inner ear. Our focus is the neurotransmission in vestibular hair cells (VHCs) of the otolith organs. Vestibular epithelia in higher vertebrates contain two types of hair cells that differ in structure and function. Both encode vestibular signals at unconventional, ribbon synapses and drive some of the fastest and most accurate reflexes in the body. We are particularly interested in the function of a unique calyx synapse of the type I VHC, which has only evolved late in evolution. Patch-clamp recordings together with calcium uncaging and imaging will provide us a comprehensive analysis of excitation and secretion. We further aim at determining synaptic differences among the two types of VHCs, which will provide clues as to their precise functions in balance sensing. Current research on cochlear hair cells has started to reveal a unique, unconventional synaptic machinery of these mechanoreceptor cells (reviewed in Rutherford and Pangršič, 2012). Despite much effort, only few cochlear hair cell synaptic proteins have been identified so far and even less is known about mammalian VHCs, which might share some of the molecular specializations but clearly not all. To identify the molecular determinants that govern sensory encoding at the vestibular synapse we plan to screen the vestibular function in mice with genetic manipulations expected to affect the synaptic function by behavioural and systems physiology tests, performed in collaboration with the group of Nicola Strenzke. We are planning to investigate mutants with observed vestibular dysfunction using a combination of functional, structural and molecular approaches to gain a better understanding of the vestibular function.
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.