Our research group investigates middle ear mechanics.
Of particular interest are the smallest bones in the human body, the auditory ossicles (malleus, incus, and stapes; also known as hammer, anvil, and stirrup). The ossicles relay sound induced motions of the tympanic membrane to the oval window of the inner ear’s cochlea, compensating for the impedance difference between air and the fluid filled cochlea while maintaining fidelity. A dysfunctional ossicle chain,e.g. ossification of the stapes base-plate or of the joints, can result in severe hearing impairments. Depending on the severity and cause of the hearing loss a partial or complete surgical replacement of the ossicles can become necessary.
The primary goal of our research is to increase our understanding of middle ear impairments and diseases and the development and testing of novel middle ear ossicle prosthesis, both passive and active. An active middle ear prostheses can function similar to a regular hearing aid and is implicated when the outer ear or middle ear are impaired but he inner ear remains functional. Besides work on the middle ear, the research group is also interested in properties of the inner ear, particularly focusing on the improvement of classical, electrical cochlea implants but also on novel optical cochlea implants that are under development by other groups of the InnerEarLab.
The research lab utilizes state-of-the-art methods like laser-doppler-laser vibrometers for high-resolution contact-free measurements of middle ear mechanics simultaneously with a well-defined stimulus.
Taken together, our work group is ideally positioned to tackle challenges arising from clinical and basic research questions regarding the workings of the middle and inner ear.
Hearing encompasses the reception, translation and subsequent perception of acoustic signals. Acoustic signals, or sound, possess two major attributes: i) sound particle velocity (m/s) and ii) sound pressure (dB SPL). While sound particle velocity disperses rapidly with distance to the source, sound pressure can travel large distances in the form of oscillating pressure waves. Where i.e. insects can possess sound particle velocity and/or pressure sensitive ears, vertebrates only possess sound pressure sensitive ears. In general terms, sound particle velocity sensitive ears are only seen were short range acoustic signalling is employed, where sound pressure sensitive ears premise a certain animal size and are utilized for short to long range acoustic signalling.
Regardless of the type of ear, the key-step of sound perception is the translation of mechanical forces into electrochemical signals. This process, termed mechano-electrical-transduction (MET), is performed by dedicated MET-channels. MET-channels allow for the inflow of cations into sensory cells, depolarizing those cells in the process, and resulting in either neurotransmitter release (vertebrates) or the firing of action potentials (insects).
We utilize insect (Drosophila melanogaster)and mammalian model organisms (mice/rats) to understand the inner workings of MET. Our goal is to decipher how structures, cells and processes have been specialized during evolution to achieve the high sensitivity and fidelity we find today in hearing organs. To achieve said goal, we employ the following methods and techniques:
• Whole-cell patch clamping (Drosophila S2 cells and mammalian inner ear hair cells)
• Extracellular compound action potential (CAP) recordings
• Ca 2+ imaging
• Chemical manipulation of the cell membrane composition
• Utilizing lipid metabolism mutants
We closely collaborate with groups of the InnerEarLab and other groups at the University of Göttingen and beyond.
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?"
Our brains must make sense of the myriad sensations that all of us face each and every day to successfully find one’s bearings in life. We need to categorize, learn, remember, adapt, pay attention etc. in order to appropriately react and act in our environments. The auditory system is a particularly fascinating example of these feats as we e.g. use speech heavily to infer about our social environment and to interact with others. Our lab is interested in the neurobiological underpinnings of these cognitive aspects of hearing.
To tackle our experimental questions we employ behavioral and neurophysiological methods. A guiding principle for our research is a distinctly interactive view on brain function. Traditionally the brain has been thought off simply as a feedforward system. While this approach has been tremendously successful it is neglecting the massive and rich feedback individual brain structures maintain with each other. We believe that a comprehensive, mechanistic understanding of cognitive aspects of hearing requires elucidating the orchestrated function of various brain systems.
The participation in the optogenetic cochlea implant research program of the Institute for Auditory Neuroscience at the University Medical Center (link) and the program on Auditory Neuroscience and Optogenetics at the German Primate Center presents a large part of our experimental efforts. Disabling hearing loss affects a large number of people worldwide. Hearing can be partially restored by electrical cochlear implants which directly stimulate the auditory nerve. While electrical cochlea implants provide the majority of users with open speech comprehension in quiet a number of limitations remain. These limitations are linked to the poor frequency resolution of electrical stimulation that is caused by the large current spread around each electrode contact and presents a fundamental physical barrier. Optogenetic cochlea implants promise greatly enhanced frequency resolution as light can be conveniently focused. However, genetic manipulation of neurons within the auditory nerve is required (see Rankovic group, link). Work on rodents by the optogenetic cochlea implant research program of the Institute for Auditory Neuroscience has provided proof of principle for optogenetic manipulation of neurons via viral gene transfer, activation of the auditory pathway and auditory percepts by optical cochlear implants. However, before optogenetic cochlea implants can be tested in human patients, experiments in non-human primates need to be conducted to establish the efficacy and safety of optogenetic cochlea stimulation. Furthermore, the quality of sound encoding by optogenetic means has to be compared with acoustic and electrical stimulation to establish superiority of optogenetic over electrical stimulation. In this regard, experiments in non-human primates – in particular the common marmoset – are of critical importance for at least three reasons: 1) marmosets have larger cochlea than most rodents which makes a comparison and extrapolation of frequency resolution to humans much more direct, 2) the non-human primate immune system is much closer related to the human immune system allowing more adequate conclusions about optogenetic manipulation in humans and 3) common marmosets rely – similar to humans – on vocal communication for social behavior. Within the cochlea optogenetics program, we contribute behavioral and neurophysiological experiments investigating and comparing hearing with normal acoustic in contrast to artificial electric and optical stimulation in marmosets.
The aim of our group is to investigate, optimize and provide reliable gene transfer to cochlear structures by combining molecular biology, protein biochemistry, immunochemistry, and optogenetics. We collaborate with physiologists applying optogenetics and studying hearing and deafness. Our group focuses on the design, optimization and testing of various channelrhodopsins (ChRs) for cochlear optogenetics. Moreover, we devise strategies for gene therapeutic approaches to deafness. Our group provides research grade adeno-associated viral (AAV) vector preparations and related services for in vitro and in vivo applications. Challenges are the large size of some of the cDNA transcripts, identifying viral serotypes with specific tropism for auditory neurons and the efficiency of AAV transduction, as well as adjusting appropriate expression levels of genes of interest. Using AAV gene transfer, we aim to restore the hearing by designing the ChRs specific to spiral ganglion neurons (SGNs) that will be efficiently used in combination with optical stimulation by tens of microscale light emitters along the tonotopic axis of the cochlea (cochlear optogenetics). In addition, we aim to restore hearing in mouse models of human genetic deafness. For both applications we already have proof of principle in rodents. In the future we aim to develop cochlear optogenetics and hearing restoration by gene editing in larger animals such as non-human primates. Last but not the least, the group is interested in providing biosafety information after alien gene transfer (mainly ChRs) into cochlear structure as a part of larger inter-disciplinary study as required for future late preclinical and early clinical trials.
Within Institute for Auditory Neuroscience we extensively collaborate with the Moser Group and the Jeschke Group in development of cochlear optogenetics and future translation to non-human primates and with Moser, Vogl and Pangršič groups on gene therapeutic strategies targeting inner hair cells. Further collaborators include Dr. Volker Buskamp (Technische Universität Dresden, CRTD), Prof. Dr. Wolfram Zimmermann (UMG, Göttingen) and Dr. Maria-Patapia Zafeiriou (UMG, Göttingen).
Major research directions of the group:
1. Optimization and manufacturing of the viral vectors for the cochlear genomics
2. Development of viral transfer of suitable ChR variants into SGNs
3. Development of viral transfer of suitable rescue variants or the genes related to synaptic physiology into IHCs
4. The study of the biology of, and host response to alien gene transfer
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.
Ribbon synapses of auditory inner hair cells (IHCs) are capable of transmitting acoustic information with exquisite temporal precision. Here, sound encoding – i.e. the translation of a physical signal into neural code – is achieved by highly regulated presynaptic exocytosis of glutamate-filled synaptic vesicles onto postsynaptic fibers of spiral ganglion neurons (SGNs). In this context, it is commonly believed that the presynaptic ribbon itself plays a major role in orchestrating not only synaptic release (e.g. by clustering presynaptic voltage-gated Ca2+ channels and other components of the release machinery), but also rapid vesicular replenishment that ensures a constant supply of releasable vesicles and hence avoid synaptic fatigue.
Within the past decade, a vast range of molecular scaffolds and key regulators of these presynaptic complexes could be identified and have deepened our understanding of this unconventional synapse. Moreover, a large body of work has investigated mechanisms of synaptogenesis within the organ of Corti, providing insights into neuronal pathfinding within the tissue and synapse formation. However, fundamental aspects of synaptogenesis from a presynaptic perspective (i.e. "presynaptogenesis") – thereby linking the presynaptic molecular complex and the processes involved in synapse formation with the postsynapse – have so far been neglected. In this context, some of the most important questions that still require clarification are the following: (i) which molecular mechanisms determine presynaptic active zone formation, (ii) where do ribbon bodies originate from, (iii) how are they targeted subcellularly to their designated presynaptic active zones, (iv) what is the mode of trafficking within the cytosol and finally, (v) which molecular cues mediate the identification of the correct destination. Moreover, after the presynaptic site is established during embryonic and early postnatal development, extensive morphological and functional maturation processes take place prior to the onset of hearing that are still not fully understood. To date, with the advent of super-resolution microscopy and the capability of performing long-term fluorescent single particle tracking experiments in living cells, many of these open questions can now be adequately addressed.
Genetic manipulation of auditory hair cells remains a challenging task as liposome-based gene transfer methods have proven ineffective in the organ of Corti, and global electroporation produces highly variable and non-specific transfection rates. Here, adeno-associated viruses (AAVs) are valuable tools that achieve high IHC transduction rates; however, virus production remains expensive, time-consuming and inflexible when a range of different constructs need to be evaluated in a timely manner. To circumvent this issue, we make use of an organotypic culture approach of early postnatal organs of Corti (Vogl et al., 2015) and have established a microinjection-based in situ electroporation system (i.e. "injectoporation", Xiong et al., 2014), in which site-targeted transfection is accomplished by local delivery of a plasmid solution into the interstitial space surrounding the hair cells during electroporation. By using this system, we can visualize fluorescently-labeled protein components of the IHC presynapse to identify and characterize the involved trafficking pathways that mediate ribbon targeting to the active zone in live-cell imaging experiments.
Methodologically, we employ confocal long-term live-cell imaging as well as immunohistochemical analysis and super-resolution microscopy (STED) to monitor active zone morphological plasticity and ribbon dynamics during hair cell development and identify key players in this intricate framework with nanoscale resolution. Additionally, we combine electrophysiological and optogenetic stimulation of IHCs to better understand the impact of synaptic activity on presynaptic morphology.
Our ongoing collaborations with the Wichmann Group, who provides ultrastructural support (i.e. TEM and EM tomography), and the lab of Prof. Lukas Kapitein from Utrecht University, who develops molecular tools for acute manipulation of ribbon availability at the active zone, will further assist in clarifying the modes of initial presynapse formation, subsequent maturation and ultimately add another piece in the puzzle to understand the function of the ribbon in vesicular replenishment.
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.
Our group focuses on methods to recover hearing and their clinical translation.
One field of activity is the optogenetic manipulation of spiral ganglion neurons – first neurons of the hearing system – to enable future optical cochlear implants. In collaboration with the Institute of auditory neuroscience, headed by Prof. Dr. T. Moser, optogenetically manipulated spiral ganglion neurons in adult rodents and their optical cochlear stimulation with consequent auditory activity and triggered behavior could recently be established.
This provides a broad basis for further investigations of and insights into auditory system and for developing optical cochlear implants with high frequency and intensity resolution. Other fields of activity are targeted, e. g. recovering the auditory nerve in the global degenerated cochlea.