Publications

Edward C. and Amy H. Sewall Professor in the School of Medicine and Professor of Otolaryngology - Head & Neck Surgery (OHNS) and, by courtesy of Molecular and Cellular Physiology

Bio

Anthony Ricci, PhD got his PhD in Neuroscience at Tulane University School of Medicine where he was studying the peripheral vestibular system. He did a postdoctoral fellowship with Manning Correia at UTMB and then at the University of Wisconsin with Robert Fettiplace. His work has focused upon hair cell function, using electrophysiological and imaging tools for this work. Dr. Ricci has contributed at many levels to understanding signal processing from the periphery to the CNS. He also has a translational component to his work where he is collaborating to develop non ototoxic antibiotics, developing new drug delivery systems for the ear to facilitate gene therapy treatments and more recently investigating how hearing loss impacts cognitive function. As a PI, Dr. Ricci has trained numerous students both medical and graduate. He has also trained postdocs and residents. Dr. Ricci served as the director of the Neuroscience Graduate Program at Stanford for almost eight years. He was a co-founder of the ADVANCE Summer Institute, an onboarding program for incoming bioscience graduate students from underserved backgrounds. He has been the faculty lead for this program for the past 9 years. He is presently the faculty lead on a new postdoctoral fellows program, Propel, that targets scholars form underrepresented communities who are interested in academic careers. Most recently, Dr. Ricci is part of a team launching a new postbaccalaureate program REACH that provides a strong research based opportunity to scholars from underrepresented backgrounds interested in a research or medical career. Dr. Ricci is presently the Associate Dean of Graduate Education and Postdoctoral Affairs and the Director of Research for the Department of Otolaryngology.

Publications

  • The Cousa objective: a long-working distance air objective for multiphoton imaging in vivo. Nature methods Yu, C. H., Yu, Y., Adsit, L. M., Chang, J. T., Barchini, J., Moberly, A. H., Benisty, H., Kim, J., Young, B. K., Heng, K., Farinella, D. M., Leikvoll, A., Pavan, R., Vistein, R., Nanfito, B. R., Hildebrand, D. G., Otero-Coronel, S., Vaziri, A., Goldberg, J. L., Ricci, A. J., Fitzpatrick, D., Cardin, J. A., Higley, M. J., Smith, G. B., Kara, P., Nielsen, K. J., Smith, I. T., Smith, S. L. 2023

    Abstract

    Multiphoton microscopy can resolve fluorescent structures and dynamics deep in scattering tissue and has transformed neural imaging, but applying this technique in vivo can be limited by the mechanical and optical constraints of conventional objectives. Short working distance objectives can collide with compact surgical windows or other instrumentation and preclude imaging. Here we present an ultra-long working distance (20 mm) air objective called the Cousa objective. It is optimized for performance across multiphoton imaging wavelengths, offers a more than 4 mm2 field of view with submicrometer lateral resolution and is compatible with commonly used multiphoton imaging systems. A novel mechanical design, wider than typical microscope objectives, enabled this combination of specifications. We share the full optical prescription, and report performance including in vivo two-photon and three-photon imaging in an array of species and preparations, including nonhuman primates. The Cousa objective can enable a range of experiments in neuroscience and beyond.

    View details for DOI 10.1038/s41592-023-02098-1

    View details for PubMedID 38129618

    View details for PubMedCentralID 8211867

  • Large-scale annotated dataset for cochlear hair cell detection and classification. bioRxiv : the preprint server for biology Buswinka, C. J., Rosenberg, D. B., Simikyan, R. G., Osgood, R. T., Fernandez, K., Nitta, H., Hayashi, Y., Liberman, L. W., Nguyen, E., Yildiz, E., Kim, J., Jarysta, A., Renauld, J., Wesson, E., Thapa, P., Bordiga, P., McMurtry, N., Llamas, J., Kitcher, S. R., López-Porras, A. I., Cui, R., Behnammanesh, G., Bird, J. E., Ballesteros, A., Vélez-Ortega, A. C., Edge, A. S., Deans, M. R., Gnedeva, K., Shrestha, B. R., Manor, U., Zhao, B., Ricci, A. J., Tarchini, B., Basch, M., Stepanyan, R. S., Landegger, L. D., Rutherford, M., Liberman, M. C., Walters, B. J., Kros, C. J., Richardson, G. P., Cunningham, L. L., Indzhykulian, A. A. 2023

    Abstract

    Our sense of hearing is mediated by cochlear hair cells, localized within the sensory epithelium called the organ of Corti. There are two types of hair cells in the cochlea, which are organized in one row of inner hair cells and three rows of outer hair cells. Each cochlea contains a few thousands of hair cells, and their survival is essential for our perception of sound because they are terminally differentiated and do not regenerate after insult. It is often desirable in hearing research to quantify the number of hair cells within cochlear samples, in both pathological conditions, and in response to treatment. However, the sheer number of cells along the cochlea makes manual quantification impractical. Machine learning can be used to overcome this challenge by automating the quantification process but requires a vast and diverse dataset for effective training. In this study, we present a large collection of annotated cochlear hair-cell datasets, labeled with commonly used hair-cell markers and imaged using various fluorescence microscopy techniques. The collection includes samples from mouse, human, pig and guinea pig cochlear tissue, from normal conditions and following in-vivo and in-vitro ototoxic drug application. The dataset includes over 90'000 hair cells, all of which have been manually identified and annotated as one of two cell types: inner hair cells and outer hair cells. This dataset is the result of a collaborative effort from multiple laboratories and has been carefully curated to represent a variety of imaging techniques. With suggested usage parameters and a well-described annotation procedure, this collection can facilitate the development of generalizable cochlear hair cell detection models or serve as a starting point for fine-tuning models for other analysis tasks. By providing this dataset, we aim to supply other groups within the hearing research community with the opportunity to develop their own tools with which to analyze cochlear imaging data more fully, accurately, and with greater ease.

    View details for DOI 10.1101/2023.08.30.553559

    View details for PubMedID 37693382

    View details for PubMedCentralID PMC10491224

  • GIPC3 couples to MYO6 and PDZ domain proteins and shapes the hair cell apical region. Journal of cell science Chatterjee, P., Morgan, C. P., Krey, J. F., Benson, C., Goldsmith, J., Bateschell, M., Ricci, A. J., Barr-Gillespie, P. G. 2023

    Abstract

    GIPC3 has been implicated in auditory function. Initially localized to the cytoplasm of inner and outer hair cells of the cochlea, GIPC3 increasingly concentrated in cuticular plates and at cell junctions during postnatal development. Early postnatal Gipc3KO/KO mice had mostly normal mechanotransduction currents, but had no auditory brainstem response at one month of age. Cuticular plates of Gipc3KO/KO hair cells did not flatten during development as did those of controls; moreover, hair bundles were squeezed along the cochlear axis in mutant hair cells. Junctions between inner hair cells and adjacent inner phalangeal cells were also severely disrupted in Gipc3KO/KO cochleas. GIPC3 bound directly to MYO6, and the loss of MYO6 led to altered distribution of GIPC3. Immunoaffinity purification of GIPC3 from chicken inner ear extracts identified co-precipitating proteins associated with adherens junctions, intermediate filament networks, and the cuticular plate. Several of immunoprecipitated proteins contained GIPC-family consensus PDZ binding motifs (PBMs), including MYO18A, which binds directly to the PDZ domain of GIPC3. We propose that GIPC3 and MYO6 couple to PBMs of cytoskeletal and cell-junction proteins to shape the cuticular plate. (180 words; 180 maximum).

    View details for DOI 10.1242/jcs.261100

    View details for PubMedID 37096733

  • Cholesterol as a tool to probe the role of membrane in cochlear hair cell mechanotransduction George, S., Effertz, T., Ricci, A. J. CELL PRESS. 2023: 91A-92A
  • Tmc regulates membrane viscosity in mammalian cochlear hair cells. Biophysical journal Sam George, S., El Bahloul-Jaziri, A., Ricci, A. J. 2023; 122 (3S1): 91a

    View details for DOI 10.1016/j.bpj.2022.11.692

    View details for PubMedID 36785083

  • Coupling between the stereocilia of rat sensory inner-hair-cell hair bundles is weak, shaping their sensitivity to stimulation. The Journal of neuroscience : the official journal of the Society for Neuroscience Scharr, A. L., Maoileidigh, D. O., Ricci, A. J. 2023

    Abstract

    The hair bundle is the universal mechanosensory organelle of auditory, vestibular, and lateral-line systems. A bundle comprises mechanically coupled stereocilia, whose displacements in response to stimulation activate a receptor current. The similarity of stereociliary displacements within a bundle regulates fundamental properties of the receptor current like its speed, magnitude, and sensitivity. However, the dynamics of individual stereocilia from the mammalian cochlea in response to a known bundle stimulus has not been quantified. We developed a novel high-speed system, which dynamically stimulates and tracks individual inner-hair-cell stereocilia from male and female rats. Stimulating 2-3 of the tallest stereocilia within a bundle (nonuniform stimulation) caused dissimilar stereociliary displacements. Stereocilia further from the stimulator moved less, but with little delay, implying that there is little slack in the system. Along the axis of mechanical sensitivity, stereocilium displacements peaked and reversed direction in response to a step stimulus. A viscoelastic model explained the observed displacement dynamics, which implies that coupling between the tallest stereocilia is effectively viscoelastic. Coupling elements between the tallest inner-hair-cell stereocilia were 2-3 times stronger than elements anchoring stereocilia to the cell's surface but were 10-10,000 times weaker than those of a well-studied non-cochlear hair bundle. Coupling was too weak to ensure that stereocilia move similarly in response to nonuniform stimulation at auditory frequencies. Our results imply that more uniform stimulation across the tallest stereocilia of an inner-hair-cell bundle in vivo is required to ensure stereociliary displacement similarity, increasing the speed, sensitivity, and magnitude of the receptor current.SIGNIFICANCE STATEMENT:Generation of the hair cell's receptor current is the first step in electrically encoding auditory information in the hearing organs of all vertebrates. The receptor current is shaped by mechanical coupling between stereocilia in each hair cell's hair bundle. Here we provide foundational information on the mechanical coupling between stereocilia of cochlear inner hair cells. In contrast to other types of hair cell, coupling between inner hair cell stereocilia is weak, causing slower, smaller, and less sensitive receptor currents in response to stimulation of few, rather than many, stereocilia. Our results imply that inner hair cells need many stereocilia to be stimulated in vivo to ensure fast, large, and sensitive receptor currents.

    View details for DOI 10.1523/JNEUROSCI.1588-22.2023

    View details for PubMedID 36746628

  • A chemo-mechanical cochleostomy preserves hearing for the in vivo functional imaging of cochlear cells. Nature protocols Kim, J., Ricci, A. J. 2023

    Abstract

    In vivo and real-time multicellular imaging enables the decoding of sensory circuits and the tracking of systemic drug uptake. However, in vivo imaging of the auditory periphery remains technically challenging owing to the deep location, mechanosensitivity and fluid-filled, bone-encased nature of the cochlear structure. Existing methods that expose the cochlea invariably cause irreversible damage to auditory function, severely limiting the experimental measurements possible in living animals. Here we present an in vivo surgical protocol that permits the imaging of cochlear cells in hearing mice. Our protocol describes a ventro-lateral approach for preserving external and middle ear structures while performing surgery, the correct mouse positioning for imaging cochlear cells with effective sound transmission into the ear, the chemo-mechanical cochleostomy for creating the imaging window in the otic capsule bone that prevents intracochlear fluid leakage by maintaining an intact endosteum, and the release of intracochlear pressure that separates the endosteum from the otic capsule bone while creating an imaging window. The procedure thus preserves hearing thresholds. Individual inner and outer hair cells, supporting cells and nerve fibers can be visualized in vivo while hearing function is preserved. This approach may enable future original investigations, such as the real-time tracking of ototoxic drug transport into the cochleae. The technique may be applied to the monitoring of sound-evoked functional activity in multiple cochlear cells, in combination with optogenetic tools, and may help to improve cochlear implantation in humans. The cochleostomy takes ~1 h and requires experience in surgery.

    View details for DOI 10.1038/s41596-022-00786-4

    View details for PubMedID 36599963

  • ANKRD24 organizes TRIOBP to reinforce stereocilia insertion points. The Journal of cell biology Krey, J. F., Liu, C., Belyantseva, I. A., Bateschell, M., Dumont, R. A., Goldsmith, J., Chatterjee, P., Morrill, R. S., Fedorov, L. M., Foster, S., Kim, J., Nuttall, A. L., Jones, S. M., Choi, D., Friedman, T. B., Ricci, A. J., Zhao, B., Barr-Gillespie, P. G. 2022; 221 (4)

    Abstract

    The stereocilia rootlet is a key structure in vertebrate hair cells, anchoring stereocilia firmly into the cell's cuticular plate and protecting them from overstimulation. Using superresolution microscopy, we show that the ankyrin-repeat protein ANKRD24 concentrates at the stereocilia insertion point, forming a ring at the junction between the lower and upper rootlets. Annular ANKRD24 continues into the lower rootlet, where it surrounds and binds TRIOBP-5, which itself bundles rootlet F-actin. TRIOBP-5 is mislocalized in Ankrd24KO/KO hair cells, and ANKRD24 no longer localizes with rootlets in mice lacking TRIOBP-5; exogenous DsRed-TRIOBP-5 restores endogenous ANKRD24 to rootlets in these mice. Ankrd24KO/KO mice show progressive hearing loss and diminished recovery of auditory function after noise damage, as well as increased susceptibility to overstimulation of the hair bundle. We propose that ANKRD24 bridges the apical plasma membrane with the lower rootlet, maintaining a normal distribution of TRIOBP-5. Together with TRIOBP-5, ANKRD24 organizes rootlets to enable hearing with long-term resilience.

    View details for DOI 10.1083/jcb.202109134

    View details for PubMedID 35175278

  • Identifying targets to prevent aminoglycoside ototoxicity. Molecular and cellular neurosciences Kim, J., Hemachandran, S., Cheng, A. G., Ricci, A. J. 2022: 103722

    Abstract

    Aminoglycosides are potent antibiotics that are commonly prescribed worldwide. Their use carries significant risks of ototoxicity by directly causing inner ear hair cell degeneration. Despite their ototoxic side effects, there are currently no approved antidotes. Here we review recent advances in our understanding of aminoglycoside ototoxicity, mechanisms of drug transport, and promising sites for intervention to prevent ototoxicity.

    View details for DOI 10.1016/j.mcn.2022.103722

    View details for PubMedID 35341941

  • In vivo real-time imaging reveals megalin as the aminoglycoside gentamicin transporter into cochlea whose inhibition is otoprotective. Proceedings of the National Academy of Sciences of the United States of America Kim, J., Ricci, A. J. 2022; 119 (9)

    Abstract

    Aminoglycosides (AGs) are commonly used antibiotics that cause deafness through the irreversible loss of cochlear sensory hair cells (HCs). How AGs enter the cochlea and then target HCs remains unresolved. Here, we performed time-lapse multicellular imaging of cochlea in live adult hearing mice via a chemo-mechanical cochleostomy. The invivo tracking revealed that systemically administered Texas Red-labeled gentamicin (GTTR) enters the cochlea via the stria vascularis and then HCs selectively. GTTR uptake into HCs was completely abolished in transmembrane channel-like protein 1 (TMC1) knockout mice, indicating mechanotransducer channel-dependent AG uptake. Blockage of megalin, the candidate AG transporter in the stria vascularis, by binding competitor cilastatin prevented GTTR accumulation in HCs. Furthermore, cilastatin treatment markedly reduced AG-induced HC degeneration and hearing loss invivo. Together, our invivo real-time tracking of megalin-dependent AG transport across the blood-labyrinth barrier identifies new therapeutic targets for preventing AG-induced ototoxicity.

    View details for DOI 10.1073/pnas.2117946119

    View details for PubMedID 35197290

  • The functional role of connectors in outer-hair-cell hair bundles Zhu, Z., Ricci, A. J., Maoileidigh, D. O. CELL PRESS. 2022: 436A
  • A two-photon FRAP protocol to measure the stereociliary membrane diffusivity in rat cochlear hair cells. STAR protocols George, S. S., Steele, C. R., Ricci, A. J. 2021; 2 (3): 100637

    Abstract

    Fluorescence recovery after photobleaching (FRAP) has been widely used to monitor membrane properties by measuring the lateral diffusion of fluorescent particles. This protocol describes how to perform two-photon FRAP on the stereocilia of live cochlear inner hair cells using a lipophilic dye, di-3-ANEPPDHQ, to assess the stereociliary membrane diffusivity. We also detail two-photon FRAP microscope setup and calibration, as well as FRAP parameter setting and data analysis. For complete details on the use and execution of this protocol, please refer to George etal. (2020).

    View details for DOI 10.1016/j.xpro.2021.100637

    View details for PubMedID 34258597

  • Functional subgroups of cochlear inner hair cell ribbon synapses differently modulate their EPSC properties in response to stimulation. Journal of neurophysiology Niwa, M., Young, E. D., Glowatzki, E., Ricci, A. J. 2021

    Abstract

    Spiral ganglion neurons (SGNs) form single synapses on Inner Hair Cells (IHCs), transforming sound induced IHC receptor potentials into trains of action potentials. SGN neurons are classified by spontaneous firing rates as well as their threshold response to sound intensity levels. We investigated the hypothesis that synaptic specializations underlie mouse SGN response properties and vary with pillar versus modiloar synapse location around the hair cell. Depolarizing hair cells with 40 mM K+ increased the rate of postsynaptic responses. Pillar synapses matured later than modiolar synapses. EPSC amplitude, area and number of underlying events per EPSC were similar between synapse locations at steady-state. However, modiolar synapses produced larger monophasic EPSCs when EPSC rates were low and EPSCs became more multiphasic and smaller in amplitude when rates were higher, while pillar synapses produced more monophasic and larger EPSCs when the release rates were higher. We propose that pillar and modiolar synapses have different operating points. Our data provide insight into underlying mechanisms regulating EPSC generation.

    View details for DOI 10.1152/jn.00452.2020

    View details for PubMedID 33949873

  • Loxhd1 mutations cause mechanotransduction defects in cochlear hair cells. The Journal of neuroscience : the official journal of the Society for Neuroscience Trouillet, A., Miller, K. K., George, S. S., Wang, P., Ali, N., Ricci, A., Grillet, N. 2021

    Abstract

    Sound detection happens in the inner ear via the mechanical deflection of the hair bundle of cochlear hair cells. The hair bundle is an apical specialization consisting of actin-filled membrane protrusions (called stereocilia) connected by tip links (TLs) that transfer the deflection force to gate the mechanotransduction channels. Here, we identified the hearing loss-associated Loxhd1/DFNB77 gene as being required for the mechanotransduction process. LOXHD1 consists of 15 polycystin lipoxygenase alpha-toxin (PLAT) repeats, which in other proteins can bind lipids and proteins. LOXHD1 was distributed along the length of the stereocilia. Two LOXHD1 mouse models with mutations in the 10th PLAT repeat exhibited mechanotransduction defects (in both sexes). While mechanotransduction currents in mutant inner hair cells (IHCs) were similar to wild-type (WT) levels in the first postnatal week, they were severely affected by postnatal day 11. The onset of the MET phenotype was consistent with the temporal progression of postnatal LOXHD1 expression/localization in the hair bundle. The mechanotransduction defect observed in Loxhd1-mutant IHCs was not accompanied by a morphological defect of the hair bundle or a reduction in TL number. Using immunolocalization, we found that two proteins of the upper and lower TL protein complexes (Harmonin and LHFPL5) were maintained in the mutants, suggesting that the mechanotransduction machinery was present but not activatable. This work identified a novel LOXHD1-dependent step in hair bundle development that is critical for mechanotransduction in mature hair cells as well as for normal hearing function in mice and humans.SIGNIFICANCE STATEMENT:Hair cells detect sound-induced forces via the hair bundle, which consists of membrane protrusions connected by tip links. The mechanotransduction machinery forms protein complexes at the tip-link ends. The current study showed that LOXHD1, a multi-repeat protein responsible for hearing loss in humans and mice when mutated, was required for hair-cell mechanotransduction, but only after the first postnatal week. Using immunochemistry, we demonstrated that this defect was not caused by the mislocalization of the tip-link complex proteins Harmonin or LHFPL5, suggesting that the mechanotransduction protein complexes were maintained. This work identified a new step in hair bundle development, which is critical for both hair-cell mechanotransduction and hearing.

    View details for DOI 10.1523/JNEUROSCI.0975-20.2021

    View details for PubMedID 33707295

  • Fluid Jet Stimulation of Auditory Hair Bundles Reveal Spatial Non-uniformities and Two Viscoelastic-Like Mechanisms. Frontiers in cell and developmental biology Peng, A. W., Scharr, A. L., Caprara, G. A., Nettles, D., Steele, C. R., Ricci, A. J. 2021; 9: 725101

    Abstract

    Hair cell mechanosensitivity resides in the sensory hair bundle, an apical protrusion of actin-filled stereocilia arranged in a staircase pattern. Hair bundle deflection activates mechano-electric transduction (MET) ion channels located near the tops of the shorter rows of stereocilia. The elicited macroscopic current is shaped by the hair bundle motion so that the mode of stimulation greatly influences the cell's output. We present data quantifying the displacement of the whole outer hair cell bundle using high-speed imaging when stimulated with a fluid jet. We find a spatially non-uniform stimulation that results in splaying, where the hair bundle expands apart. Based on modeling, the splaying is predominantly due to fluid dynamics with a small contribution from hair bundle architecture. Additionally, in response to stimulation, the hair bundle exhibited a rapid motion followed by a slower motion in the same direction (creep) that is described by a double exponential process. The creep is consistent with originating from a linear passive system that can be modeled using two viscoelastic processes. These viscoelastic mechanisms are integral to describing the mechanics of the mammalian hair bundle.

    View details for DOI 10.3389/fcell.2021.725101

    View details for PubMedID 34513845