Auditory Hair Cells and Spiral Ganglion Neurons Regenerate Synapses with Refined Release Properties In Vitro.

Ribbon synapses between inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs) in the inner ear are damaged by noise trauma and with aging, causing 'synaptopathy 'and hearing loss. Co-cultures of neonatal denervated organs of Corti and newly introduced SGNs have been developed to find strategies for improving IHC synapse regeneration, but evidence of the physiological normality of regenerated synapses is missing. This study utilizes IHC optogenetic stimulation and SGN recordings, showing that newly formed IHC synapses are indeed functional, exhibiting glutamatergic excitatory postsynaptic currents. When older organs of Corti were plated, synaptic activity probed by deconvolution, showed more mature release properties, closer to the highly specialized mode of IHC synaptic transmission that is crucial for coding the sound signal. This newly developed functional assessment of regenerated IHC synapses provides a powerful tool for testing approaches to improve synapse regeneration.

Molecular signatures define subtypes of auditory afferents with distinct peripheral projection patterns and physiological properties.

Type I spiral ganglion neurons (SGNs) are the auditory afferents that transmit sound information from cochlear inner hair cells (IHCs) to the brainstem. These afferents consist of physiological subtypes that differ in their spontaneous firing rate (SR), activation threshold, and dynamic range and have been described as low, medium, and high SR fibers. Lately, single-cell RNA sequencing experiments have revealed three molecularly defined type I SGN subtypes. The extent to which physiological type I SGN subtypes correspond to molecularly defined subtypes is unclear. To address this question, we have generated mouse lines expressing CreERT2 in SGN subtypes that allow for a physiological assessment of molecular subtypes. We show that Lypd1-CreERT2 expressing SGNs represent a well-defined group of neurons that preferentially innervate the IHC modiolar side and exhibit a narrow range of low SRs. In contrast, Calb2-CreERT2 expressing SGNs preferentially innervate the IHC pillar side and exhibit a wider range of SRs, thus suggesting that a strict stratification of all SGNs into three molecular subclasses is not obvious, at least not with the CreERT2 tools used here. Genetically marked neuronal subtypes refine their innervation specificity onto IHCs postnatally during the time when activity is required to refine their molecular phenotype. Type I SGNs thus consist of genetically defined subtypes with distinct physiological properties and innervation patterns. The molecular subtype-specific lines characterized here will provide important tools for investigating the role of the physiologically distinct type I SGNs in encoding sound signals.

Engineering olivocochlear inhibition to reduce acoustic trauma.

Efferent brain-stem neurons release acetylcholine to desensitize cochlear hair cells and can protect the inner ear from acoustic trauma. That protection is absent from knockout mice lacking efferent inhibition and is stronger in mice with a gain-of-function point mutation of the hair cell-specific nicotinic acetylcholine receptor. The present work uses viral transduction of gain-of-function receptors to restore acoustic prophylaxis to the knockout mice. Widespread postsynaptic expression of the transgene was visualized in excised tissue with a fluorophore-conjugated peptide toxin that binds selectively to hair cell acetylcholine receptors. Viral transduction into efferent knockout mice reduced the temporary hearing loss measured 1 day post acoustic trauma. The acoustic evoked-response waveform (auditory brain-stem response) recovered more rapidly in treated mice than in control mice. Thus, both cochlear amplification by outer hair cells (threshold shift) and afferent signaling (evoked-response amplitude) in knockout mice were protected by viral transduction of hair cell acetylcholine receptors. Gene therapy to strengthen efferent cochlear feedback could be complementary to existing and future therapies to prevent hearing loss, including ear coverings, hearing aids, single-gene repair, or small-molecule therapies.

Rationally Designed Magnetic Nanoparticles for Cochlear Drug Delivery: Synthesis, Characterization, and In Vitro Biocompatibility in a Murine Model.

Hypothesis: Magnetic nanoparticles (MNPs) for cochlear drug delivery can be precisely engineered for biocompatibility in the cochlea. Background: MNPs are promising drug delivery vehicles that can enhance the penetration of both small and macromolecular therapeutics into the cochlea. However, concerns exist regarding the application of oxidative, metal-based nanomaterials to delicate sensory tissues of the inner ear. Translational development of MNPs for cochlear drug deliver requires specifically tuned nanoparticles that are not cytotoxic to inner ear tissues. We describe the synthesis and characterization of precisely tuned MNP vehicles, and their in vitro biocompatibility in murine organ of Corti organotypic cultures. Methods: MNPs were synthesized via 2-phase ligand transfer process with precise control of nanoparticle size. Core and hydrodynamic sizes of nanoparticles were characterized using electron microscopy and dynamic light scattering, respectively. In vitro biocompatibility was assayed via mouse organ of Corti organotypic cultures with and without an external magnetic field gradient. Imaging was performed using immunohistochemical labeling and confocal microscopy. Outer hair cell, inner hair cell, and spiral ganglion neurites were individually quantified. Results: Monocore PEG-MNPs of 45 and 148 nm (mean hydrodynamic diameter) were synthesized. Organ of Corti cultures demonstrated preserved outer hair cell, inner hair cell, and neurite counts across 2 MNP sizes and doses, and irrespective of external magnetic field gradient. Conclusion: MNPs can be custom-synthesized with precise coating, size, and charge properties specific for cochlear drug delivery while also demonstrating biocompatibility in vitro.

Cy3-RgIA-5727 Labels and Inhibits α9-Containing nAChRs of Cochlear Hair Cells.

Efferent cholinergic neurons inhibit sensory hair cells of the vertebrate inner ear through the combined action of calcium-permeable α9α10-containing nicotinic acetylcholine receptors (nAChRs) and associated calcium-dependent potassium channels. The venom of cone snails is a rich repository of bioactive peptides, many with channel blocking activities. The conopeptide analog, RgIA-5474, is a specific and potent antagonist of α9α10-containing nAChRs. We added an alkyl functional group to the N-terminus of the RgIA-5474, to enable click chemistry addition of the fluorescent cyanine dye, Cy3. The resulting peptide, Cy3-RgIA-5727, potently blocked mouse α9α10 nAChRs expressed in Xenopus oocytes (IC50 23 pM), with 290-fold less activity on α7 nAChRs and 40,000-fold less activity on all other tested nAChR subtypes. The tight binding of Cy3-RgIA-5727 provided robust visualization of hair cell nAChRs juxtaposed to cholinergic efferent terminals in excised, unfixed cochlear tissue from mice. Presumptive postsynaptic sites on outer hair cells (OHCs) were labeled, but absent from inner hair cells (IHCs) and from OHCs in cochlear tissue from α9-null mice and in cochlear tissue pre-incubated with non-Cy3-conjugated RgIA-5474. In cochlear tissue from younger (postnatal day 10) mice, Cy3-RgIA-5727 also labeled IHCs, corresponding to transient efferent innervation at that age. Cy3 puncta in Kölliker's organ remained in the α9-null tissue. Pre-exposure with non-Cy3-conjugated RgIA-5474 or bovine serum albumin reduced this non-specific labeling to variable extents in different preparations. Cy3-RgIA-5727 and RgIA-5474 blocked the native hair cell nAChRs, within the constraints of application to the excised cochlear tissue. Cy3-RgIA-5727 or RgIA-5474 block of efferent synaptic currents in young IHCs was not relieved after 50 min washing, so effectively irreversible.

Optogenetically transduced human ES cell-derived neural progenitors and their neuronal progenies: Phenotypic characterization and responses to optical stimulation.

Optogenetically engineered human neural progenitors (hNPs) are viewed as promising tools in regenerative neuroscience because they allow the testing of the ability of hNPs to integrate within nervous system of an appropriate host not only structurally, but also functionally based on the responses of their differentiated progenies to light. Here, we transduced H9 embryonic stem cell-derived hNPs with a lentivirus harboring human channelrhodopsin (hChR2) and differentiated them into a forebrain lineage. We extensively characterized the fate and optogenetic functionality of hChR2-hNPs in vitro with electrophysiology and immunocytochemistry. We also explored whether the in vivo phenotype of ChR2-hNPs conforms to in vitro observations by grafting them into the frontal neocortex of rodents and analyzing their survival and neuronal differentiation. Human ChR2-hNPs acquired neuronal phenotypes (TUJ1, MAP2, SMI-312, and synapsin 1 immunoreactivity) in vitro after an average of 70 days of coculturing with CD1 astrocytes and progressively displayed both inhibitory and excitatory neurotransmitter signatures by immunocytochemistry and whole-cell patch clamp recording. Three months after transplantation into motor cortex of naïve or injured mice, 60-70% of hChR2-hNPs at the transplantation site expressed TUJ1 and had neuronal cytologies, whereas 60% of cells also expressed ChR2. Transplant-derived neurons extended axons through major commissural and descending tracts and issued synaptophysin+ terminals in the claustrum, endopiriform area, and corresponding insular and piriform cortices. There was no apparent difference in engraftment, differentiation, or connectivity patterns between injured and sham subjects. Same trends were observed in a second rodent host, i.e. rat, where we employed longer survival times and found that the majority of grafted hChR2-hNPs differentiated into GABAergic neurons that established dense terminal fields and innervated mostly dendritic profiles in host cortical neurons. In physiological experiments, human ChR2+ neurons in culture generated spontaneous action potentials (APs) 100-170 days into differentiation and their firing activity was consistently driven by optical stimulation. Stimulation generated glutamatergic and GABAergic postsynaptic activity in neighboring ChR2- cells, evidence that hChR2-hNP-derived neurons had established functional synaptic connections with other neurons in culture. Light stimulation of hChR2-hNP transplants in vivo generated complicated results, in part because of the variable response of the transplants themselves. Our findings show that we can successfully derive hNPs with optogenetic properties that are fully transferrable to their differentiated neuronal progenies. We also show that these progenies have substantial neurotransmitter plasticity in vitro, whereas in vivo they mostly differentiate into inhibitory GABAergic neurons. Furthermore, neurons derived from hNPs have the capacity of establishing functional synapses with postsynaptic neurons in vitro, but this outcome is technically challenging to explore in vivo. We propose that optogenetically endowed hNPs hold great promise as tools to explore de novo circuit formation in the brain and, in the future, perhaps launch a new generation of neuromodulatory therapies.

Clustered Ca2+ Channels Are Blocked by Synaptic Vesicle Proton Release at Mammalian Auditory Ribbon Synapses.

A Ca2+ current transient block (ICaTB) by protons occurs at some ribbon-type synapses after exocytosis, but this has not been observed at mammalian hair cells. Here we show that a robust ICaTB occurs at post-hearing mouse and gerbil inner hair cell (IHC) synapses, but not in immature IHC synapses, which contain non-compact active zones, where Ca2+ channels are loosely coupled to the release sites. Unlike ICaTB at other ribbon synapses, ICaTB in mammalian IHCs displays a surprising multi-peak structure that mirrors the EPSCs seen in paired recordings. Desynchronizing vesicular release with intracellular BAPTA or by deleting otoferlin, the Ca2+ sensor for exocytosis, greatly reduces ICaTB, whereas enhancing release synchronization by raising Ca2+ influx or temperature increases ICaTB. This suggests that ICaTB is produced by fast multivesicular proton-release events. We propose that ICaTB may function as a submillisecond feedback mechanism contributing to the auditory nerve's fast spike adaptation during sound stimulation.

Different CaV1.3 Channel Isoforms Control Distinct Components of the Synaptic Vesicle Cycle in Auditory Inner Hair Cells.

The mechanisms orchestrating transient and sustained exocytosis in auditory inner hair cells (IHCs) remain largely unknown. These exocytotic responses are believed to mobilize sequentially a readily releasable pool of vesicles (RRP) underneath the synaptic ribbons and a slowly releasable pool of vesicles (SRP) at farther distance from them. They are both governed by Cav1.3 channels and require otoferlin as Ca2+ sensor, but whether they use the same Cav1.3 isoforms is still unknown. Using whole-cell patch-clamp recordings in posthearing mice, we show that only a proportion (∼25%) of the total Ca2+ current in IHCs displaying fast inactivation and resistance to 20 µm nifedipine, a l-type Ca2+ channel blocker, is sufficient to trigger RRP but not SRP exocytosis. This Ca2+ current is likely conducted by short C-terminal isoforms of Cav1.3 channels, notably Cav1.342A and Cav1.343S, because their mRNA is highly expressed in wild-type IHCs but poorly expressed in Otof-/- IHCs, the latter having Ca2+ currents with considerably reduced inactivation. Nifedipine-resistant RRP exocytosis was poorly affected by 5 mm intracellular EGTA, suggesting that the Cav1.3 short isoforms are closely associated with the release site at the synaptic ribbons. Conversely, our results suggest that Cav1.3 long isoforms, which carry ∼75% of the total IHC Ca2+ current with slow inactivation and confer high sensitivity to nifedipine and to internal EGTA, are essentially involved in recruiting SRP vesicles. Intracellular Ca2+ imaging showed that Cav1.3 long isoforms support a deep intracellular diffusion of Ca2+SIGNIFICANCE STATEMENT Auditory inner hair cells (IHCs) encode sounds into nerve impulses through fast and indefatigable Ca2+-dependent exocytosis at their ribbon synapses. We show that this synaptic process involves long and short C-terminal isoforms of the Cav1.3 Ca2+ channel that differ in the kinetics of their Ca2+-dependent inactivation and their relative sensitivity to the l-type Ca2+ channel blocker nifedipine. The short C-terminal isoforms, having fast inactivation and low sensitivity to nifedipine, mainly control the fast fusion of the readily releasable pool (RRP); that is, they encode the phasic exocytotic component. The long isoforms, with slow inactivation and great sensitivity to nifedipine, mainly regulate the vesicular replenishment of the RRP; that is, the sustained or tonic exocytosis.

Exocytotic machineries of vestibular type I and cochlear ribbon synapses display similar intrinsic otoferlin-dependent Ca2+ sensitivity but a different coupling to Ca2+ channels.

The hair cell ribbon synapses of the mammalian auditory and vestibular systems differ greatly in their anatomical organization and firing properties. Notably, vestibular Type I hair cells (VHC-I) are surrounded by a single calyx-type afferent terminal that receives input from several ribbons, whereas cochlear inner hair cells (IHCs) are contacted by several individual afferent boutons, each facing a single ribbon. The specificity of the presynaptic molecular mechanisms regulating transmitter release at these different sensory ribbon synapses is not well understood. Here, we found that exocytosis during voltage activation of Ca(2+) channels displayed higher Ca(2+) sensitivity, 10 mV more negative half-maximum activation, and a smaller dynamic range in VHC-I than in IHCs. VHC-I had a larger number of Ca(2+) channels per ribbon (158 vs 110 in IHCs), but their Ca(2+) current density was twofold smaller because of a smaller open probability and unitary conductance. Using confocal and stimulated emission depletion immunofluorescence microscopy, we showed that VHC-I had fewer synaptic ribbons (7 vs 17 in IHCs) to which Cav1.3 channels are more tightly organized than in IHCs. Gradual intracellular Ca(2+) uncaging experiments revealed that exocytosis had a similar intrinsic Ca(2+) sensitivity in both VHC-I and IHCs (KD of 3.3 ± 0.6 µM and 4.0 ± 0.7 µM, respectively). In otoferlin-deficient mice, exocytosis was largely reduced in VHC-I and IHCs. We conclude that VHC-I and IHCs use a similar micromolar-sensitive otoferlin Ca(2+) sensor and that their sensory encoding specificity is essentially determined by a different functional organization of Ca(2+) channels at their synaptic ribbons.