VDR Regulates BNP Promoting Neurite Growth and Survival of Cochlear Spiral Ganglion Neurons through cGMP-PKG Signaling Pathway.

Spiral ganglion neurons (SGNs) are important for hearing, and their peripheral and central processes connect sensory cells of the Corti organ to the central nervous system. The resulting network forms a point-to-point auditory conduction. As a cardiac hormone, brain natriuretic peptide (BNP) binds to natriuretic peptide receptor type A leading to diuresis, vasodilatation, inhibition of renin and aldosterone production, and cardiac and vascular myocyte growth. This study primarily aimed to explore the expression and function of BNP in the rat's inner ear and elucidate its regulatory mechanism. We determined the expression and function of BNP and found that the vitamin D receptor (VDR) could upregulate the expression of BNP and enhance its function. In SGNs of the rat inner ear, BNP promotes neuron survival and prolongs neurite length through the cGMP-PKG signaling pathway, which could be regulated by VDR and provide a novel approach for neuronal regeneration therapy. To the best of our knowledge, this is the first study to report this potential transcriptional regulatory relationship and will act as a reference for research on neuronal regeneration therapy for SGNs injury.

Salicylate Induced GABAAR Internalization by Dopamine D1-Like Receptors Involving Protein Kinase C (PKC) in Spiral Ganglion Neurons.

BACKGROUND Sodium salicylate (SS) induces excitotoxicity of spiral ganglion neurons (SGNs) by inhibiting the response of γ-aminobutyric acid type A receptors (GABAARs). Our previous studies have shown that SS can increase the internalization of GABAARs on SGNs, which involves dopamine D1-like receptors (D1Rs) and related signaling pathways. In this study, we aimed to explore the role of D1Rs and their downstream molecule protein kinase C (PKC) in the process of SS inhibiting GABAARs. MATERIAL AND METHODS The expression of D1Rs and GABARγ2 on rat cochlear SGNs cultured in vitro was tested by immunofluorescence. Then, the SGNs were exposed to SS, D1R agonist (SKF38393), D1R antagonist (SCH23390), clathrin/dynamin-mediated endocytosis inhibitor (dynasore), and PKC inhibitor (Bisindolylmaleimide I). Western blotting and whole-cell patch clamp technique were used to assess the changes of surface and total protein of GABARγ2 and GABA-activated currents. RESULTS Immunofluorescence showed that D1 receptors (DRD1) were expressed on SGNs. Data from western blotting showed that SS promoted the internalization of cell surface GABAARs, and activating D1Rs had the same result. Inhibiting D1Rs and PKC decreased the internalization of GABAARs. Meanwhile, the phosphorylation level of GABAARγ2 S327 affected by PKC was positively correlated with the degree of internalization of GABAARs. Moreover, whole-cell patch clamp recording showed that inhibition of D1Rs or co-inhibition of D1Rs and PKC attenuated the inhibitory effect of SS on GABA-activated currents. CONCLUSIONS D1Rs mediate the GABAAR internalization induced by SS via a PKC-dependent manner and participate in the excitotoxic process of SGNs.

Dynamic Heterogeneity Shapes Patterns of Spiral Ganglion Activity.

Neural response properties that typify primary sensory afferents are critical to fully appreciate because they establish and, ultimately represent, the fundamental coding design used for higher-level processing. Studies illuminating the center-surround receptive fields of retinal ganglion cells, for example, were ground-breaking because they determined the foundation of visual form detection. For the auditory system, a basic organizing principle of the spiral ganglion afferents is their extensive electrophysiological heterogeneity establishing diverse intrinsic firing properties in neurons throughout the spiral ganglion. Moreover, these neurons display an impressively large array of neurotransmitter receptor types that are responsive to efferent feedback. Thus, electrophysiological diversity and its neuromodulation are a fundamental encoding mechanism contributed by the primary afferents in the auditory system. To place these features into context, we evaluated the effects of hyperpolarization and cAMP on threshold level as indicators of overall afferent responsiveness in CBA/CaJ mice of either sex. Hyperpolarization modified threshold gradients such that distinct voltage protocols could shift the relationship between sensitivity and stimulus input to reshape resolution. This resulted in an "accordion effect" that appeared to stretch, compress, or maintain responsivity across the gradient of afferent thresholds. cAMP targeted threshold and kinetic shifts to rapidly adapting neurons, thus revealing multiple cochleotopic properties that could potentially be independently regulated. These examples of dynamic heterogeneity in primary auditory afferents not only have the capacity to shift the range, sensitivity, and resolution, but to do so in a coordinated manner that appears to orchestrate changes with a seemingly unlimited repertoire.SIGNIFICANCE STATEMENT How do we discriminate the more nuanced qualities of the sound around us? Beyond the basics of pitch and loudness, aspects, such as pattern, distance, velocity, and location, are all attributes that must be used to encode acoustic sensations effectively. While higher-level processing is required for perception, it would not be unexpected if the primary auditory afferents optimized receptor input to expedite neural encoding. The findings reported herein are consistent with this design. Neuromodulation compressed, expanded, shifted, or realigned intrinsic electrophysiological heterogeneity to alter neuronal responses selectively and dynamically. This suggests that diverse spiral ganglion phenotypes provide a rich substrate to support an almost limitless array of coding strategies within the first neural element of the auditory pathway.

A cell-type-specific atlas of the inner ear transcriptional response to acoustic trauma.

Noise-induced hearing loss (NIHL) results from a complex interplay of damage to the sensory cells of the inner ear, dysfunction of its lateral wall, axonal retraction of type 1C spiral ganglion neurons, and activation of the immune response. We use RiboTag and single-cell RNA sequencing to survey the cell-type-specific molecular landscape of the mouse inner ear before and after noise trauma. We identify induction of the transcription factors STAT3 and IRF7 and immune-related genes across all cell-types. Yet, cell-type-specific transcriptomic changes dominate the response. The ATF3/ATF4 stress-response pathway is robustly induced in the type 1A noise-resilient neurons, potassium transport genes are downregulated in the lateral wall, mRNA metabolism genes are downregulated in outer hair cells, and deafness-associated genes are downregulated in most cell types. This transcriptomic resource is available via the Gene Expression Analysis Resource (gEAR; https://umgear.org/NIHL) and provides a blueprint for the rational development of drugs to prevent and treat NIHL.

Analog transmission of action potential fine structure in spiral ganglion axons.

Action potential waveforms generated at the axon initial segment (AIS) are specialized between and within neuronal classes. But is the fine structure of each electrical event retained when transmitted along myelinated axons or is it rapidly and uniformly transmitted to be modified again at the axon terminal? To address this issue, action potential axonal transmission was evaluated in a class of primary sensory afferents that possess numerous types of voltage-gated ion channels underlying a complex repertoire of endogenous firing patterns. In addition to their signature intrinsic electrophysiological heterogeneity, spiral ganglion neurons are uniquely designed. The bipolar, myelinated somata of type I neurons are located within the conduction pathway, requiring that action potentials generated at the first heminode must be conducted through their electrically excitable membrane. We used this unusual axonal-like morphology to serve as a window into action potential transmission to compare locally evoked action potential profiles to those generated peripherally at their glutamatergic synaptic connections with hair cell receptors. These comparisons showed that the distinctively shaped somatic action potentials were highly correlated with the nodally generated, invading ones for each neuron. This result indicates that the fine structure of the action potential waveform is maintained axonally, thus supporting the concept that analog signaling is incorporated into each digitally transmitted action potential in the specialized primary auditory afferents.NEW & NOTEWORTHY Diverse action potential shapes and kinetics resulting from dynamic heterogeneity in spiral ganglion neurons are axonally transmitted as multiplexed signals that retain the fine structure of each distinctive waveform within a digital code.

Cochlear SGN neurons elevate pain thresholds in response to music.

The C-tactile (CLTM) peripheral nervous system is involved in social bonding in primates and humans through its capacity to trigger the brain's endorphin system. Since the mammalian cochlea has an unusually high density of similar neurons (type-II spiral ganglion neurons, SGNs), we hypothesise that their function may have been exploited for social bonding by co-opting head movements in response to music and other rhythmic movements of the head in social contexts. Music provides one of many cultural behavioural mechanisms for 'virtual grooming' in that it is used to trigger the endorphin system with many people simultaneously so as to bond both dyadic relationships and large groups. Changes in pain threshold across an activity are a convenient proxy assay for endorphin uptake in the brain, and we use this, in two experiments, to show that pain thresholds are higher when nodding the head than when sitting still.

Neurite Extension and Orientation of Spiral Ganglion Neurons Can Be Directed by Superparamagnetic Iron Oxide Nanoparticles in a Magnetic Field.

INTRODUCTION: Neuroregeneration is a major challenge in neuroscience for treating degenerative diseases and for repairing injured nerves. Numerous studies have shown the importance of physical stimulation for neuronal growth and development, and here we report an approach for the physical guidance of neuron orientation and neurite growth using superparamagnetic iron oxide (SPIO) nanoparticles and magnetic fields (MFs). METHODS: SPIO nanoparticles were synthesized by classic chemical co-precipitation methods and then characterized by transmission electron microscope, dynamic light scattering, and vibrating sample magnetometer. The cytotoxicity of the prepared SPIO nanoparticles and MF was determined using CCK-8 assay and LIVE/DEAD assay. The immunofluorescence images were captured by a laser scanning confocal microscopy. Cell migration was evaluated using the wound healing assay. RESULTS: The prepared SPIO nanoparticles showed a narrow size distribution, low cytotoxicity, and superparamagnetism. SPIO nanoparticles coated with poly-L-lysine could be internalized by spiral ganglion neurons (SGNs) and showed no cytotoxicity at concentrations less than 300 µg/mL. The neurite extension of SGNs was promoted after internalizing SPIO nanoparticles with or without an external MF, and this might be due to the promotion of growth cone development. It was also confirmed that SPIO can regulate cell migration and can direct neurite outgrowth in SGNs preferentially along the direction imposed by an external MF. CONCLUSION: Our results provide a fundamental understanding of the regulation of cell behaviors under physical cues and suggest alternative treatments for sensorineural hearing loss caused by the degeneration of SGNs.

Exosomes derived from cochlear spiral ganglion progenitor cells prevent cochlea damage from ischemia-reperfusion injury via inhibiting the inflammatory process.

Ischemia-reperfusion injury (I/R)-induced inflammatory process can mediate cochlea damage-related hearing loss; whether cochlear spiral ganglion progenitor cell-derived exosomes (CSGPC-exos) play a protective role by carrying functional microRNAs into recipient cells is unknown. Different doses of CSGPC-exos (0.1 µg/µl, 0.2 µg/µl, 0.5 µg/µl, 1.0 µg/µl) were administrated into the cochleae of the I/R group induced with 30-min occlusion of the bilateral vertebral arteries and sham surgery group. The speech-evoked auditory brainstem response (ABR) test was utilized to estimate the auditory threshold shift. The relative expression of proinflammatory cytokines was detected with RT-PCR and Western blot, while parvalbumin and caspase-3 expression were detected by immunofluorescence staining in the cochleae. The relative expression of microRNAs (miR-21-5p, miR-26a-5p, and miR-181a-5p) was detected in the cochleae. I/R significantly up-regulated ABR threshold shift and promoted hair cell apoptosis indicated by parvalbumin and caspase-3 staining, while CSGPC-exos (0.5 µg/µl, 1.0 µg/µl) could diminish such damages with downregulated proinflammatory factors (IL-6, IL-1ß, TNF-α, and Cox-2) and upregulated anti-inflammatory miRNAs (miR-21-5p, miR-26a-5p, and miR-181a-5p) expression in the cochleae. CSGPC-exos could protect cochleae damage from I/R, probably via inhibiting the inflammatory process.

Chromatin remodeler CHD7 is critical for cochlear morphogenesis and neurosensory patterning.

Epigenetic regulation of gene transcription by chromatin remodeling proteins has recently emerged as an important contributing factor in inner ear development. Pathogenic variants in CHD7, the gene encoding Chromodomain Helicase DNA binding protein 7, cause CHARGE syndrome, which presents with malformations in the developing ear. Chd7 is broadly expressed in the developing mouse otocyst and mature auditory epithelium, yet the pathogenic effects of Chd7 loss in the cochlea are not well understood. Here we characterized cochlear epithelial phenotypes in mice with deletion of Chd7 throughout the otocyst (using Foxg1Cre/+ and Pax2Cre), in the otic mesenchyme (using TCre), in hair cells (using Atoh1Cre), in developing neuroblasts (using NgnCre), or in spiral ganglion neurons (using ShhCre/+). Pan-otic deletion of Chd7 resulted in shortened cochleae with aberrant projections and axonal looping, disorganized, supernumerary hair cells at the apical turn and a narrowed epithelium with missing hair cells in the middle region. Deletion of Chd7 in the otic mesenchyme had no effect on overall cochlear morphology. Loss of Chd7 in hair cells did not disrupt their formation or organization of the auditory epithelium. Similarly, absence of Chd7 in spiral ganglion neurons had no effect on axonal projections. In contrast, deletion of Chd7 in developing neuroblasts led to smaller spiral ganglia and disorganized cochlear neurites. Together, these observations reveal dosage-, tissue-, and time-sensitive cell autonomous roles for Chd7 in cochlear elongation and cochlear neuron organization, with minimal functions for Chd7 in hair cells. These studies provide novel information about roles for Chd7 in development of auditory neurons.

CI in single-sided deafness.

The cochlear implant (CI) as a treatment option for single-sided deafness (SSD) started with a clinical study looking in to the influence of cochlear implantation with a MED-EL device on incapacitating unilateral tinnitus in SSD. The study began in 2003 and was conducted by P. Van de Heyning and his team in Antwerp, Belgium. The first CI in SSD without tinnitus in Germany was implanted by J. Mueller and R. Jacob in Koblenz in 2005. Translational research activities took place since then to evaluate the CI as a treatment option for SSD not only in adults but also in children. They assessed the hearing performance of SSD patients implanted with CI, importance of long electrode arrays in SSD patients, degree of acceptance of CI by SSD children, importance of early CI implantation in SSD children in developing language skills, music enjoyment by hearing with two ears and evidence on spiral ganglion cell body distribution. In 2013, MED-EL was the first CI manufacturer to receive the CE mark for the indication of SSD and asymmetric hearing loss (AHL) in adults and children. In 2019, MED-EL was the first CI manufacturer to get its CI device approved for patients over the age of five with SSD and AHL, by the FDA in the USA. This article covers the milestones of translational research from the first concept to the widespread clinical use of CI in SSD.

A human induced pluripotent stem cell-based modular platform to challenge sensorineural hearing loss.

The sense of hearing depends on a specialized sensory organ in the inner ear, called the cochlea, which contains the auditory hair cells (HCs). Noise trauma, infections, genetic factors, side effects of ototoxic drugs (ie, some antibiotics and chemotherapeutics), or simply aging lead to the loss of HCs and their associated primary neurons. This results in irreversible sensorineural hearing loss (SNHL) as in mammals, including humans; the inner ear lacks the capacity to regenerate HCs and spiral ganglion neurons. SNHL is a major global health problem affecting millions of people worldwide and provides a growing concern in the aging population. To date, treatment options are limited to hearing aids and cochlear implants. A major bottleneck for development of new therapies for SNHL is associated to the lack of human otic cell bioassays. Human induced pluripotent stem cells (hiPSCs) can be induced in two-dimensional and three-dimensional otic cells in vitro models that can generate inner ear progenitors and sensory HCs and could be a promising preclinical platform from which to work toward restoring SNHL. We review the potential applications of hiPSCs in the various biological approaches, including disease modeling, bioengineering, drug testing, and autologous stem cell based-cell therapy, that offer opportunities to understand the pathogenic mechanisms of SNHL and identify novel therapeutic strategies.

Electron Microscopic Reconstruction of Neural Circuitry in the Cochlea.

Recent studies reveal great diversity in the structure, function, and efferent innervation of afferent synaptic connections between the cochlear inner hair cells (IHCs) and spiral ganglion neurons (SGNs), which likely enables audition to process a wide range of sound pressures. By performing an extensive electron microscopic (EM) reconstruction of the neural circuitry in the mature mouse organ of Corti, we demonstrate that afferent SGN dendrites differ in abundance and composition of efferent innervation in a manner dependent on their afferent synaptic connectivity with IHCs. SGNs that sample glutamate release from several presynaptic ribbons receive more efferent innervation from lateral olivocochlear projections than those driven by a single ribbon. Next to the prevailing unbranched SGN dendrites, we found branched SGN dendrites that can contact several ribbons of 1-2 IHCs. Unexpectedly, medial olivocochlear neurons provide efferent innervation of SGN dendrites, preferring those forming single-ribbon, pillar-side synapses. We propose a fine-tuning of afferent and efferent SGN innervation.

Decreasing auditory input induces neurogenesis impairment in the hippocampus.

Hearing loss is associated with cognitive decline and dementia risk. Sensorineural hearing loss suppresses hippocampal neurogenesis, resulting in cognitive decline. However, the underlying mechanism of impaired neurogenesis and the role of microglial activation and stress responses related to hearing loss in the hippocampus remains unknown. Using a conductive hearing loss (CHL) model, we investigated whether a decrease in sound level could induce impairment of hippocampal neurogenesis and examined the differences between unilateral CHL (uCHL) and bilateral CHL (bCHL). To establish the CHL mouse model, ears were unilaterally or bilaterally occluded for five weeks by auditory canal ligation. Although hearing thresholds were significantly increased following CHL, CHL mice exhibited no significant loss of spiral ganglion or hippocampal neurons. Hippocampal neurogenesis was significantly and equally decreased in both sides following uCHL. More severe decreases in hippocampal neurogenesis were observed in both sides in bCHL mice compared with that in uCHL mice. Furthermore, microglial invasion significantly increased following CHL. Serum cortisol levels, which indicate stress response, significantly increased following bCHL. Therefore, auditory deprivation could lead to increased microglial invasion and stress responses and might be a risk factor for hippocampal neurogenesis impairment.

Contrasting mechanisms for hidden hearing loss: Synaptopathy vs myelin defects.

Hidden hearing loss (HHL) is an auditory neuropathy characterized by normal hearing thresholds but reduced amplitudes of the sound-evoked auditory nerve compound action potential (CAP). In animal models, HHL can be caused by moderate noise exposure or aging, which induces loss of inner hair cell (IHC) synapses. In contrast, recent evidence has shown that transient loss of cochlear Schwann cells also causes permanent auditory deficits in mice with similarities to HHL. Histological analysis of the cochlea after auditory nerve remyelination showed a permanent disruption of the myelination patterns at the heminode of type I spiral ganglion neuron (SGN) peripheral terminals, suggesting that this defect could be contributing to HHL. To shed light on the mechanisms of different HHL scenarios observed in animals and to test their impact on type I SGN activity, we constructed a reduced biophysical model for a population of SGN peripheral axons whose activity is driven by a well-accepted model of cochlear sound processing. We found that the amplitudes of simulated sound-evoked SGN CAPs are lower and have greater latencies when heminodes are disorganized, i.e. they occur at different distances from the hair cell rather than at the same distance as in the normal cochlea. These results confirm that disruption of heminode positions causes desynchronization of SGN spikes leading to a loss of temporal resolution and reduction of the sound-evoked SGN CAP. Another mechanism resulting in HHL is loss of IHC synapses, i.e., synaptopathy. For comparison, we simulated synaptopathy by removing high threshold IHC-SGN synapses and found that the amplitude of simulated sound-evoked SGN CAPs decreases while latencies remain unchanged, as has been observed in noise exposed animals. Thus, model results illuminate diverse disruptions caused by synaptopathy and demyelination on neural activity in auditory processing that contribute to HHL as observed in animal models and that can contribute to perceptual deficits induced by nerve damage in humans.

Divergent membrane properties of mouse cochlear glial cells around hearing onset.

Spiral ganglion neurons (SGNs) are the primary afferent neurons of the auditory system, and together with their attendant glia, form the auditory nerve. Within the cochlea, satellite glial cells (SGCs) encapsulate the cell body of SGNs, whereas Schwann cells (SCs) wrap their peripherally- and centrally-directed neurites. Despite their likely importance in auditory nerve function and homeostasis, the physiological properties of auditory glial cells have evaded description. Here, we characterized the voltage-activated membrane currents of glial cells from the mouse cochlea. We identified a prominent weak inwardly rectifying current in SGCs within cochlear slice preparations (postnatal day P5-P6), which was also present in presumptive SGCs within dissociated cultures prepared from the cochleae of hearing mice (P14-P15). Pharmacological block by Ba2+ and desipramine suggested that channels belonging to the Kir4 family mediated the weak inwardly rectifying current, and post hoc immunofluorescence implicated the involvement of Kir4.1 subunits. Additional electrophysiological profiles were identified for glial cells within dissociated cultures, suggesting that glial subtypes may have specific membrane properties to support distinct physiological roles. Immunofluorescence using fixed cochlear sections revealed that although Kir4.1 is restricted to SGCs after the onset of hearing, these channels are more widely distributed within the glial population earlier in postnatal development (i.e., within both SGCs and SCs). The decrease in Kir4.1 immunofluorescence during SC maturation was coincident with a reduction of Sox2 expression and advancing neurite myelination. The data suggest a diversification of glial properties occurs in preparation for sound-driven activity in the auditory nerve.

Directed differentiation and direct reprogramming: Applying stem cell technologies to hearing research.

Hearing loss is the most widely spread sensory disorder in our society. In the majority of cases, it is caused by the loss or malfunctioning of cells in the cochlea: the mechanosensory hair cells, which act as primary sound receptors, and the connecting auditory neurons of the spiral ganglion, which relay the signal to upper brain centers. In contrast to other vertebrates, where damage to the hearing organ can be repaired through the activity of resident cells, acting as tissue progenitors, in mammals, sensory cell damage or loss is irreversible. The understanding of gene and cellular functions, through analysis of different animal models, has helped to identify causes of disease and possible targets for hearing restoration. Translation of these findings to novel therapeutics is, however, hindered by the lack of cellular assays, based on human sensory cells, to evaluate the conservation of molecular pathways across species and the efficacy of novel therapeutic strategies. In the last decade, stem cell technologies enabled to generate human sensory cell types in vitro, providing novel tools to study human inner ear biology, model disease, and validate therapeutics. This review focuses specifically on two technologies: directed differentiation of pluripotent stem cells and direct reprogramming of somatic cell types to sensory hair cells and neurons. Recent development in the field are discussed as well as how these tools could be implemented to become routinely adopted experimental models for hearing research.

SGN nerve filaments develop synapses with IHCs earlier than with OHCs in C57BL/6 mouse inner ear.

OBJECTIVE: To explore the connections between hair cells and spiral ganglion neurons (SGNs) during the development of the C57BL/6 mouse inner ear. MATERIALS AND METHODS: The specimens of C57BL/6 mouse inner ear, from E15 (embryo day 15) to adult mouse, were collected; immunohistochemistry was employed to explore the frozen sections of specimens. RESULTS: The development of cochlea starts sequentially from the basal turn to the apex turn. Morphological development of SGNs occurs mainly from E16 to P12 (postnatal day 12). Hair cells appear from E18 to P12, and inner hair cells (IHCs) develop earlier than outer hair cells (OHCs). The connections between hair cells and SGNs begin to develop during E18-P1, morphologically resemble mature synapses during P8-P12, and completely mature in adult mice. CONCLUSIONS: The genesis of auditory ribbon synapse occurs from E18 to P1. Synchronized with the development of SGNs and hair cells, the functional filaments remain connected to hair cells, while the spare ones get disconnected from the surface of hair cells. Connections between SGN nerve filaments and IHCs occur earlier than those between SGN nerve filaments and OHCs.

Neuronal processes and glial precursors form a scaffold for wiring the developing mouse cochlea.

In the developing nervous system, axons navigate through complex terrains that change depending on when and where outgrowth begins. For instance, in the developing cochlea, spiral ganglion neurons extend their peripheral processes through a growing and heterogeneous environment en route to their final targets, the hair cells. Although the basic principles of axon guidance are well established, it remains unclear how axons adjust strategies over time and space. Here, we show that neurons with different positions in the spiral ganglion employ different guidance mechanisms, with evidence for both glia-guided growth and fasciculation along a neuronal scaffold. Processes from neurons in the rear of the ganglion are more directed and grow faster than those from neurons at the border of the ganglion. Further, processes at the wavefront grow more efficiently when in contact with glial precursors growing ahead of them. These findings suggest a tiered mechanism for reliable axon guidance.

Atrial Natriuretic Peptide Improves Neurite Outgrowth from Spiral Ganglion Neurons In Vitro through a cGMP-Dependent Manner.

The spiral ganglion neurons (SGNs) are the primary afferent neurons in the spiral ganglion (SG), while their degeneration or loss would cause sensorineural hearing loss. As a cardiac-derived hormone, atrial natriuretic peptide (ANP) plays a critical role in cardiovascular homeostasis through binding to its functional receptors (NPR-A and NPR-C). ANP and its receptors are widely expressed in the mammalian nervous system where they could be implicated in the regulation of multiple neural functions. Although previous studies have provided direct evidence for the presence of ANP and its functional receptors in the inner ear, their presence within the cochlear SG and their regulatory roles during auditory neurotransmission and development remain largely unknown. Based on our previous findings, we investigated the expression patterns of ANP and its receptors in the cochlear SG and dissociated SGNs and determined the influence of ANP on neurite outgrowth in vitro by using organotypic SG explants and dissociated SGN cultures from postnatal rats. We have demonstrated that ANP and its receptors are expressed in neurons within the cochlear SG of postnatal rat, while ANP may promote neurite outgrowth of SGNs via the NPR-A/cGMP/PKG pathway in a dose-dependent manner. These results indicate that ANP would play a role in normal neuritogenesis of SGN during cochlear development and represents a potential therapeutic candidate to enhance regeneration and regrowth of SGN neurites.

Altered expression of genes regulating inflammation and synaptogenesis during regrowth of afferent neurons to cochlear hair cells.

The spiral ganglion neurons constitute the primary connection between auditory hair cells and the brain. The spiral ganglion afferent fibers and their synapse with hair cells do not regenerate to any significant degree in adult mammalian ears after damage. We have investigated gene expression changes after kainate-induced disruption of the synapses in a neonatal cochlear explant model in which peripheral fibers and the afferent synapse do regenerate. We compared gene expression early after damage, during regeneration of the fibers and synapses, and after completion of in vitro regeneration. These analyses revealed a total of 2.5% differentially regulated transcripts (588 out of 24,000) based on a threshold of p<0.005. Inflammatory response genes as well as genes involved in regeneration of neural circuits were upregulated in the spiral ganglion neurons and organ of Corti, where the hair cells reside. Prominent genes upregulated at several time points included genes with roles in neurogenesis (Elavl4 and Sox21), neural outgrowth (Ntrk3 and Ppp1r1c), axonal guidance (Rgmb and Sema7a), synaptogenesis (Nlgn2 and Psd2), and synaptic vesicular function (Syt8 and Syn1). Immunohistochemical and in situ hybridization analysis of genes that had not previously been described in the cochlea confirmed their cochlear expression. The time course of expression of these genes suggests that kainate treatment resulted in a two-phase response in spiral ganglion neurons: an acute response consistent with inflammation, followed by an upregulation of neural regeneration genes. Identification of the genes activated during regeneration of these fibers suggests candidates that could be targeted to enhance regeneration in adult ears.