isolating-culturing-vestibular-spiral-ganglion-somata-from-neonatal

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...

University of Southern California

Invariant distortion product otoacoustic emission (DPOAE) phase elucidates scaling symmetry in the cochlea. Below some low-frequency boundary, DPOAE phase slope steepens. The origin of this break in phase invariance is not clear. Stimulus frequency (SF)OAE delays computed from the slope of phase also manifest discontinuities at low frequencies, though the relationship between the breaking of cochlear scaling as defined by SFOAE and DPOAE metrics has not been examined. In this study, OAEs were recorded in normal-hearing human adults to probe cochlear scaling and its breaking and to examine the correspondence between two OAE metrics of scaling. Results indicate: (1) the apical break in DPOAE phase invariance cannot be explained by contributions from the reflection-source component; (2) DPOAE phase signals a break from scaling near 1.5 kHz and (3) DPOAE and SFOAE metrics of cochlear scaling produce phase discontinuities within approximately one-quarter octave of each other and show comparable rates of breaking, suggesting a common underlying origin.

Deviations from Scaling Symmetry in the Apical Half of the Human Cochlea.

This study presents the first reported measurements of stimulus frequency emissions (SFOAEs) in 15 human newborns and compares their magnitudes and phase-gradient delays to those reported in adults. SFOAEs in newborns were measured at stimulus levels as low as 15 dB sound pressure level (SPL). Responses were compared between adults and newborns at stimulus levels where SFOAEs in both age groups demonstrated approximately linear growth (<40 dB SPL for newborns, <25 dB SPL for adults). Neonates had adult-like SFOAE delays when compared in this fashion, which compensates for newborn middle ear inefficiencies.

Stimulus-frequency otoacoustic emissions in human newborns.

The vestibular nerve is characterized by two broad groups of neurons that differ in the timing of their interspike intervals; some fire at highly regular intervals, whereas others fire at highly irregular intervals. Heterogeneity in ion channel properties has been proposed as shaping these firing patterns (Highstein SM, Politoff AL. Brain Res 150: 182-187, 1978; Smith CE, Goldberg JM. Biol Cybern 54: 41-51, 1986). Kalluri et al. (J Neurophysiol 104: 2034-2051, 2010) proposed that regularity is controlled by the density of low-voltage-activated potassium currents (IKL). To examine the impact of IKL on spike timing regularity, we implemented a single-compartment model with three conductances known to be present in the vestibular ganglion: transient sodium (gNa), low-voltage-activated potassium (gKL), and high-voltage-activated potassium (gKH). Consistent with in vitro observations, removing gKL depolarized resting potential, increased input resistance and membrane time constant, and converted current step-evoked firing patterns from transient (1 spike at current onset) to sustained (many spikes). Modeled neurons were driven with a time-varying synaptic conductance that captured the random arrival times and amplitudes of glutamate-driven synaptic events. In the presence of gKL, spiking occurred only in response to large events with fast onsets. Models without gKL exhibited greater integration by responding to the superposition of rapidly arriving events. Three synaptic conductance were modeled, each with different kinetics to represent a variety of different synaptic processes. In response to all three types of synaptic conductance, models containing gKL produced spike trains with irregular interspike intervals. Only models lacking gKL when driven by rapidly arriving small excitatory postsynaptic currents were capable of generating regular spiking.

A biophysical model examining the role of low-voltage-activated potassium currents in shaping the responses of vestibular ganglion neurons.

Two distinct processes generate otoacoustic emissions (OAEs).  emissions, here recorded as stimulus frequency OAEs, are optimally informative at low sound levels and are more sensitive to slight hearing loss; they have been linked to cochlear amplifier gain and tuning.  emissions are strongest at moderate-high sound levels and persist despite mild hearing loss; they likely originate in the nonlinear process of hair cell transduction. In this preliminary study, we exploit the unique features of each by generating a combined reflection-distortion OAE profile in normal hearing and hearing-impaired ears. Distortion-product (DP) and stimulus-frequency (SF) OAEs were recorded over a broad range of stimulus levels and frequencies. Individual I/O and transfer functions were generated for both emission types in each ear, and OAE peak strength, compression threshold, and rate of compression were calculated. These combined SFOAE and DPOAE features in normal and hearing-impaired ears may provide a potentially informative and novel index of hearing loss. This is an initial step toward utilizing OAE source in characterizing cochlear function and dysfunction.

Exploiting Dual Otoacoustic Emission Sources.

The size and locations of pre-synaptic ribbons and glutamate receptors within and around inner hair cells are correlated with auditory afferent response features such as the spontaneous discharge rate (SR), threshold, and dynamic range of sound intensity representation (the so-called SR-groups). To test if the development of these spatial gradients requires experience with sound intensity, we quantified the size and spatial distribution of synaptic ribbons from the inner hair cells of neonatal rats before and after the onset of hearing (from post-natal day (P) 3 to P33). To quantify ribbon size, we used high resolution fluorescence confocal microscopy and 3-D reconstructions of immunolabeled ribbons. The size, density, and spatial distribution of ribbons changed during development. At P3, ribbons were densely clustered near the basal/modiolar face of the hair cell where low SR-groups preferentially contact adult hair cells. By P12, the disparity in ribbon count was less striking and ribbons were equally likely to occupy both faces. At all ages before P12, ribbons were larger on the modiolar face than on the pillar face. These differences initially grew larger with age but collapsed around the onset of hearing. Between P12 and P33, the spatial gradients remained small and began to re-emerge around P33. Even by P12, we did not find spatial gradients in the size of the post-synaptic glutamate receptors as is found on afferent terminals contacting adult inner hair cells. These results suggest that spatial gradients in ribbon size develop in the absence of sensory experience.

Spatial Gradients in the Size of Inner Hair Cell Ribbons Emerge Before the Onset of Hearing in Rats.

Otoacoustic emissions (OAEs) provide salient information about cochlear function and dysfunction. Two broad classes of emissions, linear reflection and nonlinear distortion, arise via distinct cochlear processes and hence, appear to provide independent information about cochlear health and hearing. Considered in combination, these two OAE types may characterize sensory hearing loss most effectively. In this study, the level-dependent growth of stimulus-frequency OAEs (a reflection-type emission) and distortion-product OAEs (a distortion-type emission) were measured in ten normal-hearing ears and eight ears with slight-to-moderate sensorineural hearing loss. Metrics of OAE strength and compression were derived from OAE input/output functions and then considered in a combined fashion. Results indicate that SFOAEs and DPOAEs differ significantly in their strength and compression features. When SFOAE and DPOAE metrics are displayed together on a two-dimensional plot, relatively well-defined data clusters describe their normative relationship. In hearing-impaired ears, this relationship is disrupted but not in a uniform way across ears; ears with similar audiograms showed differently altered joint-OAE profiles. Hearing loss sometimes affected only one OAE or one more than the other. Results suggest a joint-OAE profile is promising and warrants study in a large group of subjects with sensory hearing loss of varied etiologies.

Towards a joint reflection-distortion otoacoustic emission profile: Results in normal and impaired ears.

Vestibular ganglion neurons (VGNs) transmit information along parallel neuronal pathways whose signature distinction is variability in spike-timing; some fire at regular intervals while others fire at irregular intervals. The mechanisms driving timing differences are not fully understood but two opposing (but not mutually exclusive) hypotheses have emerged. In the first, regular-spiking is inversely correlated to the density of low-voltage-gated potassium currents (). In the second, regular spiking is directly correlated to the density of hyperpolarization-activated cyclic nucleotide-sensitive currents (). Supporting the idea that variations in ion channel composition shape spike-timing, VGNs from the first postnatal week respond to synaptic-noise-like current injections with irregular-firing patterns if they have  and with more regular firing patterns if they do not. However,  firing patterns are not as regular as those  Here we considered whether highly-regular spiking requires  currents and whether this dependence emerges later in development after channel expression matures. We recorded from rat VGN somata of either sex aged postnatal day (P)9-P21. Counter to expectation,  firing patterns were less diverse, more transient-spiking, and more irregular at older ages than at younger ages. Resting potentials hyperpolarized and resting conductance increased, consistent with developmental upregulation of  Activation of  (by increasing intracellular cAMP) increased spike rates but not spike-timing regularity. In a model, we found that activating  counter-intuitively suppressed regularity by recruiting  Developmental upregulation in  appears to overwhelm  These results counter previous hypotheses about how  shapes vestibular afferent responses. Vestibular sensory information is conveyed on parallel neuronal pathways with irregularly-firing neurons encoding information using a temporal code and regularly-firing neurons using a rate code. This is a striking example of spike-timing statistics influencing information coding. Previous studies from immature vestibular ganglion neurons (VGNs) identified hyperpolarization-activated mixed cationic currents () as driving highly-regular spiking and proposed that this influence grows with the current during maturation. We found that  becomes less influential, likely because maturing VGNs also acquire low-voltage-gated potassium currents (), whose inhibitory influence opposes  Because efferent activity can partly close , VGN firing patterns may become more receptive to extrinsic control. Spike-timing regularity likely relies on dynamic ion channel properties and complementary specializations in synaptic connectivity.

Enhanced Activation of HCN Channels Reduces Excitability and Spike-Timing Regularity in Maturing Vestibular Afferent Neurons.

The mechanoreceptive sensory hair cells in the inner ear are selectively vulnerable to numerous genetic and environmental insults. In mammals, hair cells lack regenerative capacity, and their death leads to permanent hearing loss and vestibular dysfunction. Their paucity and inaccessibility has limited the search for otoprotective and regenerative strategies. Growing hair cells in vitro would provide a route to overcome this experimental bottleneck. We report a combination of four transcription factors (, and ) that can convert mouse embryonic fibroblasts, adult tail-tip fibroblasts and postnatal supporting cells into induced hair cell-like cells (iHCs). iHCs exhibit hair cell-like morphology, transcriptomic and epigenetic profiles, electrophysiological properties, mechanosensory channel expression, and vulnerability to ototoxin in a high-content phenotypic screening system. Thus, direct reprogramming provides a platform to identify causes and treatments for hair cell loss, and may help identify future gene therapy approaches for restoring hearing.

Generation of inner ear hair cells by direct lineage conversion of primary somatic cells.

Sound intensity is encoded by auditory neuron subgroups that differ in thresholds and spontaneous rates. Whether variations in neuronal biophysics contributes to this functional diversity is unknown. Because intensity thresholds correlate with synaptic position on sensory hair cells, we combined patch clamping with fiber labeling in semi-intact cochlear preparations in neonatal rats from both sexes. The biophysical properties of auditory neurons vary in a striking spatial gradient with synaptic position. Neurons with high thresholds to injected currents contact hair cells at synaptic positions where neurons with high thresholds to sound-intensity are found in vivo. Alignment between in vitro and in vivo thresholds suggests that biophysical variability contributes to intensity coding. Biophysical gradients were evident at all ages examined, indicating that cell diversity emerges in early post-natal development and persists even after continued maturation. This stability enabled a remarkably successful model for predicting synaptic position based solely on biophysical properties.

Gradients in the biophysical properties of neonatal auditory neurons align with synaptic contact position and the intensity coding map of inner hair cells.

The membranes of auditory and vestibular afferent neurons each contain diverse groups of ion channels that lead to heterogeneity in their intrinsic biophysical properties. Pioneering work in both auditory- and vestibular-ganglion physiology have individually examined this remarkable diversity, but there are few direct comparisons between the two ganglia. Here the firing patterns recorded by whole-cell patch-clamping in neonatal vestibular- and spiral ganglion neurons are compared. Indicative of an overall heterogeneity in ion channel composition, both ganglia exhibit qualitatively similar firing patterns ranging from sustained-spiking to transient-spiking in response to current injection. The range of resting potentials, voltage thresholds, current thresholds, input-resistances, and first-spike latencies are similarly broad in both ganglion groups. The covariance between several biophysical properties (e.g., resting potential to voltage threshold and their dependence on postnatal age) was similar between the two ganglia. Cell sizes were on average larger and more variable in VGN than in SGN. One sub-group of VGN stood out as having extra-large somata with transient-firing patterns, very low-input resistance, fast first-spike latencies, and required large current amplitudes to induce spiking. Despite these differences, the input resistance per unit area of the large-bodied transient neurons was like that of smaller-bodied transient-firing neurons in both VGN and SGN, thus appearing to be size-scaled versions of other transient-firing neurons. Our analysis reveals that although auditory and vestibular afferents serve very different functions in distinct sensory modalities, their biophysical properties are more closely related by firing pattern and cell size than by sensory modality.

Similarities in the Biophysical Properties of Spiral-Ganglion and Vestibular-Ganglion Neurons in Neonatal Rats.

Spiral ganglion neurons (SGN) are the primary neuronal pathway for transmitting sensory information from the inner ear to the brainstem. Recent studies have revealed significant biophysical and molecular diversity indicating that auditory neurons are comprised of sub-groups whose intrinsic properties contribute to their diverse functions. Previous approaches for studying the intrinsic biophysical properties of spiral ganglion neurons relied on patch-clamp and molecular analysis of cultured somata that were disconnected from their pre-synaptic hair cell partners. In the absence of the information provided by cell-to-cell connectivity, such studies could not associate biophysical diversity with functional sub-groups. Here we describe a protocol for preparing, recording, and labeling spiral ganglion neurons in a semi-intact ex-vivo preparation. In these preparations, the cell bodies of spiral ganglion neurons remain connected to their hair cell partners. The recordings are completed within 4 hours of euthanasia, alleviating concerns about whether long culture times and culture conditions change the intrinsic properties of neurons.

Patch-clamp Recordings and Single Fiber Labeling from Spiral Ganglion Somata in Acutely Prepared Semi-intact Cochleae from Neonatal Rats.

Efferent modulation of vestibular afferent excitability is linked to muscarinic signaling cascades that close low-voltage-gated potassium channels (i.e., KCNQ). Here, we show that muscarinic signaling cascades also depolarize the activation range of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels. We compared the voltage activation range and kinetics of HCN channels and induced firing patterns before and after administering the muscarinic acetylcholine receptor (mAChR) agonist oxotremorine-M (Oxo-M) in dissociated vestibular ganglion neurons (VGNs) from rats of either sex using perforated whole-cell patch-clamp methods. Oxo-M depolarized HCN channels' half-activation voltage ( ) and sped up the rate of activation near resting potential twofold. HCN channels in large-diameter and/or transient firing VGN (putative cell bodies of irregular firing neuron from central epithelial zones) had relatively depolarized   in control solution and were less sensitive to mAChR activation than those found in small-diameter VGN with sustained firing patterns (putatively belonging to regular firing afferents). The impact of mAChR on HCN channels is not a direct consequence of closing KCNQ channels since pretreating the cells with Linopirdine, a KCNQ channel blocker, did not prevent HCN channel depolarization by Oxo-M. Efferent signaling promoted ion channel configurations that were favorable to highly regular spiking in some VGN, but not others. This is consistent with previous observations that low-voltage gated potassium currents in VGN are conducted by mAChR agonist-sensitive and -insensitive channels. Connecting efferent signaling to HCN channels is significant because of the channel's impact on spike-timing regularity and nonchemical transmission between Type I hair cells and vestibular afferents. Vestibular afferents express a diverse complement of ion channels.  studies identified low-voltage activated potassium channels and hyperpolarization-activated cyclic-nucleotide gated (HCN) channels as crucial for shaping the timing and sensitivity of afferent responses. Moreover, a network of acetylcholine-releasing efferent neurons controls afferent excitability by closing a subgroup of low-voltage activated potassium channels on the afferent neuron. This work shows that these efferent signaling cascades also enhance the activation of HCN channels by depolarizing their voltage activation range. The size of this effect varies depending on the endogenous properties of the HCN channel and on cell type (as determined by discharge patterns and cell size). Simultaneously controlling two ion-channel groups gives the vestibular efferent system exquisite control over afferent neuron activity.

Muscarinic Acetylcholine Receptors Modulate HCN Channel Properties in Vestibular Ganglion Neurons.

Nuclear position is precisely orchestrated during cell division, migration, and maturation of cells and tissues. Here we report a previously unrecognized, programmed movement of the nucleus in rat and mouse cochlear inner hair cells (IHCs) coinciding with the functional maturation of inner hair cells around the onset of hearing.

Article	Year
Deviations from Scaling Symmetry in the Apical Half of the Human Cochlea. AIP conference proceedings\| PubMed ID: 22745514	2011
Stimulus-frequency otoacoustic emissions in human newborns. The Journal of the Acoustical Society of America\| PubMed ID: 25618103	2015
A biophysical model examining the role of low-voltage-activated potassium currents in shaping the responses of vestibular ganglion neurons. Journal of neurophysiology\| PubMed ID: 27121577	2016
Exploiting Dual Otoacoustic Emission Sources. AIP conference proceedings\| PubMed ID: 27695142	2015
Spatial Gradients in the Size of Inner Hair Cell Ribbons Emerge Before the Onset of Hearing in Rats. Journal of the Association for Research in Otolaryngology : JARO\| PubMed ID: 28361374	2017
Towards a joint reflection-distortion otoacoustic emission profile: Results in normal and impaired ears. The Journal of the Acoustical Society of America\| PubMed ID: 28863614	2017
Enhanced Activation of HCN Channels Reduces Excitability and Spike-Timing Regularity in Maturing Vestibular Afferent Neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 30696730	2019
Generation of inner ear hair cells by direct lineage conversion of primary somatic cells. eLife\| PubMed ID: 32602462	2020
Gradients in the biophysical properties of neonatal auditory neurons align with synaptic contact position and the intensity coding map of inner hair cells. eLife\| PubMed ID: 32639234	2020
Similarities in the Biophysical Properties of Spiral-Ganglion and Vestibular-Ganglion Neurons in Neonatal Rats. Frontiers in neuroscience\| PubMed ID: 34712112	2021
Patch-clamp Recordings and Single Fiber Labeling from Spiral Ganglion Somata in Acutely Prepared Semi-intact Cochleae from Neonatal Rats. Bio-protocol\| PubMed ID: 35118173	2022
Muscarinic Acetylcholine Receptors Modulate HCN Channel Properties in Vestibular Ganglion Neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 36604171	2023
Nuclear Translocation Triggered at the Onset of Hearing in Cochlear Inner Hair Cells of Rats and Mice. Journal of the Association for Research in Otolaryngology : JARO\| PubMed ID: 36932316	2023

Radha Kalluri

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