Arrays of MicroLEDs and Astrocytes: Biological Amplifiers to Optogenetically Modulate Neuronal Networks Reducing Light Requirement

In the modern view of synaptic transmission, astrocytes are no longer confined to the role of merely supportive cells. Although they do not generate action potentials, they nonetheless exhibit electrical activity and can influence surrounding neurons through gliotransmitter release. In this work, we explored whether optogenetic activation of glial cells could act as an amplification mechanism to optical neural stimulation via gliotransmission to the neural network. We studied the modulation of gliotransmission by selective photo-activation of channelrhodopsin-2 (ChR2) and by means of a matrix of individually addressable super-bright microLEDs (μLEDs) with an excitation peak at 470 nm. We combined Ca²⁺ imaging techniques and concurrent patch-clamp electrophysiology to obtain subsequent glia/neural activity. First, we tested the μLEDs efficacy in stimulating ChR2-transfected astrocyte. ChR2-induced astrocytic current did not desensitize overtime, and was linearly increased and prolonged by increasing μLED irradiance in terms of intensity and surface illumination. Subsequently, ChR2 astrocytic stimulation by broad-field LED illumination with the same spectral profile, increased both glial cells and neuronal calcium transient frequency and sEPSCs suggesting that few ChR2-transfected astrocytes were able to excite surrounding not-ChR2-transfected astrocytes and neurons. Finally, by using the μLEDs array to selectively light stimulate ChR2 positive astrocytes we were able to increase the synaptic activity of single neurons surrounding it. In conclusion, ChR2-transfected astrocytes and μLEDs system were shown to be an amplifier of synaptic activity in mixed corticalneuronal and glial cells culture.     

A comparison of electrically evoked and channel rhodopsin-evoked postsynaptic potentials in the pharyngeal system of Caenorhabditis elegans

Dissecting the function of neural circuits requires the capability to stimulate and record from the component neurones. Optimally, the methods employed should enable precise activation of distinct elements within the circuit and high-fidelity readout of the neuronal response. Here we compare two methods for neural stimulation in the pharyngeal system of Caenorhabditis elegans by evoking postsynaptic potentials (PSPs) either by electrical stimulation or by expression of the channelrhodopsin [ChR2(gf)] in cholinergic neurones of the pharyngeal circuit. Using a dissection that isolates the pharynx and its embedded neural system of 20 neurones permits analysis of the neurotransmitter pathways within this microcircuit. We describe protocols for selective electrically evoked or ChR2-mediated cholinergic synaptic events in this circuit. The latter was achieved by generating strains, punc-17::ChR2(gf);yfp, that express ChR2(gf) in cholinergic neurones. PSPs evoked by both electrical and light stimulation exhibited a rapid time-course and were blocked by cholinergic receptor antagonists and rapidly reversed on cessation of the stimulus. Electrically evoked PSPs were also reduced in a hypomorphic mutant for the synaptic vesicle acetylcholine transporter, unc-17, further indicating they are nicotinic cholinergic PSPs. The pharyngeal nervous system is exquisitely sensitive to both electrical and light activation. For the latter, short light pulses of 200 μs delivered to punc-17::ChR2(gf);yfp are capable of generating full muscle action potentials. We conclude that the application of optogenetic approaches to the C. elegans isolated pharynx preparation opens the way for a precise molecular dissection of synaptic events in the pharyngeal microcircuit by providing a molecular and system level analysis of the synapses that control the feeding behaviour of C. elegans.     

Role of nitric oxide on purinergic signalling in the cochlea

In the inner ear, there is considerable evidence that extracellular adenosine 5′-triphosphate (ATP) plays an important role in auditory neurotransmission as a neurotransmitter or a neuromodulator, although the potential role of adenosine signalling in the modulation of auditory neurotransmission has also been reported. The activation of ligand-gated ionotropic P2X receptors and G protein-coupled metabotropic P2Y receptors has been reported to induce an increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons (SGNs), and supporting cells in the cochlea. ATP may participate in auditory neurotransmission by modulating [Ca²⁺]_i in the cochlear cells. Recent studies showed that extracellular ATP induced nitric oxide (NO) production in IHCs, OHCs, and SGNs, which affects the ATP-induced Ca²⁺ response via the NO-cGMP-PKG pathway in those cells by a feedback mechanism. A cross-talk between NO and ATP may therefore exist in the auditory signal transduction. In the present article, I review the role of NO on the ATP-induced Ca²⁺ signalling in IHCs and OHCs. I also consider the possible role of NO in the ATP-induced Ca²⁺ signalling in SGNs and supporting cells.     

Optogenetic versus Electrical Stimulation of Human Cardiomyocytes: Modeling Insights

Optogenetics provides an alternative to electrical stimulation to manipulate membrane voltage, and trigger or modify action potentials (APs) in excitable cells. We compare biophysically and energetically the cellular responses to direct electrical current injection versus optical stimulation mediated by genetically expressed light-sensitive ion channels, e.g., Channelrhodopsin-2 (ChR2). Using a computational model of ChR2(H134R mutant), we show that both stimulation modalities produce similar-in-morphology APs in human cardiomyocytes, and that electrical and optical excitability vary with cell type in a similar fashion. However, whereas the strength-duration curves for electrical excitation in ventricular and atrial cardiomyocytes closely follow the theoretical exponential relationship for an equivalent RC circuit, the respective optical strength-duration curves significantly deviate, exhibiting higher nonlinearity. We trace the origin of this deviation to the waveform of the excitatory current—a nonrectangular self-terminating inward current produced in optical stimulation due to ChR2 kinetics and voltage-dependent rectification. Using a unifying charge measure to compare energy needed for electrical and optical stimulation, we reveal that direct electrical current injection (rectangular pulse) is more efficient at short pulses, whereas voltage-mediated negative feedback leads to self-termination of ChR2 current and renders optical stimulation more efficient for long low-intensity pulses. This applies to cardiomyocytes but not to neuronal cells (with much shorter APs). Furthermore, we demonstrate the cell-specific use of ChR2 current as a unique modulator of intrinsic activity, allowing for optical control of AP duration in atrial and, to a lesser degree, in ventricular myocytes. For self-oscillatory cells, such as Purkinje, constant light at extremely low irradiance can be used for fine control of oscillatory frequency, whereas constant electrical stimulation is not feasible due to electrochemical limitations. Our analysis offers insights for designing future new energy-efficient stimulation strategies in heart or brain.     

White light requirements for stimulation of plant growth by malformin

Over a 3-day period, the minimum white fluorescent light intensity required for malformin-induced growth stimulation of etiolated and green cuttings of Phaseolus aureus was approximately 2.6 × 10³ and 0.4 × 10³ ergs/cm² · sec, respectively. High light intensities were unable to inhibit the ability of malformin to stimulate growth. Over 3 days, the minimum photoperiod for malformin-induced growth stimulation using etiolated and green cuttings and a light intensity of 13.5 × 10³ ergs/cm² · sec was 4 hours and 1 hour, respectively. Malformin must be present in the area of growth stimulation during the time of light treatment. Those changes induced by light and required for malformin-induced growth stimulation were estimated to undergo almost complete decay within 1 hour in the dark. By manipulating the experimental technique, it was possible to stimulate the growth of green cuttings with malformin with a 10-min light treatment (13.5 × 10³ ergs/cm² · sec). Although low light intensities and short photoperiods did not allow growth stimulation by malformin using etiolated cuttings, they prevented or alleviated growth inhibition induced by malformin in the dark.     

Purinergic signaling in cochleovestibular hair cells and afferent neurons

Purinergic signaling in the mammalian cochleovestibular hair cells and afferent neurons is reviewed. The scope includes P2 and P1 receptors in the inner hair cells (IHCs) of the cochlea, the type I spiral ganglion neurons (SGNs) that convey auditory signals from IHCs, the vestibular hair cells (VHCs) in the vestibular end organs (macula in the otolith organs and crista in the semicircular canals), and the vestibular ganglion neurons (VGNs) that transmit postural and rotatory information from VHCs. Various subtypes of P2X ionotropic receptors are expressed in IHCs as well as P2Y metabotropic receptors that mobilize intracellular calcium. Their functional roles still remain speculative, but adenosine 5′-triphosphate (ATP) could regulate the spontaneous activity of the hair cells during development and the receptor potentials of mature hair cells during sound stimulation. In SGNs, P2Y metabotropic receptors activate a nonspecific cation conductance that is permeable to large cations as NMDG⁺ and TEA⁺. Remarkably, this depolarizing nonspecific conductance in SGNs can also be activated by other metabotropic processes evoked by acetylcholine and tachykinin. The molecular nature and the role of this depolarizing channel are unknown, but its electrophysiological properties suggest that it could lie within the transient receptor potential channel family and could regulate the firing properties of the afferent neurons. Studies on the vestibular partition (VHC and VGN) are sparse but have also shown the expression of P2X and P2Y receptors. There is still little evidence of functional P1 (adenosine) receptors in the afferent system of the inner ear.     

Glutamate residue 90 in the predicted transmembrane domain 2 is crucial for cation flux through channelrhodopsin 2

Channelrhodopsin 2 (ChR2) is a microbial-type rhodopsin with a putative heptahelical structure that binds all-trans-retinal. Blue light illumination of ChR2 activates an intrinsic leak channel conductive for cations. Sequence comparison of ChR2 with the related ChR1 protein revealed a cluster of charged amino acids within the predicted transmembrane domain 2 (TM2), which includes glutamates E90, E97 and E101. Charge inversion substitutions of these residues significantly altered ChR2 function as revealed by two-electrode voltage-clamp recordings of light-induced currents from Xenopus laevis oocytes expressing the respective mutant proteins. Specifically, replacement of E90 by lysine or alanine resulted in differential effects on H⁺- and Na⁺-mediated currents. Our results are consistent with this glutamate side chain within the proposed TM2 contributing to ion flux through and the cation selectivity of ChR2.     

In Vivo Optogenetic Control of Striatal and Thalamic Neurons in Non-Human Primates

Electrical and pharmacological stimulation methods are commonly used to study neuronal brain circuits in vivo, but are problematic, because electrical stimulation has limited specificity, while pharmacological activation has low temporal resolution. A recently developed alternative to these methods is the use of optogenetic techniques, based on the expression of light sensitive channel proteins in neurons. While optogenetics have been applied in in vitro preparations and in in vivo studies in rodents, their use to study brain function in nonhuman primates has been limited to the cerebral cortex. Here, we characterize the effects of channelrhodopsin-2 (ChR2) transfection in subcortical areas, i.e., the putamen, the external globus pallidus (GPe) and the ventrolateral thalamus (VL) of rhesus monkeys. Lentiviral vectors containing the ChR2 sequence under control of the elongation factor 1α promoter (pLenti-EF1α -hChR2(H134R)-eYFP-WPRE, titer 10⁹ particles/ml) were deposited in GPe, putamen and VL. Four weeks later, a probe combining a conventional electrode and an optic fiber was introduced in the previously injected brain areas. We found light-evoked responses in 31.5% and 32.7% of all recorded neurons in the striatum and thalamus, respectively, but only in 2.5% of recorded GPe neurons. As expected, most responses were time-locked increases in firing, but decreases or mixed responses were also seen, presumably via ChR2-mediated activation of local inhibitory connections. Light and electron microscopic analyses revealed robust expression of ChR2 on the plasma membrane of cell somas, dendrites, spines and terminals in the striatum and VL. This study demonstrates that optogenetic experiments targeting the striatum and basal ganglia-related thalamic nuclei can be successfully achieved in monkeys. Our results indicate important differences of the type and magnitude of responses in each structure. Experimental conditions such as the vector used, the number and rate of injections, or the light stimulation conditions have to be optimized for each structure studied.     

Functional Significance of K+ Channel β-Subunit KCNE3 in Auditory Neurons

The KCNE3 β-subunit interacts with and regulates the voltage-dependent gating, kinetics, and pharmacology of a variety of Kv channels in neurons. Because a single neuron may express multiple KCNE3 partners, it is impossible to predict the overall functional relevance of the single transmembrane domain peptide on the pore-forming K⁺ channel subunits with which it associates. In the inner ear, the role of KCNE3 is undefined, despite its association with Meniere disease and tinnitus. To gain insights on the functional significance of KCNE3 in auditory neurons, we examined the properties of spiral ganglion neurons (SGNs) in Kcne3 null mutant neurons relative to their age-matched controls. We demonstrate that null deletion of Kcne3 abolishes characteristic wide variations in the resting membrane potentials of SGNs and yields age-dependent alterations in action potential and firing properties of neurons along the contour of the cochlear axis, in comparison with age-matched wild-type neurons. The properties of basal SGNs were markedly altered in Kcne3^−/− mice compared with the wild-type controls; these include reduced action potential latency, amplitude, and increased firing frequency. Analyses of the underlying conductance demonstrate that null mutation of Kcne3 results in enhanced outward K⁺ currents, which is sufficient to explain the ensuing membrane potential changes. Additionally, we have demonstrated that KCNE3 may regulate the activity of Kv4.2 channels in SGNs. Finally, there were developmentally mediated compensatory changes that occurred such that, by 8 weeks after birth, the electrical properties of the null mutant neurons were virtually indistinguishable from the wild-type neurons, suggesting that ion channel remodeling in auditory neurons progresses beyond hearing onset.     

The Effect of 2,4,6-Trinitro-m-Cresol on Cation and Anion Transport in Sheep Red Blood Cells

2,4,6-Trinitro-3-methyl-phenol (trinitrocresol, H⁺TNC^-) was found to inhibit anion and stimulate cation movements across the membranes of both high potassium (HK) and low potassium (LK) sheep red blood cells. The concentration of TNC^- required to inhibit SO₄ ^- and Cl^- efflux (10^-5-10^-3 M) was less than that required to increase Na⁺ and K⁺ leakage (10^-3-10^-2 M). Both the inhibition of anion and stimulation of cation permeation were reversed if TNC^- was washed from the red cells. The cation leak caused by TNC^- was much greater at 0° and 37°C than at room temperature (23°C). In sheep red cells, TNC^- was found to be about 20 times more effective than salicylate and about 40 times more effective than thiocyanate in increasing cation leak. TNC^- also inhibited the ouabain-sensitive potassium influx.     

Impact of Morphometry,Myelinization and Synaptic Current Strength on Spike Conduction in Human and Cat Spiral Ganglion Neurons

Background

Our knowledge about the neural code in the auditory nerve is based to a large extent on experiments on cats. Several anatomical differences between auditory neurons in human and cat are expected to lead to functional differences in speed and safety of spike conduction.

Methodology/Principal Findings

Confocal microscopy was used to systematically evaluate peripheral and central process diameters, commonness of myelination and morphology of spiral ganglion neurons (SGNs) along the cochlea of three human and three cats. Based on these morphometric data, model analysis reveales that spike conduction in SGNs is characterized by four phases: a postsynaptic delay, constant velocity in the peripheral process, a presomatic delay and constant velocity in the central process. The majority of SGNs are type I, connecting the inner hair cells with the brainstem. In contrast to those of humans, type I neurons of the cat are entirely myelinated. Biophysical model evaluation showed delayed and weak spikes in the human soma region as a consequence of a lack of myelin. The simulated spike conduction times are in accordance with normal interwave latencies from auditory brainstem response recordings from man and cat. Simulated 400 pA postsynaptic currents from inner hair cell ribbon synapses were 15 times above threshold. They enforced quick and synchronous spiking. Both of these properties were not present in type II cells as they receive fewer and much weaker (^∼26 pA) synaptic stimuli.

Conclusions/Significance

Wasting synaptic energy boosts spike initiation, which guarantees the rapid transmission of temporal fine structure of auditory signals. However, a lack of myelin in the soma regions of human type I neurons causes a large delay in spike conduction in comparison with cat neurons. The absent myelin, in combination with a longer peripheral process, causes quantitative differences of temporal parameters in the electrically stimulated human cochlea compared to the cat cochlea.     

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)¹. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue². Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (>97% spike detection efficiency), has a low rate of false positive spike detection (< 0.003 spikes/sec), and a high temporal precision (about 3 msec) ³. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo^3,4.     

A sensory cell diversifies its output by varying Ca2+ influx‐release coupling among active zones

The cochlea encodes sound pressures varying over six orders of magnitude by collective operation of functionally diverse spiral ganglion neurons (SGNs). The mechanisms enabling this functional diversity remain elusive. Here, we asked whether the sound intensity information, contained in the receptor potential of the presynaptic inner hair cell (IHC), is fractionated via heterogeneous synapses. We studied the transfer function of individual IHC synapses by combining patch‐clamp recordings with dual‐color Rhod‐FF and iGluSnFR imaging of presynaptic Ca²⁺ signals and glutamate release. Synapses differed in the voltage dependence of release: Those residing at the IHC'' pillar side activated at more hyperpolarized potentials and typically showed tight control of release by few Ca²⁺ channels. We conclude that heterogeneity of voltage dependence and release site coupling of Ca²⁺ channels among the synapses varies synaptic transfer within individual IHCs and, thereby, likely contributes to the functional diversity of SGNs. The mechanism reported here might serve sensory cells and neurons more generally to diversify signaling even in close‐by synapses.     

Optimizing the spatial resolution of Channelrhodopsin-2 activation

Over the past few years, the light-gated cation channel Channelrhodopsin-2 (ChR2) has seen a remarkable diversity of applications in neuroscience. However, commonly used wide-field illumination provides poor spatial selectivity for cell stimulation. We explored the potential of focal laser illumination to map photocurrents of individual neurons in sparsely transfected hippocampal slice cultures. Interestingly, the best spatial resolution of photocurrent induction was obtained at the lowest laser power. By adjusting the light intensity to a neuron's spike threshold, we were able to trigger action potentials with a spatial selectivity of less than 30 microm. Experiments with dissociated hippocampal cells suggested that the main factor limiting the spatial resolution was ChR2 current density rather than scattering of the excitation light. We conclude that subcellular resolution can be achieved only in cells with a high ChR2 expression level and that future improved variants of ChR2 are likely to extend the spatial resolution of photocurrent induction to the level of single dendrites.     

Lignin synthesis: The generation of hydrogen peroxide and superoxide by horseradish peroxidase and its stimulation by manganese (II) and phenols

The enzyme horseradish peroxidase (EC 1.11.1.7) catalyses oxidation of NADH. NADH oxidation is prevented by addition of the enzyme superoxide dismutase (EC 1.15.1.1) to the reaction mixture before adding peroxidase but addition of dismutase after peroxidase has little inhibitory effect. Catalase (EC 1.11.1.6) inhibits peroxidase-catalysed NADH oxidation when added at any time during the reaction. Apparently the peroxidase uses hydrogen peroxide (H₂O₂) generated by non-enzymic breakdown of NADH to catalyse oxidation of NADH to a free-radical, NAD., which reduces oxygen to the superoxide free-radical ion, O₂ ^.-. Some of the O₂ ^.- reacts with peroxidase to give peroxidase compound III, which is catalytically inactive in NADH oxidation. The remaining O₂ ^.- undergoes dismutation to O₂ and H₂O₂. O₂ ^.- does not react with NADH at significant rates. Mn²⁺ or lactate dehydrogenase stimulate NADH oxidation by peroxidase because they mediate a reaction between O₂ ^.- and NADH. 2,4-Dichlorophenol, p-cresol and 4-hydroxycinnamic acid stimulate NADH oxidation by peroxidase, probably by breaking down compound III and so increasing the amount of active peroxidase in the reaction mixture. Oxidation in the presence of these phenols is greatly increased by adding H₂O₂. The rate of NADH oxidation by peroxidase is greatest in the presence of both Mn²⁺ and those phenols which interact with compound III. Both O₂ ^.- and H₂O₂ are involved in this oxidation, which plays an important role in lignin synthesis.     

Characterization of Engineered Channelrhodopsin Variants with Improved Properties and Kinetics

Channelrhodopsin 2 (ChR2), a light-activated nonselective cationic channel from Chlamydomonas reinhardtii, has become a useful tool to excite neurons into which it is transfected. The other ChR from Chlamydomonas, ChR1, has attracted less attention because of its proton-selective permeability. By making chimeras of the transmembrane domains of ChR1 and ChR2, combined with site-directed mutagenesis, we developed a ChR variant, named ChEF, that exhibits significantly less inactivation during persistent light stimulation. ChEF undergoes only 33% inactivation, compared with 77% for ChR2. Point mutation of Ile¹⁷⁰ of ChEF to Val (yielding “ChIEF”) accelerates the rate of channel closure while retaining reduced inactivation, leading to more consistent responses when stimulated above 25 Hz in both HEK293 cells and cultured hippocampal neurons. In addition, these variants have altered spectral responses, light sensitivity, and channel selectivity. ChEF and ChIEF allow more precise temporal control of depolarization, and can induce action potential trains that more closely resemble natural spiking patterns.     

Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives from their characteristic apical bundle of stereocilia, a critical element for detection and transduction of sound energy ¹. OHCs are able to change shape —elongate, shorten and bend— in response to electrical, mechanical and chemical stimulation, a motor response considered crucial for cochlear amplification of acoustic signals ².OHC stimulation induces two different motile responses: i) electromotility, a.k.a fast motility, changes in length in the microsecond range derived from electrically-driven conformational changes in motor proteins densely packed in OHC plasma membrane, and ii) slow motility, shape changes in the millisecond to seconds range involving cytoskeletal reorganization ^{2, 3}. OHC bending is associated with electromotility, and result either from an asymmetric distribution of motor proteins in the lateral plasma membrane, or asymmetric electrical stimulation of those motor proteins (e.g., with an electrical field perpendicular to the long axis of the cells) ⁴. Mechanical and chemical stimuli induce essentially slow motile responses, even though changes in the ionic conditions of the cells and/or their environment can also stimulate the plasma membrane-embedded motor proteins ^{5, 6}. Since OHC motile responses are an essential component of the cochlear amplifier, the qualitative and quantitative analysis of these motile responses at acoustic frequencies (roughly from 20 Hz to 20 kHz in humans) is a very important matter in the field of hearing research ⁷.The development of new imaging technology combining high-speed videocameras, LED-based illumination systems, and sophisticated image analysis software now provides the ability to perform reliable qualitative and quantitative studies of the motile response of isolated OHCs to an external alternating electrical field (EAEF) ⁸. This is a simple and non-invasive technique that circumvents most of the limitations of previous approaches ^9-11. Moreover, the LED-based illumination system provides extreme brightness with insignificant thermal effects on the samples and, because of the use of video microscopy, optical resolution is at least 10-fold higher than with conventional light microscopy techniques ¹². For instance, with the experimental setup described here, changes in cell length of about 20 nm can be routinely and reliably detected at frequencies of 10 kHz, and this resolution can be further improved at lower frequencies. We are confident that this experimental approach will help to extend our understanding of the cellular and molecular mechanisms underlying OHC motility.Download video file.^{(62M, mov)}     

EphA7 regulates spiral ganglion innervation of cochlear hair cells

During the development of periphery auditory circuitry, spiral ganglion neurons (SGNs) form a spatially precise pattern of innervation of cochlear hair cells (HCs), which is an essential structural foundation for central auditory processing. However, molecular mechanisms underlying the developmental formation of this precise innervation pattern remain not well understood. Here, we specifically examined the involvement of Eph family members in cochlear development. By performing RNA‐sequencing for different types of cochlear cell, in situ hybridization, and immunohistochemistry, we found that EphA7 was strongly expressed in a large subset of SGNs. In EphA7 deletion mice, there was a reduction in the number of inner radial bundles originating from SGNs and projecting to HCs as well as in the number of ribbon synapses on inner hair cells (IHCs), as compared with wild‐type or heterozygous mutant mice, attributable to fewer type I afferent fibers. The overall activity of the auditory nerve in EphA7 deletion mice was also reduced, although there was no significant change in the hearing intensity threshold. In vitro analysis further suggested that the reduced innervation of HCs by SGNs could be attributed to a role of EphA7 in regulating outgrowth of SGN neurites as knocking down EphA7 in SGNs resulted in diminished SGN fibers. In addition, suppressing the activity of ERK1/2, a potential downstream target of EphA7 signaling, either with specific inhibitors in cultured explants or by knocking out Prkg1, also resulted in reduced SGN fibers. Together, our results suggest that EphA7 plays an important role in the developmental formation of cochlear innervation pattern through controlling SGN fiber ontogeny. Such regulation may contribute to the salience level of auditory signals presented to the central auditory system. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 452–469, 2016