Spiking Pattern of the Mouse Developing Inner Hair Cells Is Mostly Invariant Along the Tonotopic Axis.
ABSTRACT: During development, the sensory cells of the cochlea, the inner hair cells (IHCs), fire spontaneous calcium action potentials. This activity at the pre-hearing stage allows the IHCs to autonomously excite the auditory nerve fibers and hence, represents an efficient mechanism to shape the tonotopic organization along the ascending auditory pathway. Using calcium imaging, we show that the activity in the developing cochlea consists of calcium waves that propagate across the supporting and sensory cells. Both basal and apical IHCs were characterized by similar spontaneous calcium transients interspaced with silent periods, consistent with bursts of action potentials recorded in patch-clamp. In addition, adjacent auditory hair cells tend to have a synchronized [Ca2+]i activity, irrespective of their location along the base-to-apex gradient of the cochlea. Finally, we show that the mechanical ablation of the inner phalangeal cells (IPCs), a class of supporting cells, reduces the synchronized [Ca2+]i activity between neighboring sensory cells. These findings support the hypothesis that the tonotopic map refinement in higher auditory centers would depend on the synchronization of a discrete number of auditory sensory cells.
Project description:Inner hair cells (IHCs) are the primary transducer for sound encoding in the cochlea. In contrast to the graded receptor potential of adult IHCs, immature hair cells fire spontaneous calcium action potentials during the first postnatal week. This spiking activity has been proposed to shape the tonotopic map along the ascending auditory pathway. Using perforated patch-clamp recordings, we show that developing IHCs fire spontaneous bursts of action potentials and that this pattern is indistinguishable along the basoapical gradient of the developing cochlea. In both apical and basal IHCs, the spiking behavior undergoes developmental changes, where the bursts of action potential tend to occur at a regular time interval and have a similar length toward the end of the first postnatal week. Although disruption of purinergic signaling does not interfere with the action potential firing pattern, pharmacological ablation of the ?9?10 nicotinic receptor elicits an increase in the discharge rate. We therefore suggest that in addition to carrying place information to the ascending auditory nuclei, the IHCs firing pattern controlled by the ?9?10 receptor conveys a temporal signature of the cochlear development.
Project description:Spontaneous action potential activity is crucial for mammalian sensory system development. In the auditory system, patterned firing activity has been observed in immature spiral ganglion and brain-stem neurons and is likely to depend on cochlear inner hair cell (IHC) action potentials. It remains uncertain whether spiking activity is intrinsic to developing IHCs and whether it shows patterning. We found that action potentials were intrinsically generated by immature IHCs of altricial rodents and that apical IHCs showed bursting activity as opposed to more sustained firing in basal cells. We show that the efferent neurotransmitter acetylcholine fine-tunes the IHC's resting membrane potential (V(m)), and as such is crucial for the bursting pattern in apical cells. Endogenous extracellular ATP also contributes to the V(m) of apical and basal IHCs by triggering small-conductance Ca(2+)-activated K(+) (SK2) channels. We propose that the difference in firing pattern along the cochlea instructs the tonotopic differentiation of IHCs and auditory pathway.
Project description:In the mature auditory system, inner hair cells (IHCs) convert sound-induced vibrations into electrical signals that are relayed to the central nervous system via auditory afferents. Before the cochlea can respond to normal sound levels, developing IHCs fire calcium-based action potentials that disappear close to the onset of hearing. Action potential firing triggers transmitter release from the immature IHC that in turn generates experience-independent firing in auditory neurons. These early signaling events are thought to be essential for the organization and development of the auditory system and hair cells. A critical component of the action potential is the rise in intracellular calcium that activates both small conductance potassium channels essential during membrane repolarization, and triggers transmitter release from the cell. Whether this calcium signal is generated by calcium influx or requires calcium-induced calcium release (CICR) is not yet known. IHCs can generate CICR, but to date its physiological role has remained unclear. Here, we used high and low concentrations of ryanodine to block or enhance CICR to determine whether calcium release from intracellular stores affected action potential waveform, interspike interval, or changes in membrane capacitance during development of mouse IHCs. Blocking CICR resulted in mixed action potential waveforms with both brief and prolonged oscillations in membrane potential and intracellular calcium. This mixed behavior is captured well by our mathematical model of IHC electrical activity. We perform two-parameter bifurcation analysis of the model that predicts the dependence of IHCs firing patterns on the level of activation of two parameters, the SK2 channels activation and CICR rate. Our data show that CICR forms an important component of the calcium signal that shapes action potentials and regulates firing patterns, but is not involved directly in triggering exocytosis. These data provide important insights into the calcium signaling mechanisms involved in early developmental processes.
Project description:Neurons in the developing auditory system fire bursts of action potentials before the onset of hearing. This spontaneous activity promotes the survival and maturation of auditory neurons and the refinement of synaptic connections in auditory nuclei; however, the mechanisms responsible for initiating this activity remain uncertain. Previous studies indicate that inner supporting cells (ISCs) in the developing cochlea periodically release ATP, which depolarizes inner hair cells (IHCs), leading to bursts of action potentials in postsynaptic spiral ganglion neurons (SGNs). To determine when purinergic signaling appears in the developing cochlea and whether it is responsible for initiating auditory neuron activity throughout the prehearing period, we examined spontaneous activity from ISCs, IHCs, and SGNs in cochleae acutely isolated from rats during the first three postnatal weeks. We found that ATP was released from ISCs within the cochlea from birth until the onset of hearing, which led to periodic inward currents, Ca(2+) transients, and morphological changes in these supporting cells. This spontaneous release of ATP also depolarized IHCs and triggered bursts of action potentials in SGNs for most of the postnatal prehearing period, beginning a few days after birth as IHCs became responsive to ATP, until the onset of hearing when ATP was no longer released from ISCs. When IHCs were not subject to purinergic excitation, SGNs exhibited little or no activity. These results suggest that supporting cells in the cochlea provide the primary excitatory stimulus responsible for initiating bursts of action potentials in auditory nerve fibers before the onset of hearing.
Project description:Mutations in the genes encoding for gap junction proteins connexin 26 (Cx26) and connexin 30 (Cx30) have been linked to syndromic and nonsyndromic hearing loss in mice and humans. The release of ATP from connexin hemichannels in cochlear nonsensory cells has been proposed to be the main trigger for action potential activity in immature sensory inner hair cells (IHCs), which is crucial for the refinement of the developing auditory circuitry. Using connexin knock-out mice, we show that IHCs fire spontaneous action potentials even in the absence of ATP-dependent intercellular Ca2+ signaling in the nonsensory cells. However, this signaling from nonsensory cells was able to increase the intrinsic IHC firing frequency. We also found that connexin expression is key to IHC functional maturation. In Cx26 conditional knock-out mice (Cx26Sox10-Cre), the maturation of IHCs, which normally occurs at approximately postnatal day 12, was partially prevented. Although Cx30 has been shown not to be required for hearing in young adult mice, IHCs from Cx30 knock-out mice exhibited a comprehensive brake in their development, such that their basolateral membrane currents and synaptic machinery retain a prehearing phenotype. We propose that IHC functional differentiation into mature sensory receptors is initiated in the prehearing cochlea provided that the expression of either connexin reaches a threshold level. As such, connexins regulate one of the most crucial functional refinements in the mammalian cochlea, the disruption of which contributes to the deafness phenotype observed in mice and DFNB1 patients. SIGNIFICANCE STATEMENT:The correct development and function of the mammalian cochlea relies not only on the sensory hair cells, but also on the surrounding nonsensory cells. Although the nonsensory cells have been largely implicated in the general homeostasis in the mature cochlea, their involvement in the initial functional differentiation of the sensory inner hair cells is less clear. Using mutant mouse models for the most common form of congenital deafness in humans, which are knock-outs for the gap-junction channels connexin 26 and connexin 30 genes, we show that defects in nonsensory cells prevented the functional maturation of inner hair cells. In connexin knock-outs, inner hair cells remained stuck at a prehearing stage of development and, as such, are unable to process sound information.
Project description:The perception of sound relies on sensory hair cells in the cochlea that convert the mechanical energy of sound into release of glutamate onto postsynaptic auditory nerve fibers. The hair cell receptor potential regulates the strength of synaptic transmission and is shaped by a variety of voltage-dependent conductances. Among these conductances, the Ca2+- and voltage-activated large conductance Ca2+-activated K+ channel (BK) current is prominent, and in mammalian inner hair cells (IHCs) displays unusual properties. First, BK currents activate at unprecedentedly negative membrane potentials (-60 mV) even in the absence of intracellular Ca2+ elevations. Second, BK channels are positioned in clusters away from the voltage-dependent Ca2+ channels that mediate glutamate release from IHCs. Here, we test the contributions of two recently identified leucine-rich-repeat-containing (LRRC) regulatory ? subunits, LRRC26 and LRRC52, to BK channel function and localization in mouse IHCs. Whereas BK currents and channel localization were unaltered in IHCs from Lrrc26 knockout (KO) mice, BK current activation was shifted more than +200 mV in IHCs from Lrrc52 KO mice. Furthermore, the absence of LRRC52 disrupted BK channel localization in the IHCs. Given that heterologous coexpression of LRRC52 with BK ? subunits shifts BK current gating about -90 mV, to account for the profound change in BK activation range caused by removal of LRRC52, we suggest that additional factors may help define the IHC BK gating range. LRRC52, through stabilization of a macromolecular complex, may help retain some other components essential both for activation of BK currents at negative membrane potentials and for appropriate BK channel positioning.
Project description:Sensory transduction in auditory and vestibular hair cells requires expression of transmembrane channel-like (Tmc) 1 and 2 genes, but the function of these genes is unknown. To investigate the hypothesis that TMC1 and TMC2 proteins are components of the mechanosensitive ion channels that convert mechanical information into electrical signals, we recorded whole-cell and single-channel currents from mouse hair cells that expressed Tmc1, Tmc2, or mutant Tmc1. Cells that expressed Tmc2 had high calcium permeability and large single-channel currents, while cells with mutant Tmc1 had reduced calcium permeability and reduced single-channel currents. Cells that expressed Tmc1 and Tmc2 had a broad range of single-channel currents, suggesting multiple heteromeric assemblies of TMC subunits. The data demonstrate TMC1 and TMC2 are components of hair cell transduction channels and contribute to permeation properties. Gradients in TMC channel composition may also contribute to variation in sensory transduction along the tonotopic axis of the mammalian cochlea.
Project description:The development of neural circuits relies on spontaneous electrical activity that occurs during immature stages of development. In the developing mammalian auditory system, spontaneous calcium action potentials are generated by inner hair cells (IHCs), which form the primary sensory synapse. It remains unknown whether this electrical activity is required for the functional maturation of the auditory system. We found that sensory-independent electrical activity controls synaptic maturation in IHCs. We used a mouse model in which the potassium channel SK2 is normally overexpressed, but can be modulated in vivo using doxycycline. SK2 overexpression affected the frequency and duration of spontaneous action potentials, which prevented the development of the Ca(2+)-sensitivity of vesicle fusion at IHC ribbon synapses, without affecting their morphology or general cell development. By manipulating the in vivo expression of SK2 channels, we identified the "critical period" during which spiking activity influences IHC synaptic maturation. Here we provide direct evidence that IHC development depends upon a specific temporal pattern of calcium spikes before sound-driven neuronal activity.
Project description:Spontaneous electrical activity of neurons in developing sensory systems promotes their maturation and proper connectivity. In the auditory system, spontaneous activity of cochlear inner hair cells (IHCs) is initiated by the release of ATP from glia-like inner supporting cells (ISCs), facilitating maturation of central pathways before hearing onset. Here, we find that ATP stimulates purinergic autoreceptors in ISCs, triggering Cl(-) efflux and osmotic cell shrinkage by opening TMEM16A Ca(2+)-activated Cl(-) channels. Release of Cl(-) from ISCs also forces K(+) efflux, causing transient depolarization of IHCs near ATP release sites. Genetic deletion of TMEM16A markedly reduces the spontaneous activity of IHCs and spiral ganglion neurons in the developing cochlea and prevents ATP-dependent shrinkage of supporting cells. These results indicate that supporting cells in the developing cochlea have adapted a pathway used for fluid secretion in other organs to induce periodic excitation of hair cells.
Project description:Spontaneous bursts of activity in developing sensory pathways promote maturation of neurons, refinement of neuronal connections, and assembly of appropriate functional networks. In the developing auditory system, inner hair cells (IHCs) spontaneously fire Ca(2+) spikes, each of which is transformed into a mini-burst of action potentials in spiral ganglion neurons (SGNs). Here we show that NMDARs are expressed in SGN dendritic terminals and play a critical role during transmission of activity from IHCs to SGNs before hearing onset. NMDAR activation enhances glutamate-mediated Ca(2+) influx at dendritic terminals, promotes repetitive firing of individual SGNs in response to each synaptic event, and enhances coincident activity of neighboring SGNs that will eventually encode similar frequencies of sound. Loss of NMDAR signaling from SGNs reduced their survival both in vivo and in vitro, revealing that spontaneous activity in the prehearing cochlea promotes maturation of auditory circuitry through periodic activation of NMDARs in SGNs.