Noise-induced hearing loss vulnerability in type III intermediate filament peripherin gene knockout mice.

In the post-natal mouse cochlea, type II spiral ganglion neurons (SGNs) innervating the electromotile outer hair cells (OHCs) of the 'cochlear amplifier' selectively express the type III intermediate filament peripherin gene (Prph). Immunolabeling showed that Prph knockout (KO) mice exhibited disruption of this (outer spiral bundle) afferent innervation, while the radial fiber (type I SGN) innervation of the inner hair cells (~95% of the SGN population) was retained. Functionality of the medial olivocochlear (MOC) efferent innervation of the OHCs was confirmed in the PrphKO, based on suppression of distortion product otoacoustic emissions (DPOAEs) via direct electrical stimulation. However, "contralateral suppression" of the MOC reflex neural circuit, evident as a rapid reduction in cubic DPOAE when noise is presented to the opposite ear in wildtype mice, was substantially disrupted in the PrphKO. Auditory brainstem response (ABR) measurements demonstrated that hearing sensitivity (thresholds and growth-functions) were indistinguishable between wildtype and PrphKO mice. Despite this comparability in sound transduction and strength of the afferent signal to the central auditory pathways, high-intensity, broadband noise exposure (108 dB SPL, 1 h) produced permanent high frequency hearing loss (24-32 kHz) in PrphKO mice but not the wildtype mice, consistent with the attenuated contralateral suppression of the PrphKO. These data support the postulate that auditory neurons expressing Prph contribute to the sensory arm of the otoprotective MOC feedback circuit.

Onset kinetics of noise-induced purinergic adaptation of the 'cochlear amplifier'.

A major component of slowly reversible hearing loss which develops with sustained exposure to noise has been attributed to release of ATP in the cochlea activating P2X2 receptor (P2X2R) type ATP-gated ion channels. This purinergic humoral adaptation is thought to enable the highly sensitive hearing organ to maintain function with loud sound, protecting the ear from acoustic overstimulation. In the study that established this hearing adaptation mechanism as reported by Housley et al. (Proc Natl Acad Sci U S A 110:7494-7499, 2013), the activation kinetics were determined in mice from auditory brainstem response (ABR) threshold shifts with sustained noise presentation at time points beyond 10 min. The present study was designed to achieve finer resolution of the onset kinetics of purinergic hearing adaptation, and included the use of cubic (2f1-f2) distortion product otoacoustic emissions (DPOAEs) to probe whether the active mechanical outer hair cell 'cochlear amplifier' contributed to this process. We show that the ABR and DPOAE threshold shifts were largely complete within the first 7.5 min of moderate broadband noise (85 dB SPL) in wildtype C57Bl/6J mice. The ABR and DPOAE adaptation rates were both best fitted by a single exponential function with ~ 3 min time constants. ABR and DPOAE threshold shifts with this noise were minimal in mice null for the P2rx2 gene encoding the P2X2R. The findings demonstrate a considerably faster purinergic hearing adaptation to noise than previously appreciated. Moreover, they strongly implicate the outer hair cell as the site of action, as the DPOAEs stem from active cochlear electromotility.

Human Brain Region-Specific Alternative Splicing of TRPC3, the Type 3 Canonical Transient Receptor Potential Non-Selective Cation Channel.

Canonical transient receptor potential (TRPC) non-selective cation channels are broadly expressed by neurons, glia and the microvasculature of the brain. In neurons and astrocytes, these ion channels are coupled to group I metabotropic glutamate receptors via Gαq-phospholipase C signal transduction. In the mouse cerebellar Purkinje neurons, TRPC channels assembled as tetramers of TRPC3 subunits exclusively mediate this glutamatergic signalling mechanism and regulation of alternative splicing results in dominance of a high Ca2+ conducting TRPC3c isoform. This regional control of TRPC3 transcript type likely has physiological and pathophysiological sequelae. The current study provides a quantitative comparison of the TRPC3c splice variant and the TRPC3b full-length isoform expression across seven regions of the human brain. This shows that the cerebellum has the highest expression level of both isoforms and that regulation of alternative splicing results in a higher propensity of the TRPC3c isoform in the cerebellum relative to the TRPC3b isoform (in a 1:3 ratio). This compares with the other regions (motor cortex, hippocampus, midbrain subregions, pons and medulla) where the prevalence of TRPC3c relative to TRPC3b is typically less than half as abundant. The finding here of a bias in the high-conductance TRPC3c isoform in the cerebellum is consistent with the enhanced vulnerability of the cerebellum to ischaemic injury.

Type II spiral ganglion afferent neurons drive medial olivocochlear reflex suppression of the cochlear amplifier.

The dynamic adjustment of hearing sensitivity and frequency selectivity is mediated by the medial olivocochlear efferent reflex, which suppresses the gain of the 'cochlear amplifier' in each ear. Such efferent feedback is important for promoting discrimination of sounds in background noise, sound localization and protecting the cochleae from acoustic overstimulation. However, the sensory driver for the olivocochlear reflex is unknown. Here, we resolve this longstanding question using a mouse model null for the gene encoding the type III intermediate filament peripherin (Prph). Prph((-/-)) mice lacked type II spiral ganglion neuron innervation of the outer hair cells, whereas innervation of the inner hair cells by type I spiral ganglion neurons was normal. Compared with Prph((+/+)) controls, both contralateral and ipsilateral olivocochlear efferent-mediated suppression of the cochlear amplifier were absent in Prph((-/-)) mice, demonstrating that outer hair cells and their type II afferents constitute the sensory drive for the olivocochlear efferent reflex.

ATP-gated ion channels mediate adaptation to elevated sound levels.

The sense of hearing is remarkable for its auditory dynamic range, which spans more than 10(12) in acoustic intensity. The mechanisms that enable the cochlea to transduce high sound levels without damage are of key interest, particularly with regard to the broad impact of industrial, military, and recreational auditory overstimulation on hearing disability. We show that ATP-gated ion channels assembled from P2X2 receptor subunits in the cochlea are necessary for the development of temporary threshold shift (TTS), evident in auditory brainstem response recordings as sound levels rise. In mice null for the P2RX2 gene (encoding the P2X2 receptor subunit), sustained 85-dB noise failed to elicit the TTS that wild-type (WT) mice developed. ATP released from the tissues of the cochlear partition with elevation of sound levels likely activates the broadly distributed P2X2 receptors on epithelial cells lining the endolymphatic compartment. This purinergic signaling is supported by significantly greater noise-induced suppression of distortion product otoacoustic emissions derived from outer hair cell transduction and decreased suprathreshold auditory brainstem response input/output gain in WT mice compared with P2RX2-null mice. At higher sound levels (≥95 dB), additional processes dominated TTS, and P2RX2-null mice were more vulnerable than WT mice to permanent hearing loss due to hair cell synapse disruption. P2RX2-null mice lacked ATP-gated conductance across the cochlear partition, including loss of ATP-gated inward current in hair cells. These data indicate that a significant component of TTS represents P2X2 receptor-dependent purinergic hearing adaptation that underpins the upper physiological range of hearing.

Differential actions of isoflurane and ketamine-based anaesthetics on cochlear function in the mouse.

Isoflurane is a volatile inhaled anaesthetic widely used in animal research, with particular utility for hearing research. Isoflurane has been shown to blunt hearing sensitivity compared with the awake state, but little is known about how isoflurane compares with other anaesthetics with regard to hair cell transduction and auditory neurotransmission. The current study was undertaken in C57Bl/6J and C129/SvEv strains of mice to determine whether isoflurane anaesthesia affects hearing function relative to ketamine-based anaesthesia. Cochlear function and central auditory transmission were assessed using auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE), comparing thresholds and input/output functions over time, for isoflurane vs. ketamine/xylazine/acepromazine anaesthesia. ABR thresholds at the most sensitive region of hearing (16 kHz) were initially higher under isoflurane anaesthesia. This reduced hearing sensitivity worsened over the 1 h study period, and also became evident with broadband click stimulus. Ketamine anaesthesia provided stable ABR thresholds. Although the growth functions were unchanged over time for both anaesthetics, the slopes under isoflurane anaesthesia were significantly less. Cubic (2f(1)-f(2)) DPOAE thresholds and growth functions were initially similar for both anaesthetics. After 60 min, DPOAE thresholds increased for both groups, but this effect was significantly greater with ketamine anaesthesia. The isoflurane-mediated increase in ABR thresholds over time is attributable to action on cochlear nerve activation, evident as a right-shift in the P1-N1 input/output function compared to K/X/A. The ketamine-based anaesthetic produced stable ABR thresholds and gain over time, despite a right-shift in the outer hair cell - mediated DPOAE input/output function.

Conformational changes in extracellular loop 2 associated with signal transduction in the glycine receptor.

Ligand-gated ion channels efficiently couple neurotransmitter binding to the opening of an intrinsic ion channel to generate the post-synaptic potentials that are characteristic of fast synaptic transmission. In the Cys-loop family of ligand-gated ion channels, the ligand-binding site is approximately 60 Å above the channel gate. Structural modelling of related proteins and mutagenesis studies led to the hypothesis that loops 2 and 7 of the extracellular domain may couple ligand binding to receptor activation. Mutating loop 2 residues of the glycine receptor to cysteine reveals an alternating pattern of effect upon receptor function. Mutations A52C, T54C and M56C produced a threefold right-shift in EC(50) . In contrast, a 30-fold right-shift was seen for mutations E53C, T55C and D57C. Loop 2 conformational changes associated with ligand binding were assessed by measuring the rate of covalent modification of substituted cysteines by charged methane thiosulfonate reagents. We show for the first time state-dependent differences in the rate of reaction. A52C and T54C are more accessible in the resting state and M56C is more accessible in the activated state. These results demonstrate that loop 2 does undergo a conformational change as part of the mechanism that couples ligand binding to channel opening.

Gating mechanisms in Cys-loop receptors.

The Cys-loop receptor superfamily of ligand-gated ion channels has a prominent role in neuronal signalling. These receptors are pentamers, each subunit containing ten beta-strands in the extracellular domain and four alpha-helical transmembrane domains (M1-M4). The M2 domain of each subunit lines the intrinsic ion channel pore and residues within the extracellular domain form ligand binding sites. Ligand binding initiates a conformational change that opens the ion-selective pore. The coupling between ligand binding in the extracellular domain and opening of the intrinsic ion channel pore located in the membrane is not fully understood. Several loop structures, such as loop 2, the Cys-loop, the pre-M1 region and the M2-M3 loop have been implicated in receptor activation. The current "conformational change wave" hypothesis suggests that binding of a ligand initiates a rotation of the beta-sheets around an axis that passes through the Cys-loop. Due to this rotation, the Cys-loop and loop 2 are displaced. Movement of the M2-M3 loop then twists the M2 domain leading to a separation of the helices and opening of the pore. The publication of a crystal structure of an acetylcholine binding protein and the refined structure of the Torpedo marmorata acetylcholine receptor have improved the understanding of the mechanisms and structures involved in coupling ligand binding to channel gating. In this review, the most recent findings on some of these loop structures will be reported and discussed in view of their role in the gating mechanism.