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Auditory space has been conceptualized as a matrix of systematically arranged combinations of binaural disparity cues that arise in the superior olivary complex (SOC). The computational code for interaural time and intensity differences utilizes excitatory and inhibitory projections that converge in the inferior colliculus (IC). The challenge is to determine the neural circuits underlying this convergence and to model how the binaural cues encode location. It has been shown that midbrain neurons are largely excited by sound from the contralateral ear and inhibited by sound leading at the ipsilateral ear. In this context, ascending projections from the lateral superior olive (LSO) to the IC have been reported to be ipsilaterally glycinergic and contralaterally glutamatergic. This study used CBA/CaH mice (3-6 months old) and applied unilateral retrograde tracing techniques into the IC in conjunction with immunocytochemical methods with glycine and glutamate transporters (GlyT2 and vGLUT2, respectively) to analyze the projection patterns from the LSO to the IC. Glycinergic and glutamatergic neurons were spatially intermixed within the LSO, and both types projected to the IC. For GlyT2 and vGLUT2 neurons, the average percentage of ipsilaterally and contralaterally projecting cells was similar (ANOVA, p = 0.48 ). A roughly equal number of GlyT2 and vGLUT2 neurons did not project to the IC. The somatic size and shape of these neurons match the descriptions of LSO principal cells. A minor but distinct population of small (< 40 μm 2 ) neurons that labeled for GlyT2 did not project to the IC; these cells emerge as candidates for inhibitory local circuit neurons. Our findings indicate a symmetric and bilateral projection of glycine and glutamate neurons from the LSO to the IC. The differences between our results and those from previous studies suggest that species and habitat differences have a significant role in mechanisms of binaural processing and highlight the importance of research methods and comparative neuroscience. These data will be important for modeling how excitatory and inhibitory systems converge to create auditory space in the CBA/CaH mouse., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Williams and Ryugo.)

Based on the auditory periphery and the small head size, Etruscan shrews (Suncus etruscus) approximate ancestral mammalian conditions. The auditory brainstem in this insectivore has not been investigated. Using labelling techniques, we assessed the structures of their superior olivary complex (SOC) and the nuclei of the lateral lemniscus (NLL). There, we identified the position of the major nuclei, their input pattern, transmitter content, expression of calcium binding proteins (CaBPs) and two voltage-gated ion channels. The most prominent SOC structures were the medial nucleus of the trapezoid body (MNTB), the lateral nucleus of the trapezoid body (LNTB), the lateral superior olive (LSO) and the superior paraolivary nucleus (SPN). In the NLL, the ventral (VNLL), a specific ventrolateral VNLL (VNLLvl) cell population, the intermediate (INLL) and dorsal (DNLL) nucleus, as well as the inferior colliculus's central aspect were discerned. INLL and VNLL were clearly separated by the differential distribution of various marker proteins. Most labelled proteins showed expression patterns comparable to rodents. However, SPN neurons were glycinergic and not GABAergic and the overall CaBPs expression was low. Next to the characterisation of the Etruscan shrew's auditory brainstem, our work identifies conserved nuclei and indicates variable structures in a species that approximates ancestral conditions., (© 2024. The Author(s).)

Neurexins play diverse functions as presynaptic organizers in various glutamatergic and GABAergic synapses. However, it remains unknown whether and how neurexins are involved in shaping functional properties of the glycinergic synapses, which mediate prominent inhibition in the brainstem and spinal cord. To address these issues, we examined the role of neurexins in a model glycinergic synapse between the principal neuron in the medial nucleus of the trapezoid body (MNTB) and the principal neuron in the lateral superior olive (LSO) in the auditory brainstem. Combining RNAscope with stereotactic injection of AAV-Cre in the MNTB of neurexin1/2/3 conditional triple knockout mice, we showed that MNTB neurons highly express all isoforms of neurexins although their expression levels vary remarkably. Selective ablation of all neurexins in MNTB neurons not only reduced the amplitude but also altered the kinetics of the glycinergic synaptic transmission at LSO neurons. The synaptic dysfunctions primarily resulted from an impaired Ca 2+ sensitivity of release and a loosened coupling between voltage-gated Ca 2+ channels and synaptic vesicles. Together, our current findings demonstrate that neurexins are essential in controlling the strength and temporal precision of the glycinergic synapse, which therefore corroborates the role of neurexins as key presynaptic organizers in all major types of fast chemical synapses., Competing Interests: HJ, RX, XN, ZS, XX, RP, YZ, FL No competing interests declared, (© 2024, Jiang et al.)

The auditory system comprises the auditory periphery, engaged in sound transduction and the central auditory system, implicated in auditory information processing and perception. Recently, evidence mounted that the mammalian peripheral and central auditory systems share a number of genes critical for proper development and function. This bears implication for auditory rehabilitation and evolution of the auditory system. To analyze to which extent microRNAs (miRNAs) belong to genes shared between both systems, we characterize the expression pattern of 12 cochlea-abundant miRNAs in the central auditory system. Quantitative real-time PCR (qRT-PCR) demonstrated expression of all 12 genes in the cochlea, the auditory hindbrain and the non-auditory prefrontal cortex (PFC) at embryonic stage (E)16 and postnatal stages (P)0 and P30. Eleven of them showed differences in expression between tissues and nine between the developmental time points. Hierarchical cluster analysis revealed that the temporal expression pattern in the auditory hindbrain was more similar to the PFC than to the cochlea. Spatiotemporal expression analysis by RNA in situ hybridization demonstrated widespread expression throughout the cochlear nucleus complex (CNC) and the superior olivary complex (SOC) during postnatal development. Altogether, our data indicate that miRNAs represent a relevant class of genetic factors functioning across the auditory system. Given the importance of gene regulatory network (GRN) components for development, physiology and evolution, the 12 miRNAs provide promising entry points to gain insights into their molecular underpinnings in the auditory system.

Anisotropic gap junctional coupling is a distinct feature of astrocytes in many brain regions. In the lateral superior olive (LSO), astrocytic networks are anisotropic and oriented orthogonally to the tonotopic axis. In Ca V 1.3 knock-out (KO) and otoferlin KO mice, where auditory brainstem nuclei are deprived from spontaneous cochlea-driven neuronal activity, neuronal circuitry is disturbed. So far it was unknown if this disturbance is also accompanied by an impaired topography of LSO astrocyte networks. To answer this question, we immunohistochemically analyzed the expression of astrocytic connexin (Cx) 43 and Cx30 in auditory brainstem nuclei. Furthermore, we loaded LSO astrocytes with the gap junction-permeable tracer neurobiotin and assessed the network shape and orientation. We found a strong elevation of Cx30 immunoreactivity in the LSO of Ca V 1.3 KO mice, while Cx43 levels were only slightly increased. In otoferlin KO mice, LSO showed a slight increase in Cx43 as well, whereas Cx30 levels were unchanged. The total number of tracer-coupled cells was unaltered and most networks were anisotropic in both KO strains. In contrast to the WTs, however, LSO networks were predominantly oriented parallel to the tonotopic axis and not orthogonal to it. Taken together, our data demonstrate that spontaneous cochlea-driven neuronal activity is not required per se for the formation of anisotropic LSO astrocyte networks. However, neuronal activity is required to establish the proper orientation of networks. Proper formation of LSO astrocyte networks thus necessitates neuronal input from the periphery, indicating a critical role of neuron-glia interaction during early postnatal development in the auditory brainstem.

Key Points: Loss of the calcium sensor otoferlin disrupts neurotransmission from inner hair cells. Central auditory nuclei are functionally denervated in otoferlin knockout mice (Otof KOs) via gene ablation confined to the periphery. We employed juvenile and young adult Otof KO mice (postnatal days (P)10-12 and P27-49) as a model for lacking spontaneous activity and deafness, respectively. We studied the impact of peripheral activity on synaptic refinement in the sound localization circuit from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO). MNTB in vivo recordings demonstrated drastically reduced spontaneous spiking and deafness in Otof KOs. Juvenile KOs showed impaired synapse elimination and strengthening, manifested by broader MNTB-LSO inputs, imprecise MNTB-LSO topography and weaker MNTB-LSO fibres. The impairments persisted into young adulthood. Further functional refinement after hearing onset was undetected in young adult wild-types. Collectively, activity deprivation confined to peripheral protein loss impairs functional MNTB-LSO refinement during a critical prehearing period., Abstract: Circuit refinement is critical for the developing sound localization pathways in the auditory brainstem. In prehearing mice (hearing onset around postnatal day (P)12), spontaneous activity propagates from the periphery to central auditory nuclei. At the glycinergic projection from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO) of neonatal mice, super-numerous MNTB fibres innervate a given LSO neuron. Between P4 and P9, MNTB fibres are functionally eliminated, whereas the remaining fibres are strengthened. Little is known about MNTB-LSO circuit refinement after P20. Moreover, MNTB-LSO refinement upon activity deprivation confined to the periphery is largely unexplored. This leaves a considerable knowledge gap, as deprivation often occurs in patients with congenital deafness, e.g. upon mutations in the otoferlin gene (OTOF). Here, we analysed juvenile (P10-12) and young adult (P27-49) otoferlin knockout (Otof KO) mice with respect to MNTB-LSO refinement. MNTB in vivo recordings revealed drastically reduced spontaneous activity and deafness in knockouts (KOs), confirming deprivation. As RNA sequencing revealed Otof absence in the MNTB and LSO of wild-types, Otof loss in KOs is specific to the periphery. Functional denervation impaired MNTB-LSO synapse elimination and strengthening, which was assessed by glutamate uncaging and electrical stimulation. Impaired elimination led to imprecise MNTB-LSO topography. Impaired strengthening was associated with lower quantal content per MNTB fibre. In young adult KOs, the MNTB-LSO circuit remained unrefined. Further functional refinement after P12 appeared absent in wild-types. Collectively, we provide novel insights into functional MNTB-LSO circuit maturation governed by a cochlea-specific protein. The central malfunctions in Otof KOs may have implications for patients with sensorineuronal hearing loss., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Objectives: Unilateral cochlear damage has profound effects on the central auditory pathways in the brain., Methods: We examined the effects of unilateral cochlear ablation on VGLUT1 expression in the cochlear nucleus (CN) and the superior olivary complex (SOC) in neonatal rats., Results: VGLUT1 expression in the CN subdivisions (the AVCN, the PVCN and the DCN-deep layers) and the SOC (the MnTB, the LSO and the MSO) ipsilateral to the ablated side was significantly suppressed by unilateral cochlear ablation. Interestingly, VGLUT1 expression in the PVCN and the DCN-deep layers contralateral to the ablated side was also reduced., Conclusion: Our findings indicate that unilateral cochlear ablation affects VGLUT1 expression in the central auditory pathways not only ipsilateral but also contralateral to the ablated side., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: In the mammalian superior olivary complex (SOC), synaptic inhibition contributes to the processing of binaural sound cues important for sound localization. Previous analyses demonstrated a tonotopic gradient for postsynaptic proteins mediating inhibitory neurotransmission in the lateral superior olive (LSO), a major nucleus of the SOC. To probe, whether a presynaptic molecular gradient exists as well, we investigated immunoreactivity against the vesicular inhibitory amino acid transporter (VIAAT) in the mouse auditory brainstem., Results: Immunoreactivity against VIAAT revealed a gradient in the LSO and the superior paraolivary nucleus (SPN) of NMRI mice, with high expression in the lateral, low frequency processing limb and low expression in the medial, high frequency processing limb of both nuclei. This orientation is opposite to the previously reported gradient of glycine receptors in the LSO. Other nuclei of the SOC showed a uniform distribution of VIAAT-immunoreactivity. No gradient was observed for the glycine transporter GlyT2 and the neuronal protein NeuN. Formation of the VIAAT gradient was developmentally regulated and occurred around hearing-onset between postnatal days 8 and 16. Congenital deaf Claudin14 -/- mice bred on an NMRI background showed a uniform VIAAT-immunoreactivity in the LSO, whereas cochlear ablation in NMRI mice after hearing-onset did not affect the gradient. Additional analysis of C57Bl6/J, 129/SvJ and CBA/J mice revealed a strain-specific formation of the gradient., Conclusions: Our results identify an activity-regulated gradient of VIAAT in the SOC of NRMI mice. Its absence in other mouse strains adds a novel layer of strain-specific features in the auditory system, i.e. tonotopic organization of molecular gradients. This calls for caution when comparing data from different mouse strains frequently used in studies involving transgenic animals. The presence of strain-specific differences offers the possibility of genetic mapping to identify molecular factors involved in activity-dependent developmental processes in the auditory system. This would provide an important step forward concerning improved auditory rehabilitation in cases of congenital deafness.

The superior olivary complex (SOC) is a collection of brainstem auditory nuclei which play essential roles in the localization of sound sources, temporal coding of vocalizations and descending modulation of the cochlea. Notwithstanding, the SOC nuclei vary considerably between species in accordance with the auditory needs of the animal. The canine SOC was subjected to anatomical and physiological examination nearly 50 years ago and was then virtually forgotten. Herein, we aimed to characterize the nuclei of the canine SOC using quantitative morphometrics, estimation of neuronal number, histochemistry for perineuronal nets and immunofluorescence for the calcium binding proteins calbindin and calretinin. We found the principal nuclei to be extremely well developed: the lateral superior olive (LSO) contained over 20,000 neurons and the medial superior olive (MSO) contained over 15,000 neurons. In nearly all non-chiropterian terrestrial mammals, the MSO exists as a thin, vertical column of neurons. The canine MSO was folded into a U-shaped contour and had associated with the ventromedial tip a small, round collection of neurons we termed the tail nucleus of the MSO. Further, we found evidence within the LSO, MSO and medial nucleus of the trapezoid body (MNTB) for significant morphological variations along the mediolateral or rostrocaudal axes. Finally, the majority of MNTB neurons were calbindin-immunopositive and associated with calretinin-immunopositive calyceal terminals. Together, these observations suggest the canine SOC complies with the basic plan of the mammalian SOC but possesses a number of unique anatomical features., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In mammals with good low-frequency hearing, the medial superior olive (MSO) computes sound location by comparing differences in the arrival time of a sound at each ear, called interaural time disparities (ITDs). Low-frequency sounds are not reflected by the head, and therefore level differences and spectral cues are minimal or absent, leaving ITDs as the only cue for sound localization. Although mammals with high-frequency hearing and small heads (e.g., bats, mice) barely experience ITDs, the MSO is still present in these animals. Yet, aside from studies in specialized bats, in which the MSO appears to serve functions other than ITD processing, it has not been studied in small mammals that do not hear low frequencies. Here we describe neurons in the mouse brain stem that share prominent anatomical, morphological, and physiological properties with the MSO in species known to use ITDs for sound localization. However, these neurons also deviate in some important aspects from the typical MSO, including a less refined arrangement of cell bodies, dendrites, and synaptic inputs. In vitro, the vast majority of neurons exhibited a single, onset action potential in response to suprathreshold depolarization. This spiking pattern is typical of MSO neurons in other species and is generated from a complement of K v 1, K v 3, and I H currents. In vivo, mouse MSO neurons show bilateral excitatory and inhibitory tuning as well as an improvement in temporal acuity of spiking during bilateral acoustic stimulation. The combination of classical MSO features like those observed in gerbils with more unique features similar to those observed in bats and opossums make the mouse MSO an interesting model for exploiting genetic tools to test hypotheses about the molecular mechanisms and evolution of ITD processing., (Copyright © 2016 the American Physiological Society.)

In mammals, the superior olivary complex (SOC) of the brainstem is composed of nuclei that integrate afferent auditory originating from both ears. Here, the expression of different calcium-binding proteins in subnuclei of the SOC was studied in distantly related mammals, the Mongolian gerbil (Meriones unguiculatus) and the gray short-tailed opossum (Monodelphis domestica) to get a better understanding of the basal nuclear organization of the SOC. Combined immunofluorescence labeling of the calcium-binding proteins (CaBPs) parvalbumin, calbindin-D28k, and calretinin as well as pan-neuronal markers displayed characteristic distribution patterns highlighting details of neuronal architecture of SOC nuclei. Parvalbumin was found in almost all neurons of SOC nuclei in both species, while calbindin and calretinin were restricted to specific cell types and axonal terminal fields. In both species, calbindin displayed a ubiquitous and mostly selective distribution in neurons of the medial nucleus of trapezoid body (MNTB) including their terminal axonal fields in different SOC targets. In Meriones, calretinin and calbindin showed non-overlapping expression patterns in neuron somata and terminal fields throughout the SOC. In Monodelphis, co-expression of calbindin and calretinin was observed in the MNTB, and hence both CaBPs were also co-localized in terminal fields within the adjacent SOC nuclei. The distribution patterns of CaBPs in both species are discussed with respect to the intrinsic neuronal SOC circuits as part of the auditory brainstem system that underlie the binaural integrative processing of acoustic signals as the basis for localization and discrimination of auditory objects.

Background and Objective: The auditory efferent system is a complex network of descending pathways, which mainly originate in the primary auditory cortex and are directed to several auditory subcortical nuclei. These descending pathways are connected to olivocochlear neurons, which in turn make synapses with auditory nerve neurons and outer hair cells (OHC) of the cochlea. The olivocochlear function can be studied using contralateral acoustic stimulation, which suppresses auditory nerve and cochlear responses. In the present work, we tested the proposal that the corticofugal effects that modulate the strength of the olivocochlear reflex on auditory nerve responses are produced through cholinergic synapses between medial olivocochlear (MOC) neurons and OHCs via alpha-9/10 nicotinic receptors., Methods: We used wild type (WT) and alpha-9 nicotinic receptor knock-out (KO) mice, which lack cholinergic transmission between MOC neurons and OHC, to record auditory cortex evoked potentials and to evaluate the consequences of auditory cortex electrical microstimulation in the effects produced by contralateral acoustic stimulation on auditory brainstem responses (ABR)., Results: Auditory cortex evoked potentials at 15 kHz were similar in WT and KO mice. We found that auditory cortex microstimulation produces an enhancement of contralateral noise suppression of ABR waves I and III in WT mice but not in KO mice. On the other hand, corticofugal modulations of wave V amplitudes were significant in both genotypes., Conclusion: These findings show that the corticofugal modulation of contralateral acoustic suppressions of auditory nerve (ABR wave I) and superior olivary complex (ABR wave III) responses are mediated through MOC synapses.

The energy-yielding pathways that provide the large amounts of metabolic energy required by inner ear sensorineural cells are poorly understood. Neuroglobin (Ngb) is a neuron-specific hemoprotein of the globin family, which is suggested to be involved in oxidative energy metabolism. Here, we present quantitative real-time reverse transcription PCR, in situ hybridization, immunohistochemical, and Western blot evidence that neuroglobin is highly expressed in the mouse and rat cochlea. For primary cochlea neurons, Ngb expression is limited to the subpopulation of type I spiral ganglion cells, those which innervate inner hair cells, while the subpopulation of type II spiral ganglion cells which innervate the outer hair cells do not express Ngb. We further investigated Ngb distribution in rat, mouse, and human auditory brainstem centers, and found that the cochlear nuclei and superior olivary complex (SOC) also express considerable amounts of Ngb. Notably, the majority of olivocochlear neurons, those which provide efferent innervation of outer hair cells as identified by neuronal tract tracing, were Ngb-immunoreactive. We also observed that neuroglobin in the SOC frequently co-localized with neuronal nitric oxide synthase, the enzyme responsible for nitric oxide production. Our findings suggest that neuroglobin is well positioned to play an important physiologic role in the oxygen homeostasis of the peripheral and central auditory nervous system, and provides the first evidence that Ngb signal differentiates the central projections of the inner and outer hair cells.

During development GABA and glycine synapses are initially excitatory before they gradually become inhibitory. This transition is due to a developmental increase in the activity of neuronal potassium-chloride cotransporter 2 (KCC2), which shifts the chloride equilibrium potential (ECl) to values more negative than the resting membrane potential. While the role of early GABA and glycine depolarizations in neuronal development has become increasingly clear, the role of the transition to hyperpolarization in synapse maturation and circuit refinement has remained an open question. Here we investigated this question by examining the maturation and developmental refinement of GABA/glycinergic and glutamatergic synapses in the lateral superior olive (LSO), a binaural auditory brain stem nucleus, in KCC2-knockdown mice, in which GABA and glycine remain depolarizing. We found that many key events in the development of synaptic inputs to the LSO, such as changes in neurotransmitter phenotype, strengthening and elimination of GABA/glycinergic connection, and maturation of glutamatergic synapses, occur undisturbed in KCC2-knockdown mice compared with wild-type mice. These results indicate that maturation of inhibitory and excitatory synapses in the LSO is independent of the GABA and glycine depolarization-to-hyperpolarization transition., (Copyright © 2016 the American Physiological Society.)

The neurons in the mammalian and avian auditory hindbrain nuclei share a number of significant morphological and physiological properties for fast, secure and precise neurotransmission, such as giant synapses, voltage-gated K+ channels and fast AMPA receptors. Based on the independent evolution of the middle ear in these two vertebrate lineages, on different embryonic origins of the nuclei and on marked differences on the circuit level, these similarities are assumed to reflect convergent evolution. Independent acquisition of similar phenotypes can be produced by divergent evolution of genetic mechanisms or by similar molecular mechanisms. The distinction between these two possibilities requires knowledge of the gene regulatory networks (GRNs) that orchestrate the development of auditory hindbrain structures. We therefore compared the expression pattern of GRN components, both transcription factors (TFs) and noncoding RNA, during terminal differentiation of the auditory hindbrain structures in mouse and chicken when neurons acquire their final morphological and electrophysiological properties. In general, we observed broad expression of these genes in the mouse auditory cochlear nucleus complex and the superior olivary complex at both postnatal day 4 (P4) and at P25, and for the chicken at the equivalent developmental stages, i.e. embryonic day 13 (E13) and at P14-P17. Our data are in agreement with a model based on similar molecular mechanisms underlying terminal differentiation and maintenance of neuronal cell identity in the auditory hindbrain of different vertebrate lineages. This conservation might reflect developmental constraints arising from the tagmatic organization of rhombomeres and the evolutionarily highly conserved GRNs operating in these structures., (© 2016 S. Karger AG, Basel.)

Perineuronal nets (PNs) are a unique and complex meshwork of specific extracellular matrix molecules that ensheath a subset of neurons in many regions of the central nervous system (CNS). PNs appear late in development and are supposed to restrict synaptic plasticity and to stabilize functional neuronal connections. PNs were further hypothesized to create a charged milieu around the neurons and thus, might directly modulate synaptic activity. Although PNs were first described more than 120 years ago, their exact functions still remain elusive. The purpose of the present review is to propose the nuclei of the auditory system, which are highly enriched in PN-wearing neurons, as particularly suitable structures to study the functional significance of PNs. We provide a detailed description of the distribution of PNs from the cochlear nucleus to the auditory cortex considering distinct markers for detection of PNs. We further point to the suitability of specific auditory neurons to serve as promising model systems to study in detail the contribution of PNs to synaptic physiology and also more generally to the functionality of the brain., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Electrical synapses formed by gap junctions composed of connexin36 (Cx36) are widely distributed in the mammalian central nervous system (CNS). Here, we used immunofluorescence methods to document the expression of Cx36 in the cochlear nucleus and in various structures of the auditory pathway of rat and mouse. Labeling of Cx36 visualized exclusively as Cx36-puncta was densely distributed primarily on the somata and initial dendrites of neuronal populations in the ventral cochlear nucleus, and was abundant in superficial layers of the dorsal cochlear nucleus. Other auditory centers displaying Cx36-puncta included the medial nucleus of the trapezoid body (MNTB), regions surrounding the lateral superior olivary nucleus, the dorsal nucleus of the medial lemniscus, the nucleus sagulum, all subnuclei of the inferior colliculus, and the auditory cerebral cortex. In EGFP-Cx36 transgenic mice, EGFP reporter was detected in neurons located in each of auditory centers that harbored Cx36-puncta. In the ventral cochlear nuclei and the MNTB, many neuronal somata were heavily innervated by nerve terminals containing vesicular glutamate transporter-1 (vglut1) and Cx36 was frequently localized at these terminals. Cochlear ablation caused a near total depletion of vglut1-positive terminals in the ventral cochlear nuclei, with a commensurate loss of labeling for Cx36 around most neuronal somata, but preserved Cx36-puncta at somatic neuronal appositions. The results suggest that electrical synapses formed by Cx36-containing gap junctions occur in most of the widely distributed centers of the auditory system. Further, it appears that morphologically mixed chemical/electrical synapses formed by nerve terminals are abundant in the ventral cochlear nucleus, including those at endbulbs of Held formed by cochlear primary afferent fibers, and those at calyx of Held synapses on MNTB neurons., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Hearing loss afflicts approximately 15% of the world's population, and crosses all socioeconomic boundaries. While great strides have been made in understanding the genetic components of syndromic and non-syndromic hearing loss, understanding of the mechanisms underlying noise-induced hearing loss (NIHL) have come much more slowly. NIHL is not simply a mechanism by which older individuals loose their hearing. Significantly, the incidence of NIHL is increasing, and is now involving ever younger populations. This may predict future increased occurrences of hearing loss. Current research has shown that even short-term exposures to loud sounds generating what was previously considered temporary hearing loss, actually produces an almost immediate and permanent loss of specific populations of auditory nerve fibers. Additionally, recurrent exposures to intense sound may hasten age-related hearing loss. While NIHL is a significant medical concern, to date, few compounds have delivered significant protection, arguing that new targets need to be identified. In this commentary, we will explore cellular signaling processes taking place in the cochlea believed to be involved in protection against hearing loss, and highlight new data suggestive of novel signaling not previously recognized as occurring in the cochlea, that is perhaps protective of hearing. This includes a recently described local hypothalamic-pituitary-adrenal axis (HPA)-like signaling system fully contained in the cochlea. This system may represent a local cellular stress-response system based on stress hormone release similar to the systemic HPA axis. Its discovery may hold hope for new drug therapies that can be delivered directly to the cochlea, circumventing systemic side effects., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Development and evolution of auditory hindbrain nuclei are two major unsolved issues in hearing research. Recent characterization of transgenic mice identified the rhombomeric origins of mammalian auditory nuclei and unraveled genes involved in their formation. Here, we provide an overview on these data by assembling them into rhombomere-specific gene regulatory networks (GRNs), as they underlie developmental and evolutionary processes. To explore evolutionary mechanisms, we compare the GRNs operating in the mammalian auditory hindbrain with data available from the inner ear and other vertebrate groups. Finally, we propose that the availability of genomic sequences from all major vertebrate taxa and novel genetic techniques for non-model organisms provide an unprecedented opportunity to investigate development and evolution of the auditory hindbrain by comparative molecular approaches. The dissection of the molecular mechanisms leading to auditory structures will also provide an important framework for auditory processing disorders, a clinical problem difficult to tackle so far. These data will, therefore, foster basic and clinical hearing research alike.

CaV2.1 Ca(2+) channels play a key role in triggering neurotransmitter release and mediating synaptic transmission. Familial hemiplegic migraine type-1 (FHM-1) is caused by missense mutations in the CACNA1A gene that encodes the α1A pore-forming subunit of CaV2.1 Ca(2+) channels. We used knock-in (KI) transgenic mice harbouring the pathogenic FHM-1 mutation R192Q to study inhibitory and excitatory neurotransmission in the principle neurons of the lateral superior olive (LSO) in the auditory brainstem. We tested if the R192Q FHM-1 mutation differentially affects excitatory and inhibitory synaptic transmission, disturbing the normal balance between excitation and inhibition in this nucleus. Whole cell patch-clamp was used to measure neurotransmitter elicited excitatory (EPSCs) and inhibitory (IPSCs) postsynaptic currents in wild-type (WT) and R192Q KI mice. Our results showed that the FHM-1 mutation in CaV2.1 channels has multiple effects. Evoked EPSC amplitudes were smaller whereas evoked and miniature IPSC amplitudes were larger in R192Q KI compared to WT mice. In addition, in R192Q KI mice, the release probability was enhanced compared to WT, at both inhibitory (0.53 ± 0.02 vs. 0.44 ± 0.01, P = 2.10(-5), Student's t-test) and excitatory synapses (0.60 ± 0.03 vs. 0.45 ± 0.02, P = 4 10(-6), Student's t-test). Vesicle pool size was diminished in R192Q KI mice compared to WT mice (68 ± 6 vs 91 ± 7, P = 0.008, inhibitory; 104 ± 13 vs 335 ± 30, P = 10(-6), excitatory, Student's t-test). R192Q KI mice present enhanced short-term plasticity. Repetitive stimulation of the afferent axons caused short-term depression (STD) of E/IPSCs that recovered significantly faster in R192Q KI mice compared to WT. This supports the hypothesis of a gain-of-function of the CaV2.1 channels in R192Q KI mice, which alters the balance of excitatory/inhibitory inputs and could also have implications in the altered cortical excitability responsible for FHM pathology., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In the mammalian auditory brainstem, the cochlear nuclear complex (CN) and the superior olivary complex (SOC) feature structural and functional specializations for ultrafast (<1 ms) and precise information processing. Their proteome, the basis for structure and function, has been rarely analyzed so far. Here we identified and quantified the protein profiles of three major auditory brainstem regions of adult rats, the CN, the SOC, and the inferior colliculus (IC). The rest of the brain served as a reference. Via label-free quantitative mass spectrometry and 2-D DIGE/MALDI-MS, we identified 584 and 297 proteins in the plasma membrane/synaptic vesicle proteome and the cytosolic proteome, respectively. 'Region-typical' proteins, i.e., those with higher abundance in one region than in the other three, were considered candidates for functional specializations. Key proteins were validated via Western blots and immunohistochemistry. Functional annotation clustering revealed an overrepresentation of neurofilament proteins among the CN+SOC-typical proteins. These are related to regulation of axon diameter and, thereby, conduction velocity. Interestingly, the sets of synapse-associated proteins differed between regions. For example, synaptotagmin-2 (Syt2), a Ca2+ sensor for fast exocytosis, was CN+SOC+IC-typical, whereas Syt1 was CN+SOC+IC-atypical. Together, our quantitative comparison of protein profiles has revealed several interesting candidate proteins for ultrafast and precise information processing., (Copyright © 2014. Published by Elsevier Inc.)

Neurons expressing the calcium binding proteins (CaBPs) parvalbumin (PV) and calbindin (CB) have shown age-related density changes throughout the ascending auditory system of both rodents and macaque monkeys. In the cerebral cortex, neurons expressing these CaBPs express markers of γ-aminobutyric acidergic neurotransmission, such as GAD67, and have well-understood physiological response properties. Recent evidence suggests that, in the rodent auditory brainstem, CaBP-containing cells do not express GAD67. It is unknown whether PV- and CB-containing cells in subcortical auditory structures of macaques similarly do not express GAD67, and a better understanding of the neurotransmission of neurons expressing these proteins is necessary for understanding the age-related changes in their density throughout the macaque auditory system. This was investigated with immunofluorescent double-labeling techniques to coregister PV- and CB-expressing neurons with GAD67 in the superior olivary complex and the inferior colliculus of young and aged rhesus macaques. The proportions of GAD67-expressing PV- and CB-positive neurons were computed with unbiased sampling techniques. Our results indicate that between 42% and 62% of PV- and CB-positive neurons in the auditory brainstem and midbrain express GAD67, which is significantly less than in the cerebrum. In general, fewer PV(+) neurons and more CB(+) neurons expressed GAD67 as a function of age. These results demonstrate that the inhibitory molecular profile of PV- and CB-expressing neurons can change with age in subcortical auditory structures and that these neurons are distinct from the well-described inhibitory interneurons that express these proteins in the cerebral cortex., (© 2014 Wiley Periodicals, Inc.)

Fragile X mental retardation protein (FMRP) binds select mRNAs, functions in intracellular transport of these mRNAs and represses their translation. FMRP is highly expressed in neurons and lack of FMRP has been shown to result in dendritic dysmorphology and altered synaptic function. FMRP is known to interact with mRNAs for the Kv3.1b potassium channel which is required for neurons to fire action potentials at high rates with remarkable temporal precision. Auditory brainstem neurons are known for remarkably high spike rates and expression of Kv3.1b potassium channels. Fragile X syndrome (FXS) is a genetic disorder caused by a mutation in the fragile X mental retardation 1 gene (Fmr1) resulting in decreased expression of FMRP and subsequent intellectual disability, seizures, attention deficit and hypersensitivity to auditory and other sensory stimuli. We therefore hypothesize that the auditory difficulties in FXS result, at least in part, from dysfunction of auditory brainstem neurons. To examine this hypothesis, we have studied normal human brainstem tissue with immunohistochemical techniques and confocal microscopy. Our results demonstrate that FMRP is widely expressed in cell bodies and dendritic arbors of neurons in the human cochlear nucleus and superior olivary complex and also that coincidence detector neurons of the medial superior olive colocalization of FMRP and Kv3.1b. We interpret these observations to suggest that the lower auditory brainstem is a potential site of dysfunction in FXS., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neuronal dendrites are structurally and functionally dynamic in response to changes in afferent activity. The fragile X mental retardation protein (FMRP) is an mRNA binding protein that regulates activity-dependent protein synthesis and morphological dynamics of dendrites. Loss and abnormal expression of FMRP occur in fragile X syndrome (FXS) and some forms of autism spectrum disorders. To provide further understanding of how FMRP signaling regulates dendritic dynamics, we examined dendritic expression and localization of FMRP in the reptilian and avian nucleus laminaris (NL) and its mammalian analogue, the medial superior olive (MSO), in rodents and humans. NL/MSO neurons are specialized for temporal processing of low-frequency sounds for binaural hearing, which is impaired in FXS. Protein BLAST analyses first demonstrate that the FMRP amino acid sequences in the alligator and chicken are highly similar to human FMRP with identical mRNA-binding and phosphorylation sites, suggesting that FMRP functions similarly across vertebrates. Immunocytochemistry further reveals that NL/MSO neurons have very high levels of dendritic FMRP in low-frequency hearing vertebrates including alligator, chicken, gerbil, and human. Remarkably, dendritic FMRP in NL/MSO neurons often accumulates at branch points and enlarged distal tips, loci known to be critical for branch-specific dendritic arbor dynamics. These observations support an important role for FMRP in regulating dendritic properties of binaural neurons that are essential for low-frequency sound localization and auditory scene segregation, and support the relevance of studying this regulation in nonhuman vertebrates that use low frequencies in order to further understand human auditory processing disorders., (Copyright © 2013 Wiley Periodicals, Inc.)

Positive immunoreactivity to the calcium-binding protein parvalbumin (PV) and nitric oxide synthase NADPH diaphorase (NADPHd) is well documented within neurons of the central auditory system of both rodents and primates. These proteins are thought to play roles in the regulation of auditory processing. Studies examining the age-related changes in expression of these proteins have been conducted primarily in rodents but are sparse in primate models. In the brainstem, the superior olivary complex (SOC) is crucial for the computation of sound source localization in azimuth, and one hallmark of age-related hearing deficits is a reduced ability to localize sounds. To investigate how these histochemical markers change as a function of age and hearing loss, we studied eight rhesus macaques ranging in age from 12 to 35 years. Auditory brainstem responses (ABRs) were obtained in anesthetized animals for click and tone stimuli. The brainstems of the sesame animals were then stained for PV and NADPHd reactivity. Reactive neurons in the three nuclei of the SOC were counted, and the densities of each cell type were calculated. We found that PV and NADPHd expression increased with both age and ABR thresholds in the medial superior olive but not in either the medial nucleus of the trapezoid body or the lateral superior olive. Together these results suggest that the changes in protein expression employed by the SOC may compensate for the loss of efficacy of auditory sensitivity in the aged primate.