ISL1 is necessary for auditory neuron development and contributes toward tonotopic organization.

A cardinal feature of the auditory pathway is frequency selectivity, represented in a tonotopic map from the cochlea to the cortex. The molecular determinants of the auditory frequency map are unknown. Here, we discovered that the transcription factor ISL1 regulates the molecular and cellular features of auditory neurons, including the formation of the spiral ganglion and peripheral and central processes that shape the tonotopic representation of the auditory map. We selectively knocked out Isl1 in auditory neurons using Neurod1Cre strategies. In the absence of Isl1, spiral ganglion neurons migrate into the central cochlea and beyond, and the cochlear wiring is profoundly reduced and disrupted. The central axons of Isl1 mutants lose their topographic projections and segregation at the cochlear nucleus. Transcriptome analysis of spiral ganglion neurons shows that Isl1 regulates neurogenesis, axonogenesis, migration, neurotransmission-related machinery, and synaptic communication patterns. We show that peripheral disorganization in the cochlea affects the physiological properties of hearing in the midbrain and auditory behavior. Surprisingly, auditory processing features are preserved despite the significant hearing impairment, revealing central auditory pathway resilience and plasticity in Isl1 mutant mice. Mutant mice have a reduced acoustic startle reflex, altered prepulse inhibition, and characteristics of compensatory neural hyperactivity centrally. Our findings show that ISL1 is one of the obligatory factors required to sculpt auditory structural and functional tonotopic maps. Still, upon Isl1 deletion, the ensuing central plasticity of the auditory pathway does not suffice to overcome developmentally induced peripheral dysfunction of the cochlea.

Segregation of Multimodal Inputs Into Discrete Midbrain Compartments During an Early Critical Period.

The lateral cortex of the inferior colliculus (LCIC) is a multimodal subdivision of the midbrain inferior colliculus (IC) that plays a key role in sensory integration. The LCIC is compartmentally-organized, exhibiting a series of discontinuous patches or modules surrounded by an extramodular matrix. In adult mice, somatosensory afferents target LCIC modular zones, while auditory afferents terminate throughout the encompassing matrix. Recently, we defined an early LCIC critical period (birth: postnatal day 0 to P12) based upon the concurrent emergence of its neurochemical compartments (modules: glutamic acid decarboxylase, GAD+; matrix: calretinin, CR+), matching Eph-ephrin guidance patterns, and specificity of auditory inputs for its matrix. Currently lacking are analogous experiments that address somatosensory afferent shaping and the construction of discrete LCIC multisensory maps. Combining living slice tract-tracing and immunocytochemical approaches in a developmental series of GAD67-GFP knock-in mice, the present study characterizes: (1) the targeting of somatosensory terminals for emerging LCIC modular fields; and (2) the relative separation of somatosensory and auditory inputs over the course of its established critical period. Results indicate a similar time course and progression of LCIC projection shaping for both somatosensory (corticocollicular) and auditory (intracollicular) inputs. While somewhat sparse and intermingling at birth, modality-specific projection patterns soon emerge (P4-P8), coincident with peak guidance expression and the appearance of LCIC compartments. By P12, an adult-like arrangement is in place, with fully segregated multimodal afferent arrays. Quantitative measures confirm increasingly distinct input maps, exhibiting less projection overlap with age. Potential mechanisms whereby multisensory LCIC afferent systems recognize and interface with its emerging modular-matrix framework are discussed.

Differential expression of microRNAs in the developing avian auditory hindbrain.

The avian auditory hindbrain is a longstanding model for studying neural circuit development. Information on gene regulatory network (GRN) components underlying this process, however, is scarce. Recently, the spatiotemporal expression of 12 microRNAs (miRNAs) was investigated in the mammalian auditory hindbrain. As a comparative study, we here investigated the spatiotemporal expression of the orthologous miRNAs during development of the chicken auditory hindbrain. All miRNAs were expressed both at E13, an immature stage, and P14, a mature stage of the auditory system. In most auditory nuclei, a homogeneous expression pattern was observed at both stages, like the mammalian system. An exception was the nucleus magnocellularis (NM). There, at E13, nine miRNAs showed a differential expression pattern along the cochleotopic axis with high expression at the rostromedial pole. One of them showed a gradient expression whereas eight showed a spatially selective expression at the rostral pole that reflected the different rhombomeric origins of this composite nucleus. The miRNA differential expression persisted in the NM to the mature stage, with the selective expression changed to linear gradients. Bioinformatics analysis predicted mRNA targets that are associated with neuronal developmental processes such as neurite and synapse organization, calcium and ephrin-Eph signaling, and neurotransmission. Overall, this first analysis of miRNAs in the chicken central auditory system reveals shared and strikingly distinct features between chicken and murine orthologues. The embryonic gradient expression of these GRN elements in the NM adds miRNA patterns to the list of cochleotopic and developmental gradients in the central auditory system.

Making sense of neural development by comparing wiring strategies for seeing and hearing.

The ability to perceive and interact with the world depends on a diverse array of neural circuits specialized for carrying out specific computations. Each circuit is assembled using a relatively limited number of molecules and common developmental steps, from cell fate specification to activity-dependent synaptic refinement. Given this shared toolkit, how do individual circuits acquire their characteristic properties? We explore this question by comparing development of the circuitry for seeing and hearing, highlighting a few examples where differences in each system's sensory demands necessitate different developmental strategies.

Temporal-specific roles of fragile X mental retardation protein in the development of the hindbrain auditory circuit.

Fragile X mental retardation protein (FMRP) is an RNA-binding protein abundant in the nervous system. Functional loss of FMRP leads to sensory dysfunction and severe intellectual disabilities. In the auditory system, FMRP deficiency alters neuronal function and synaptic connectivity and results in perturbed processing of sound information. Nevertheless, roles of FMRP in embryonic development of the auditory hindbrain have not been identified. Here, we developed high-specificity approaches to genetically track and manipulate throughout development of the Atoh1+ neuronal cell type, which is highly conserved in vertebrates, in the cochlear nucleus of chicken embryos. We identified distinct FMRP-containing granules in the growing axons of Atoh1+ neurons and post-migrating NM cells. FMRP downregulation induced by CRISPR/Cas9 and shRNA techniques resulted in perturbed axonal pathfinding, delay in midline crossing, excess branching of neurites, and axonal targeting errors during the period of circuit development. Together, these results provide the first in vivo identification of FMRP localization and actions in developing axons of auditory neurons, and demonstrate the importance of investigating early embryonic alterations toward understanding the pathogenesis of neurodevelopmental disorders.

Origin of acoustic-vestibular ganglionic neuroblasts in chick embryos and their sensory connections.

The inner ear is a complex three-dimensional sensory structure with auditory and vestibular functions. It originates from the otic placode, which generates the sensory elements of the membranous labyrinth and all the ganglionic neuronal precursors. Neuroblast specification is the first cell differentiation event. In the chick, it takes place over a long embryonic period from the early otic cup stage to at least stage HH25. The differentiating ganglionic neurons attain a precise innervation pattern with sensory patches, a process presumably governed by a network of dendritic guidance cues which vary with the local micro-environment. To study the otic neurogenesis and topographically-ordered innervation pattern in birds, a quail-chick chimaeric graft technique was used in accordance with a previously determined fate-map of the otic placode. Each type of graft containing the presumptive domain of topologically-arranged placodal sensory areas was shown to generate neuroblasts. The differentiated grafted neuroblasts established dendritic contacts with a variety of sensory patches. These results strongly suggest that, rather than reverse-pathfinding, the relevant role in otic dendritic process guidance is played by long-range diffusing molecules.

Npr2 null mutants show initial overshooting followed by reduction of spiral ganglion axon projections combined with near-normal cochleotopic projection.

Npr2 (natriuretic peptide receptor 2) affects bifurcation of neural crest or placode-derived afferents upon entering the brain stem/spinal cord, leading to a lack of either rostral or caudal branches. Previous work has shown that early embryonic growth of cochlear and vestibular afferents is equally affected in this mutant but later work on postnatal Npr2 point mutations suggested some additional effects on the topology of afferent projections and mild functional defects. Using multicolor lipophilic dye tracing, we show that absence of Npr2 has little to no effect on the initial patterning of inner ear afferents with respect to their dorsoventral cochleotopic-specific projections. However, in contrast to control animals, we found a variable degree of embryonic extension of auditory afferents beyond the boundaries of the anterior cochlear nucleus into the cerebellum that emanates only from apical spiral ganglion neurons. Such expansion has previously only been reported for Hox gene mutants and implies an unclear interaction of Hox codes with Npr2-mediated afferent projection patterning to define boundaries. Some vestibular ganglion neurons expand their projections to reach the cochlear apex and the cochlear nuclei, comparable to previous findings in Neurod1 mutant mice. Before birth, such expansions are reduced or lost leading to truncated projections to the anteroventral cochlear nucleus and expansion of low-frequency fibers of the apex to the posteroventral cochlear nucleus.

Effect of perinatal biotin deficiency on auditory pathway of the Wistar-Albino rats.

Aim: Severe biotin deficiency associated with biotinidase enzyme deficiency in newborns is seen as severe neurological problems and hearing loss. However, the effect on the infant of deficiencies in the maternal diet during pregnancy are not clear. Material and methods: The study included 16 female Wistar albino rats and 4 male Wistar albino rats, that were mated and then the females were separated into 4 groups. At 40 days after the birth, 3 pups were selected from each group, and these 12 pups were evaluated with DPOAE and ABR electrophysiologically and the cochlea was examined ultrastructurally with electron microscopy. Results: In the DPOAE evaluation, At 8000 and 11,000 Hz, the signal-noise ratios in the B-N and B-B groups were statistically significantly higher (p < .05). In ABR, lengthening of the latency periods was determined in all the waves at both 8 and 16 kHz in the B-B group. When the IPL periods were examined, lengthening in IPL 1-5 was statistically significant in the B-B group only at 8 kHz. Conclusions: Biotin can be said to have an effect on hearing pathways. However, specifically where on the hearing pathways that biotin is involved has not been clarified.

Conserved and divergent development of brainstem vestibular and auditory nuclei.

Vestibular function was established early in vertebrates and has remained, for the most part, unchanged. In contrast, each group of tetrapods underwent independent evolutionary processes to solve the problem of hearing on land, resulting in a remarkable mixture of conserved, divergent and convergent features that define extant auditory systems. The vestibuloacoustic nuclei of the hindbrain develop from a highly conserved ground plan and provide an ideal framework on which to address the participation of developmental processes to the evolution of neuronal circuits. We employed an electroporation strategy to unravel the contribution of two dorsoventral and four axial lineages to the development of the chick hindbrain vestibular and auditory nuclei. We compare the chick developmental map with recently established genetic fate-maps of the developing mouse hindbrain. Overall, we find considerable conservation of developmental origin for the vestibular nuclei. In contrast, a comparative analysis of the developmental origin of hindbrain auditory structures echoes the complex evolutionary history of the auditory system. In particular, we find that the developmental origin of the chick auditory interaural time difference circuit supports its emergence from an ancient vestibular network, unrelated to the analogous mammalian counterpart.

Intrinsic physiology of inhibitory neurons changes over auditory development.

During auditory development, changes in membrane properties promote the ability of excitatory neurons in the brain stem to code aspects of sound, including the level and timing of a stimulus. Some of these changes coincide with hearing onset, suggesting that sound-driven neural activity produces developmental plasticity of ion channel expression. While it is known that the coding properties of excitatory neurons are modulated by inhibition in the mature system, it is unknown whether there are also developmental changes in the membrane properties of brain stem inhibitory neurons. We investigated the primary source of inhibition in the avian auditory brain stem, the superior olivary nucleus (SON). The present studies test the hypothesis that, as in excitatory neurons, the membrane properties of these inhibitory neurons change after hearing onset. We examined SON neurons at different stages of auditory development: embryonic days 14-16 (E14-E16), a time at which cochlear ganglion neurons are just beginning to respond to sound; later embryonic stages (E18-E19); and after hatching (P0-P2). We used in vitro whole cell patch electrophysiology to explore physiological changes in SON. Age-related changes were observed at the level of a single spike and in multispiking behavior. In particular, tonic behavior, measured as a neuron's ability to sustain tonic firing over a range of current steps, became more common later in development. Voltage-clamp recordings and biophysical models were employed to examine how age-related increases in ion currents enhance excitability in SON. Our findings suggest that concurrent increases in sodium and potassium currents underlie the emergence of tonic behavior. NEW & NOTEWORTHY This article is the first to examine heterogeneity of neuronal physiology in the inhibitory nucleus of the avian auditory system and demonstrate that tonic firing here emerges over development. By pairing computer simulations with physiological data, we show that increases in both sodium and potassium channels over development are necessary for the emergence of tonic firing.

Cellular and Molecular Underpinnings of Neuronal Assembly in the Central Auditory System during Mouse Development.

During development, the organization of the auditory system into distinct functional subcircuits depends on the spatially and temporally ordered sequence of neuronal specification, differentiation, migration and connectivity. Regional patterning along the antero-posterior axis and neuronal subtype specification along the dorso-ventral axis intersect to determine proper neuronal fate and assembly of rhombomere-specific auditory subcircuits. By taking advantage of the increasing number of transgenic mouse lines, recent studies have expanded the knowledge of developmental mechanisms involved in the formation and refinement of the auditory system. Here, we summarize several findings dealing with the molecular and cellular mechanisms that underlie the assembly of central auditory subcircuits during mouse development, focusing primarily on the rhombomeric and dorso-ventral origin of auditory nuclei and their associated molecular genetic pathways.

Spiral Ganglion Neuron Projection Development to the Hindbrain in Mice Lacking Peripheral and/or Central Target Differentiation.

We investigate the importance of the degree of peripheral or central target differentiation for mouse auditory afferent navigation to the organ of Corti and auditory nuclei in three different mouse models: first, a mouse in which the differentiation of hair cells, but not central auditory nuclei neurons is compromised (Atoh1-cre; Atoh1f/f ); second, a mouse in which hair cell defects are combined with a delayed defect in central auditory nuclei neurons (Pax2-cre; Atoh1f/f ), and third, a mouse in which both hair cells and central auditory nuclei are absent (Atoh1-/-). Our results show that neither differentiated peripheral nor the central target cells of inner ear afferents are needed (hair cells, cochlear nucleus neurons) for segregation of vestibular and cochlear afferents within the hindbrain and some degree of base to apex segregation of cochlear afferents. These data suggest that inner ear spiral ganglion neuron processes may predominantly rely on temporally and spatially distinct molecular cues in the region of the targets rather than interaction with differentiated target cells for a crude topological organization. These developmental data imply that auditory neuron navigation properties may have evolved before auditory nuclei.

The effect of noise exposure during the developmental period on the function of the auditory system.

Recently, there has been growing evidence that development and maturation of the auditory system depends substantially on the afferent activity supplying inputs to the developing centers. In cases when this activity is altered during early ontogeny as a consequence of, e.g., an unnatural acoustic environment or acoustic trauma, the structure and function of the auditory system may be severely affected. Pathological alterations may be found in populations of ribbon synapses of the inner hair cells, in the structure and function of neuronal circuits, or in auditory driven behavioral and psychophysical performance. Three characteristics of the developmental impairment are of key importance: first, they often persist to adulthood, permanently influencing the quality of life of the subject; second, their manifestations are different and sometimes even contradictory to the impairments induced by noise trauma in adulthood; third, they may be 'hidden' and difficult to diagnose by standard audiometric procedures used in clinical practice. This paper reviews the effects of early interventions to the auditory system, in particular, of sound exposure during ontogeny. We summarize the results of recent morphological, electrophysiological, and behavioral experiments, discuss the putative mechanisms and hypotheses, and draw possible consequences for human neonatal medicine and noise health.

Comparative Analysis of Gene Regulatory Network Components in the Auditory Hindbrain of Mice and Chicken.

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.

Axonal Cleaved Caspase-3 Regulates Axon Targeting and Morphogenesis in the Developing Auditory Brainstem.

Caspase-3 is a cysteine protease that is most commonly associated with cell death. Recent studies have shown additional roles in mediating cell differentiation, cell proliferation and development of cell morphology. We investigated the role of caspase-3 in the development of chick auditory brainstem nuclei during embryogenesis. Immunofluorescence from embryonic days E6-13 revealed that the temporal expression of cleaved caspase-3 follows the ascending anatomical pathway. The expression is first seen in the auditory portion of VIIIth nerve including central axonal regions projecting to nucleus magnocellularis (NM), then later in NM axons projecting to nucleus laminaris (NL), and subsequently in NL dendrites. To examine the function of cleaved caspase-3 in chick auditory brainstem development, we blocked caspase-3 cleavage in developing chick embryos with the caspase-3 inhibitor Z-DEVD-FMK from E6 to E9, then examined NM and NL morphology and NM axonal targeting on E10. NL lamination in treated embryos was disorganized and the neuropil around NL contained a significant number of glial cells normally excluded from this region. Additionally, NM axons projected into inappropriate portions of NL in Z-DEVD-FMK treated embyros. We found that the presence of misrouted axons was associated with more severe NL disorganization. The effects of axonal caspase-3 inhibition on both NL morphogenesis and NM axon targeting suggest that these developmental processes are coordinated, likely through communication between axons and their targets.

Dcc Mediates Functional Assembly of Peripheral Auditory Circuits.

Proper structural organization of spiral ganglion (SG) innervation is crucial for normal hearing function. However, molecular mechanisms underlying the developmental formation of this precise organization remain not well understood. Here, we report in the developing mouse cochlea that deleted in colorectal cancer (Dcc) contributes to the proper organization of spiral ganglion neurons (SGNs) within the Rosenthal's canal and of SGN projections toward both the peripheral and central auditory targets. In Dcc mutant embryos, mispositioning of SGNs occurred along the peripheral auditory pathway with misrouted afferent fibers and reduced synaptic contacts with hair cells. The central auditory pathway simultaneously exhibited similar defective phenotypes as in the periphery with abnormal exit of SGNs from the Rosenthal's canal towards central nuclei. Furthermore, the axons of SGNs ascending into the cochlear nucleus had disrupted bifurcation patterns. Thus, Dcc is necessary for establishing the proper spatial organization of SGNs and their fibers in both peripheral and central auditory pathways, through controlling axon targeting and cell migration. Our results suggest that Dcc plays an important role in the developmental formation of peripheral and central auditory circuits, and its mutation may contribute to sensorineural hearing loss.

Development of Structure and Sensitivity of the Fish Inner Ear.

Fish represent the largest group of vertebrates and display the greatest diversity of auditory structures. However, studies addressing how the form and function of the auditory system change during development to enhance perception of the acoustic environment are rather sparse in this taxon compared to other vertebrate groups. An ontogenetic perspective of the auditory system in fishes provides a readily testable framework for understanding structure-function relationships. Additionally, studying ancestral models such as fish can convey valuable comparable information across vertebrates, as early developmental events are often evolutionary conserved. This chapter reviews the literature on the morphological development of the fish auditory system, with particular focus on the inner ear structures that evolve from an otic placode during early embryonic development and then continue to undergo differentiation and maturation in the postembryonic phase. Moreover, the chapter provides a systematic overview of how auditory sensitivity develops during ontogeny. Although most studies indicate a developmental improvement in auditory sensitivity, there is considerably species-specific variation. Lastly, the paucity of information and literature concerning the development of auditory capabilities for social communication in fishes is also discussed. Further investigation on the development of structure and function of the fish auditory system is recommended in order to obtain a deeper understanding of how ontogenetic morphological changes in the auditory pathway relate to modifications in acoustic reception, auditory processing, and the capacity to communicate acoustically.

Development of the auditory system.

Auditory development involves changes in the peripheral and central nervous system along the auditory pathways, and these occur naturally, and in response to stimulation. Human development occurs along a trajectory that can last decades, and is studied using behavioral psychophysics, as well as physiologic measurements with neural imaging. The auditory system constructs a perceptual space that takes information from objects and groups, segregates sounds, and provides meaning and access to communication tools such as language. Auditory signals are processed in a series of analysis stages, from peripheral to central. Coding of information has been studied for features of sound, including frequency, intensity, loudness, and location, in quiet and in the presence of maskers. In the latter case, the ability of the auditory system to perform an analysis of the scene becomes highly relevant. While some basic abilities are well developed at birth, there is a clear prolonged maturation of auditory development well into the teenage years. Maturation involves auditory pathways. However, non-auditory changes (attention, memory, cognition) play an important role in auditory development. The ability of the auditory system to adapt in response to novel stimuli is a key feature of development throughout the nervous system, known as neural plasticity.

The emerging framework of mammalian auditory hindbrain development.

A defining feature of the mammalian auditory system is the extensive processing of sound information in numerous ultrafast and temporally precise circuits in the hindbrain. By exploiting the experimental advantages of mouse genetics, recent years have witnessed an impressive advance in our understanding of developmental mechanisms involved in the formation and refinement of these circuits. Here, we will summarize the progress made in four major fields: the dissection of the rhombomeric origins of auditory hindbrain nuclei; the molecular repertoire involved in circuit formation such as Hox transcription factors and the Eph-ephrin signaling system; the timeline of functional circuit assembly; and the critical role of spontaneous activity for circuit refinement. In total, this information provides a solid framework for further exploration of the factors shaping auditory hindbrain circuits and their specializations. A comprehensive understanding of the developmental pathways and instructive factors will also offer important clues to the causes and consequences of hearing-loss related disorders, which represent the most common sensory impairment in humans.