Development in the Mammalian Auditory System Depends on Transcription Factors.

We review the molecular basis of several transcription factors (Eya1, Sox2), including the three related genes coding basic helix-loop-helix (bHLH; see abbreviations) proteins (Neurog1, Neurod1, Atoh1) during the development of spiral ganglia, cochlear nuclei, and cochlear hair cells. Neuronal development requires Neurog1, followed by its downstream target Neurod1, to cross-regulate Atoh1 expression. In contrast, hair cells and cochlear nuclei critically depend on Atoh1 and require Neurod1 expression for interactions with Atoh1. Upregulation of Atoh1 following Neurod1 loss changes some vestibular neurons' fate into "hair cells", highlighting the significant interplay between the bHLH genes. Further work showed that replacing Atoh1 by Neurog1 rescues some hair cells from complete absence observed in Atoh1 null mutants, suggesting that bHLH genes can partially replace one another. The inhibition of Atoh1 by Neurod1 is essential for proper neuronal cell fate, and in the absence of Neurod1, Atoh1 is upregulated, resulting in the formation of "intraganglionic" HCs. Additional genes, such as Eya1/Six1, Sox2, Pax2, Gata3, Fgfr2b, Foxg1, and Lmx1a/b, play a role in the auditory system. Finally, both Lmx1a and Lmx1b genes are essential for the cochlear organ of Corti, spiral ganglion neuron, and cochlear nuclei formation. We integrate the mammalian auditory system development to provide comprehensive insights beyond the limited perception driven by singular investigations of cochlear neurons, cochlear hair cells, and cochlear nuclei. A detailed analysis of gene expression is needed to understand better how upstream regulators facilitate gene interactions and mammalian auditory system development.

Exposures to fine particulate matter (PM2.5) and ozone above USA standards are associated with auditory brainstem dysmorphology and abnormal auditory brainstem evoked potentials in healthy young dogs.

BACKGROUND: Delayed central conduction times in the auditory brainstem have been observed in Mexico City (MC) healthy children exposed to fine particulate matter (PM2.5) and ozone (O3) above the current United States Environmental Protection Agency (US-EPA) standards. MC children have α synuclein brainstem accumulation and medial superior olivary complex (MSO) dysmorphology. The present study used a dog model to investigate the potential effects of air pollution on the function and morphology of the auditory brainstem. METHODOLOGY: Twenty-four dogs living in clean air v MC, average age 37.1 ± 26.3 months, underwent brainstem auditory evoked potential (BAEP) measurements. Eight dogs (4 MC, 4 Controls) were analysed for auditory brainstem morphology and histopathology. RESULTS: MC dogs showed ventral cochlear nuclei hypotrophy and MSO dysmorphology with a significant decrease in cell body size, decreased neuronal packing density with regions in the nucleus devoid of neurons and marked gliosis. MC dogs showed significant delayed BAEP absolute wave I, III and V latencies compared to controls. CONCLUSIONS: MC dogs show auditory nuclei dysmorphology and BAEPs consistent with an alteration of the generator sites of the auditory brainstem response waveform. This study puts forward the usefulness of BAEPs to study auditory brainstem neurodegenerative changes associated with air pollution in dogs. Recognition of the role of non-invasive BAEPs in urban dogs is warranted to elucidate novel neurodegenerative pathways link to air pollution and a promising early diagnostic strategy for Alzheimer's Disease.

Central projections of auditory nerve fibers in the western rat snake (Pantherophis obsoletus).

Despite the absence of tympanic middle ears, snakes can hear. They are thought to primarily detect substrate vibration via connections between the lower jaw and the inner ear. We used the western rat snake (Pantherophis obsoletus) to determine how vibration is processed in the brain. We measured vibration-evoked potential recordings to reveal sensitivity to low-frequency vibrations. We then used tract tracing combined with immunohistochemistry and Nissl staining to describe the central projections of the papillar branch of the VIIIth nerve. Applications of biotinylated dextran amine to the basilar papilla (homologous to the organ of Corti of mammals) labeled bouton-like terminals in two first-order cochlear nuclei, a rostrolateral nucleus angularis (NA) and a caudomedial nucleus magnocellularis (NM). NA formed a distinct dorsal eminence, consisted of heterogenous cell types, and was parvalbumin positive. NM was smaller and poorly separated from the surrounding vestibular nuclei. NM was distinguished by positive calbindin label and included fusiform and round cells. Thus, the atympanate western rat snake shares similar first-order projections to tympanate reptiles. Auditory pathways may be used for detecting vibration, not only in snakes but also potentially in atympanate early tetrapods.

Molecular identity of the lateral lemniscus nuclei in the adult mouse brain.

The dorsal (DLL), intermediate (ILL), and ventral (VLL) lateral lemniscus nuclei are relay centers in the central auditory pathway of the brainstem, commonly referred to as the lateral lemniscus nuclei (LLN). The LLN are situated in the prepontine and pontine hindbrain, from rhombomeres 1 to 4, extending from the more rostral DLL to the caudal VLL, with the ILL lying in between. These nuclei can be distinguished morphologically and by topological and connectivity criteria, and here, we set out to further characterize the molecular nature of each LLN. We searched in situ hybridization studies in the Allen Mouse Brain Atlas for genes differentially expressed along the rostrocaudal axis of the brainstem, identifying 36 genes from diverse functional families expressed in the LLN. Available information in the databases indicated that 7 of these 36 genes are either associated with or potentially related to hearing disorders. In conclusion, the LLN are characterized by specific molecular profiles that reflect their rostrocaudal organization into the three constituent nuclei. This molecular regionalization may be involved in the etiology of some hearing disorders, in accordance with previous functional studies of these genes.

Imaging Voltage Globally and in Isofrequency Lamina in Slices of Mouse Ventral Cochlear Nucleus.

The cochlear nuclei (CNs) receive sensory information from the ear and perform fundamental computations before relaying this information to higher processing centers. These computations are performed by distinct types of neurons interconnected in circuits dedicated to the specialized roles of the auditory system. In the present study, we explored the use of voltage imaging to investigate CN circuitry. We tested two approaches based on fundamentally different voltage sensing technologies. Using a voltage-sensitive dye we recorded glutamate receptor-independent signals arising predominantly from axons. The mean conduction velocity of these fibers of 0.27 m/s was rapid but in range with other unmyelinated axons. We then used a genetically-encoded hybrid voltage sensor (hVOS) to image voltage from a specific population of neurons. Probe expression was controlled using Cre recombinase linked to c-fos activation. This activity-induced gene enabled targeting of neurons that are activated when a mouse hears a pure 15-kHz tone. In CN slices from these animals auditory nerve fiber stimulation elicited a glutamate receptor-dependent depolarization in hVOS probe-labeled neurons. These cells resided within a band corresponding to an isofrequency lamina, and responded with a high degree of synchrony. In contrast to the axonal origin of voltage-sensitive dye signals, hVOS signals represent predominantly postsynaptic responses. The introduction of voltage imaging to the CN creates the opportunity to investigate auditory processing circuitry in populations of neurons targeted on the basis of their genetic identity and their roles in sensory processing.

Molecular mechanisms governing development of the hindbrain choroid plexus and auditory projection: A validation of the seminal observations of Wilhelm His.

Studies by His from 1868 to 1904 delineated the critical role of the dorsal roof plate in the development of the hindbrain choroid plexus, and of the rhombic lips in the development of hindbrain auditory centers. Modern molecular studies have confirmed these observations and placed them in a mechanistic context. Expression of the transcription factor Lmx1a/b is crucial to the development of the hindbrain choroid plexus, and also regulates the expression of Atoh1, a transcription factor that is essential for the formation of the cochlear hair cells and auditory nuclei. By contrast, development of the vestibular hair cells, vestibular ganglion and vestibular nuclei does not depend on Lmx1a/b. These findings demonstrate a common dependence on a specific gene for the hindbrain choroid plexus and the primary auditory projection from hair cells to sensory neurons to hindbrain nuclei. Thus, His' conclusions regarding the origins of specific hindbrain structures are borne out by molecular genetic experiments conducted more than a hundred years later.

Age-Related Hearing Loss: Sensory and Neural Etiology and Their Interdependence.

Age-related hearing loss (ARHL) is a common, increasing problem for older adults, affecting about 1 billion people by 2050. We aim to correlate the different reductions of hearing from cochlear hair cells (HCs), spiral ganglion neurons (SGNs), cochlear nuclei (CN), and superior olivary complex (SOC) with the analysis of various reasons for each one on the sensory deficit profiles. Outer HCs show a progressive loss in a basal-to-apical gradient, and inner HCs show a loss in a apex-to-base progression that results in ARHL at high frequencies after 70 years of age. In early neonates, SGNs innervation of cochlear HCs is maintained. Loss of SGNs results in a considerable decrease (~50% or more) of cochlear nuclei in neonates, though the loss is milder in older mice and humans. The dorsal cochlear nuclei (fusiform neurons) project directly to the inferior colliculi while most anterior cochlear nuclei reach the SOC. Reducing the number of neurons in the medial nucleus of the trapezoid body (MNTB) affects the interactions with the lateral superior olive to fine-tune ipsi- and contralateral projections that may remain normal in mice, possibly humans. The inferior colliculi receive direct cochlear fibers and second-order fibers from the superior olivary complex. Loss of the second-order fibers leads to hearing loss in mice and humans. Although ARHL may arise from many complex causes, HC degeneration remains the more significant problem of hearing restoration that would replace the cochlear implant. The review presents recent findings of older humans and mice with hearing loss.

Early Deletion of Neurod1 Alters Neuronal Lineage Potential and Diminishes Neurogenesis in the Inner Ear.

Neuronal development in the inner ear is initiated by expression of the proneural basic Helix-Loop-Helix (bHLH) transcription factor Neurogenin1 that specifies neuronal precursors in the otocyst. The initial specification of the neuroblasts within the otic epithelium is followed by the expression of an additional bHLH factor, Neurod1. Although NEUROD1 is essential for inner ear neuronal development, the different aspects of the temporal and spatial requirements of NEUROD1 for the inner ear and, mainly, for auditory neuron development are not fully understood. In this study, using Foxg1Cre for the early elimination of Neurod1 in the mouse otocyst, we showed that Neurod1 deletion results in a massive reduction of differentiating neurons in the otic ganglion at E10.5, and in the diminished vestibular and rudimental spiral ganglia at E13.5. Attenuated neuronal development was associated with reduced and disorganized sensory epithelia, formation of ectopic hair cells, and the shortened cochlea in the inner ear. Central projections of inner ear neurons with conditional Neurod1 deletion are reduced, unsegregated, disorganized, and interconnecting the vestibular and auditory systems. In line with decreased afferent input from auditory neurons, the volume of cochlear nuclei was reduced by 60% in Neurod1 mutant mice. Finally, our data demonstrate that early elimination of Neurod1 affects the neuronal lineage potential and alters the generation of inner ear neurons and cochlear afferents with a profound effect on the first auditory nuclei, the cochlear nuclei.

Neurog1, Neurod1, and Atoh1 are essential for spiral ganglia, cochlear nuclei, and cochlear hair cell development.

We review the molecular basis of three related basic helix-loop-helix (bHLH) genes (Neurog1, Neurod1, and Atoh1) and upstream regulators Eya1/Six1, Sox2, Pax2, Gata3, Fgfr2b, Foxg1, and Lmx1a/b during the development of spiral ganglia, cochlear nuclei, and cochlear hair cells. Neuronal development requires early expression of Neurog1, followed by its downstream target Neurod1, which downregulates Atoh1 expression. In contrast, hair cells and cochlear nuclei critically depend on Atoh1 and require Neurod1 and Neurog1 expression for various aspects of development. Several experiments show a partial uncoupling of Atoh1/Neurod1 (spiral ganglia and cochlea) and Atoh1/Neurog1/Neurod1 (cochlear nuclei). In this review, we integrate the cellular and molecular mechanisms that regulate the development of auditory system and provide novel insights into the restoration of hearing loss, beyond the limited generation of lost sensory neurons and hair cells.

The ion channels and synapses responsible for the physiological diversity of mammalian lower brainstem auditory neurons.

The auditory part of the brainstem is composed of several nuclei specialized in the computation of the different spectral and temporal features of the sound before it reaches the higher auditory regions. There are a high diversity of neuronal types in these nuclei, many with remarkable electrophysiological and synaptic properties unique to these structures. This diversity reflects specializations necessary to process the different auditory signals in order to extract precisely the acoustic information necessary for the auditory perception by the animal. Low threshold Kv1 channels and HCN channels are expressed in neurons that use timing clues for auditory processing, like bushy and octopus cells, in order to restrict action potential firing and reduce input resistance and membrane time constant. Kv3 channels allow principal neurons of the MNTB and pyramidal DCN neurons to fire fast trains of action potentials. Calcium channels on cartwheel DCN neurons produce complex spikes characteristic of these neurons. Calyceal synapses compensate the low input resistance of bushy and principal neurons of the MNTB by releasing hundreds of glutamate vesicles resulting in large EPSCs acting in fast ionotropic glutamate receptors, in order to reduce temporal summation of synaptic potentials, allowing more precise correspondence of pre- and post-synaptic potentials, and phase-locking. Pre-synaptic calyceal sodium channels have fast recovery from inactivation allowing extremely fast trains of action potential firing, and persistent sodium channels produce spontaneous activity of fusiform neurons at rest, which expands the dynamic range of these neurons. The unique combinations of different ion channels, ionotropic receptors and synaptic structures create a unique functional diversity of neurons extremely adapted to their complex functions in the auditory processing.

Lymphocytes B population profile in a case of multiple sclerosis presenting with sudden sensorineural hearing loss caused by a demyelinating pontine lesion.

Sudden sensorineural hearing loss (SSHL) is a rare manifestation of multiple sclerosis, typically appearing in the early stages of the disease, especially in female subjects. SSHL is produced by the involvement of auditory tract, vestibulocochlear nerve and possibly cochlear structures and rarely due to a single lesion. The authors report the case of a young woman in which the onset of multiple sclerosis presented with SSHL caused by a pontine lesion. Oligoclonal bands in the cerebrospinal fluid (CSF) were absent at the disease onset and appeared during disease progression. Immunophenotyping of cells showed low cellularity of CD19+ cells in the CSF and expression of CD38+ on the majority of CD19+, CD20+ B cells in the peripheral blood, suggesting that many of them were mature B lymphocytes.

Effect of granulocyte colony-stimulating factor on the cochlear nuclei after creation of a partial nerve lesion: an experimental study in rats.

OBJECTIVE The risk of injury of the cochlear nerve during angle (CPA) surgery is high. Granulocyte colony-stimulating factor (G-CSF) has been found in various experimental models of peripheral and CNS injury to have a neuroprotective effect by inhibiting apoptosis and inflammation. However, to the authors' knowledge, the influence of G-CSF on cochlear nerve regeneration has not been reported. This study investigated the neuroprotective effect of G-CSF after a partial cochlear nerve lesion in rats. METHODS A lesion of the right cochlear nerve in adult male Sprague-Dawley rats was created using a water-jet dissector with a pressure of 8 bar. In the first group (G-CSF-post), G-CSF was administrated on Days 1, 3, and 5 after the surgery. The second group (G-CSF-pre/post) was treated with G-CSF 1 day before and 1, 3, and 5 days after applying the nerve injury. The control group received sodium chloride after nerve injury at the various time points. Brainstem auditory evoked potentials (BAEPs) were measured directly before and after nerve injury and on Days 1 and 7 to evaluate the acoustic function of the cochlear nerve. The animals were sacrificed 1 week after the operation, and their brains were fixed in formalin. Nissl staining of the cochlear nuclei was performed, and histological sections were analyzed with a light microscope and an image-processing program. The numbers of neurons in the cochlear nuclei were assessed. RESULTS The values for Waves 2 and 4 of the BAEPs decreased abruptly in all 3 groups in the direct postoperative measurement. Although the amplitude in the control group did not recover, it increased in both treatment groups. According to 2-way ANOVA, groups treated with G-CSF had a significant increase in BAEP Wave II amplitudes on the right side (p = 0.0401) after the applied cochlear nerve injury. With respect to Wave IV, a trend toward better recovery in the G-CSF groups was found, but this difference did not reach statistical significance. In the histological analysis, higher numbers of neurons were found in the G-CSF groups. In the statistical analysis, the difference in the numbers of neurons between the control and G-CSF-post groups reached significance (p = 0.0086). The difference in the numbers of neurons between the control and G-CSF-pre/post groups and between the G-CSF-post and G-CSF-pre/post groups did not reach statistical significance. CONCLUSIONS The use of G-CSF improved the function of the eighth cranial nerve and protected cochlear nucleus cells from destruction after a controlled partial injury of the nerve. These findings might be relevant for surgery that involves CPA tumors. The use of G-CSF in patients with a lesion in the CPA might improve postoperative outcomes.

Effects of exposure to 2100MHz GSM-like radiofrequency electromagnetic field on auditory system of rats.

INTRODUCTION: The use of mobile phones has become widespread in recent years. Although beneficial from the communication viewpoint, the electromagnetic fields generated by mobile phones may cause unwanted biological changes in the human body. OBJECTIVE: In this study, we aimed to evaluate the effects of 2100MHz Global System for Mobile communication (GSM-like) electromagnetic field, generated by an electromagnetic fields generator, on the auditory system of rats by using electrophysiological, histopathologic and immunohistochemical methods. METHODS: Fourteen adult Wistar albino rats were included in the study. The rats were divided randomly into two groups of seven rats each. The study group was exposed continuously for 30days to a 2100MHz electromagnetic fields with a signal level (power) of 5.4dBm (3.47mW) to simulate the talk mode on a mobile phone. The control group was not exposed to the aforementioned electromagnetic fields. After 30days, the Auditory Brainstem Responses of both groups were recorded and the rats were sacrificed. The cochlear nuclei were evaluated by histopathologic and immunohistochemical methods. RESULTS: The Auditory Brainstem Responses records of the two groups did not differ significantly. The histopathologic analysis showed increased degeneration signs in the study group (p=0.007). In addition, immunohistochemical analysis revealed increased apoptotic index in the study group compared to that in the control group (p=0.002). CONCLUSION: The results support that long-term exposure to a GSM-like 2100MHz electromagnetic fields causes an increase in neuronal degeneration and apoptosis in the auditory system.

Developmental alterations of the auditory brainstem centers--pathogenetic implications in Sudden Infant Death Syndrome.

Sudden Infant Death Syndrome (SIDS), despite the success of campaigns to reduce its risks, is the leading cause of infant death in the Western world. Even though the pathogenesis remains unexplained, brainstem abnormalities of the neuronal network that mediates breathing and protective responses to asphyxia, particularly in the arousal phase from sleep, are believed to play a fundamental role. This is the first study to identify, in SIDS, developmental defects of specific brainstem centers involved in hearing pathways, particularly in the cochlear and vestibular nuclei, in the superior olivary complex and in the inferior colliculus, suggesting a possible influence of the acoustic system on respiratory activity. In 49 SIDS cases and 20 controls an in-depth anatomopathological examination of the autonomic nervous system was performed, with the main aim of detecting developmental alterations of brainstem structures controlling both the respiratory and auditory activities. Overall, a significantly higher incidence of cytoarchitectural alterations of both the auditory and respiratory network components were observed in SIDS victims compared with matched controls. Even if there is not sufficient evidence to presume that developmental defects of brainstem auditory structures can affect breathing, our findings, showing that developmental deficit in the control respiratory areas are frequently accompanied by alterations of auditory structures, highlight an additional important element for the understanding the pathogenetic mechanism of SIDS.

Effects of exposure to 2100 MHz GSM-like radiofrequency electromagnetic field on auditory system of rats / Efeitos da exposição a um campo eletromagnético na radiofrequência de 2100 MHz, similar ao sistema GSM, no sistema auditivo de ratos

Abstract Introduction: The use of mobile phones has become widespread in recent years. Although beneficial from the communication viewpoint, the electromagnetic fields generated by mobile phones may cause unwanted biological changes in the human body. Objective: In this study, we aimed to evaluate the effects of 2100 MHz Global System for Mobile communication (GSM-like) electromagnetic field, generated by an electromagnetic fields generator, on the auditory system of rats by using electrophysiological, histopathologic and immunohistochemical methods. Methods: Fourteen adult Wistar albino rats were included in the study. The rats were divided randomly into two groups of seven rats each. The study group was exposed continuously for 30 days to a 2100 MHz electromagnetic fields with a signal level (power) of 5.4 dBm (3.47 mW) to simulate the talk mode on a mobile phone. The control group was not exposed to the aforementioned electromagnetic fields. After 30 days, the Auditory Brainstem Responses of both groups were recorded and the rats were sacrificed. The cochlear nuclei were evaluated by histopathologic and immunohistochemical methods. Results: The Auditory Brainstem Responses records of the two groups did not differ significantly. The histopathologic analysis showed increased degeneration signs in the study group (p = 0.007). In addition, immunohistochemical analysis revealed increased apoptotic index in the study group compared to that in the control group (p = 0.002). Conclusion: The results support that long-term exposure to a GSM-like 2100 MHz electromagnetic fields causes an increase in neuronal degeneration and apoptosis in the auditory system.

Resumo Introdução: O uso de telefones celulares tornou-se generalizado nos últimos anos. Embora benéfico do ponto de vista da comunicação, os campos eletromagnéticos gerados por celulares pode causar alterações biológicas indesejáveis no corpo humano. Objetivo: Nesse estudo, o objetivo foi avaliar os efeitos do campo eletromagnético na frequência de 2.100 MHz, similar à modulação do Sistema Global para Comunicações Móveis, produzido por um gerador de campo eletromagnético, sobre o sistema auditivo de ratos usando os métodos eletrofisiológico, histopatológico e imunohistoquímico. Método: Foram incluídos no estudo catorze adultos ratos albinos Wistar. Os ratos foram divididos aleatoriamente em dois grupos de sete animais cada. O grupo de estudo foi exposto continuamente por 30 dias a um campo eletromagnético em 2100 MHz com um nível de sinal (potência) de 5,4 dBm (3,47 miliwatts) para simular o modo de conversação em um celular. O grupo controle não foi exposto ao campo eletromagnético acima mencionado. Após 30 dias, o potencial evocado auditivo de tronco encefálico de ambos os grupos foi gravado e os ratos foram sacrificados. Os núcleos cocleares foram avaliados pelos métodos histopatológico e imunohistoquímico. Resultados: Os registros do potencial evocado auditivo de tronco encefálico dos dois grupos não diferiram significativamente. A análise histopatológica mostrou aumento dos sinais de degeneração no grupo de estudo (p = 0,007). Além disso, a análise imuno-histoquímica revelou aumento do índice de apoptose no grupo de estudo em comparação com o grupo controle (p = 0,002). Conclusão: Os resultados confirmam que a exposição a longo prazo a um campo eletromagnético em 2100 MHz similar à modulação do sistema global para comunicações móveis causa um aumento na degeneração neuronal e apoptose no sistema auditivo.

The long adventurous journey of rhombic lip cells in jawed vertebrates: a comparative developmental analysis.

This review summarizes vertebrate rhombic lip and early cerebellar development covering classic approaches up to modern developmental genetics which identifies the relevant differential gene expression domains and their progeny. Most of this information is derived from amniotes. However, progress in anamniotes, particularly in the zebrafish, has recently been made. The current picture suggests that rhombic lip and cerebellar development in jawed vertebrates (gnathostomes) share many characteristics. Regarding cerebellar development, these include a ptf1a expressing ventral cerebellar proliferation (VCP) giving rise to Purkinje cells and other inhibitory cerebellar cell types, and an atoh1 expressing upper rhombic lip giving rise to an external granular layer (EGL, i.e., excitatory granule cells) and an early ventral migration into the anterior rhombencephalon (cholinergic nuclei). As for the lower rhombic lip (LRL), gnathostome commonalities likely include the formation of precerebellar nuclei (mossy fiber origins) and partially primary auditory nuclei (likely convergently evolved) from the atoh1 expressing dorsal zone. The fate of the ptf1a expressing ventral LRL zone which gives rise to (excitatory cells of) the inferior olive (climbing fiber origin) and (inhibitory cells of ) cochlear nuclei in amniotes, has not been determined in anamniotes. Special for the zebrafish in comparison to amniotes is the predominant origin of anamniote excitatory deep cerebellar nuclei homologs (i.e., eurydendroid cells) from ptf1a expressing VCP cells, the sequential activity of various atoh1 paralogs and the incomplete coverage of the subpial cerebellar plate with proliferative EGL cells. Nevertheless, the conclusion that a rhombic lip and its major derivatives evolved with gnathostome vertebrates only and are thus not an ancestral craniate character complex is supported by the absence of a cerebellum (and likely absence of its afferent and efferent nuclei) in jawless fishes.