Noise-induced hearing loss alters potassium-chloride cotransporter KCC2 and GABA inhibition in the auditory centers.

Homeostatic plasticity, the ability of neurons to maintain their averaged activity constant around a set point value, is thought to account for the central hyperactivity after hearing loss. Here, we investigated the putative role of GABAergic neurotransmission in this mechanism after a noise-induced hearing loss larger than 50 dB in high frequencies in guinea pigs. The effect of GABAergic inhibition is linked to the normal functioning of K + -Cl- co-transporter isoform 2 (KCC2) which maintains a low intracellular concentration of chloride. The expression of membrane KCC2 were investigated before and after noise trauma in the ventral and dorsal cochlear nucleus (VCN and DCN, respectively) and in the inferior colliculus (IC). Moreover, the effect of gabazine (GBZ), a GABA antagonist, was also studied on the neural activity in IC. We show that KCC2 is downregulated in VCN, DCN and IC 3 days after noise trauma, and in DCN and IC 30 days after the trauma. As expected, GBZ application in the IC of control animals resulted in an increase of spontaneous and stimulus-evoked activity. In the noise exposed animals, on the other hand, GBZ application decreased the stimulus-evoked activity in IC neurons. The functional implications of these central changes are discussed.

Cochlear Nucleus Transcriptome of a Fragile X Mouse Model Reveals Candidate Genes for Hyperacusis.

OBJECTIVE: Fragile X Syndrome (FXS) is a hereditary form of autism spectrum disorder. It is caused by a trinucleotide repeat expansion in the Fmr1 gene, leading to a loss of Fragile X Protein (FMRP) expression. The loss of FMRP causes auditory hypersensitivity: FXS patients display hyperacusis and the Fmr1- knock-out (KO) mouse model for FXS exhibits auditory seizures. FMRP is strongly expressed in the cochlear nucleus and other auditory brainstem nuclei. We hypothesize that the Fmr1-KO mouse has altered gene expression in the cochlear nucleus that may contribute to auditory hypersensitivity. METHODS: RNA was isolated from cochlear nuclei of Fmr1-KO and WT mice. Using next-generation sequencing (RNA-seq), the transcriptomes of Fmr1-KO mice and WT mice (n = 3 each) were compared and analyzed using gene ontology programs. RESULTS: We identified 270 unique, differentially expressed genes between Fmr1-KO and WT cochlear nuclei. Upregulated genes (67%) are enriched in those encoding secreted molecules. Downregulated genes (33%) are enriched in neuronal function, including synaptic pathways, some of which are ideal candidate genes that may contribute to hyperacusis. CONCLUSION: The loss of FMRP can affect the expression of genes in the cochlear nucleus that are important for neuronal signaling. One of these, Kcnab2, which encodes a subunit of the Shaker voltage-gated potassium channel, is expressed at an abnormally low level in the Fmr1-KO cochlear nucleus. Kcnab2 and other differentially expressed genes may represent pathways for the development of hyperacusis. Future studies will be aimed at investigating the effects of these altered genes on hyperacusis. LEVEL OF EVIDENCE: N/A Laryngoscope, 134:1363-1371, 2024.

Characterization of three cholinergic inputs to the cochlear nucleus.

Acetylcholine modulates responses throughout the auditory system, including at the earliest brain level, the cochlear nucleus (CN). Previous studies have shown multiple sources of cholinergic input to the CN but information about their relative contributions and the distribution of inputs from each source is lacking. Here, we used staining for cholinergic axons and boutons, retrograde tract tracing, and acetylcholine-selective anterograde tracing to characterize three sources of acetylcholine input to the CN in mice. Staining for cholinergic axons showed heavy cholinergic inputs to granule cell areas and the dorsal CN with lighter input to the ventral CN. Retrograde tract tracing revealed that cholinergic cells from the superior olivary complex, pontomesencephalic tegmentum, and lateral paragigantocellular nucleus send projections to the CN. When we selectively labeled cholinergic axons from each source to the CN, we found surprising similarities in their terminal distributions, with patterns that were overlapping rather than complementary. Each source heavily targeted granule cell areas and the dorsal CN (especially the deep dorsal CN) and sent light input into the ventral CN. Our results demonstrate convergence of cholinergic inputs from multiple sources in most regions of the CN and raise the possibility of convergence onto single CN cells. Linking sources of acetylcholine and their patterns of activity to modulation of specific cell types in the CN will be an important next step in understanding cholinergic modulation of early auditory processing.

Ptf1a expression is necessary for correct targeting of spiral ganglion neurons within the cochlear nuclei.

Two transcription factors, Atoh1 and Ptf1a, are essential for cochlear nuclei development. Atoh1 is needed to develop glutamatergic neurons, while Ptf1a is required to generate glycinergic and GABAergic neurons that migrate into the cochlear nucleus. While central projections of inner ear afferents are normal following loss of Atoh1, we wanted to know whether the loss of Ptf1a affects central projections. We found that in Ptf1a mutants, initially, afferents show a normal projection; however, a transient posterior expansion of projections to the dorsal cochlear nucleus occurs at a later stage. In addition, in older (E18.5) Ptf1a mutant mice, excessive neuronal branches form beyond the normal projection to the anterior and posterior ventral cochlear nuclei. Our results on Ptf1a null mice are comparable to that observed in loss of function Prickel1, Npr2, or Fzd3 mouse mutants. The disorganized tonotopic projections that we report in Ptf1a mutant embryos might be functionally relevant, but testing this hypothesis requires Ptf1a KO mice at postnatal stages that unfortunately cannot be performed due to their early death.

Focusing on the Emerging Role of Kainate Receptors in the Dorsal Cochlear Nucleus (DCN) and Cerebellum.

Mammals have a dorsal cochlear nucleus (DCN), which is thought to be a cerebellum-like structure with similar features in terms of structure and microcircuitry to the cerebellum. Both the DCN and cerebellum perform their functions depending on synaptic and neuronal networks mediated by various glutamate receptors. Kainate receptors (KARs) are one class of the glutamate receptor family and are strongly expressed in the hippocampus, the cerebellum, and cerebellum-like structures. The cellular distribution and the potential role of KARs in the hippocampus have been extensively investigated. However, the cellular distribution and the potential role of KARs in cerebellum-like structures, including the DCN and cerebellum, are poorly understood. In this review, we summarize the similarity between the DCN and cerebellum at the levels of structure, circuitry, and cell type as well as the investigations referring to the expression patterns of KARs in the DCN and cerebellum according to previous studies. Recent studies on the role of KARs have shown that KARs mediate a bidirectional modulatory effect at parallel fiber (PF)-Purkinje cell (PC) synapses in the cerebellum, implying insights into their roles in cerebellum-like structures, including the DCN, that remain to be explored in the coming years.

NRG1/ErbB2 axis regulated mitochondrial function and antioxidant enzymes of neural stem cells in the cochlear nucleus partially through PGC-1α.

Neuregulin-1 (NRG1)/erythroblastic leukaemia viral oncogene homologues 2 (ErbB2) pathway had been implicated in promoting differentiation and suppressing apoptosis of neuronal stem cells (NSCs) isolated from cochlear nucleus. In the current study, we aimed at determining the effects of NRG1/ErbB2 on mitochondrial (mt) function of NSCs. As expected, NRG1 increased the expression of mitofusin (Mfn) 1 and Mfn2 and decreased the expression of mitochondrial fission protein 1 (Fis1) and dynamin-related protein 1 (Drp1). However, after ErbB2 knockout, Mfn1 and Mfn2 expression decreased while Fis1 and Drp1 increased. Moreover, the increased mtDNA copy number and intracellular ATP level, elevated ATPase activities as well as decreased lactate production induced by NRG1 were partially reversed by ErbB2 knockout. Additionally, NRG1 treatment increased the activities of catalase, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and upregulated the protein expression of catalase, manganese superoxide dismutase (MnSOD), peroxisome proliferator-activated receptor-γ coactlvator-1α (PGC-1α), nuclear respiratory factor 1 (NRF1) and transcription factor A, mitochondrial (TFAM), which were also reversed by ErbB2 knockout. Furthermore, PGC-1α overexpression partially reversed the above effects of ErbB2 knockout. In conclusion, these findings suggest that the promotion of mitochondrial function of NRG1/ErbB2 axis is at least in part mediated by PGC-1α in NSCs from cochlear nucleus.

A liquid chromatography-mass spectroscopy-based untargeted metabolomic study of the rat cochlear nucleus at various stages of maturity.

The cochlear nucleus receives numerous inputs from auditory and nonauditory systems. This extensive innervation of the cochlear nucleus is involved in sound source localization and the integration of auditory signals with other sensory modalities. The dorsal cochlear nucleus may also have an important role in tinnitus. Although its gross anatomy and function have been extensively studied, the metabolome of the cochlear nucleus remains poorly understood, particularly at different stages of auditory maturity. Here, we present a protocol for untargeted metabolomics analysis of the rat cochlear nucleus, then discuss differences in the metabolome of the rat cochlear nucleus between postnatal day (PD) 14 (hearing onset) and PD60 (hearing maturation). Cochlear nucleus samples collected from rats at PD14 or PD60 were analyzed by liquid chromatography-tandem mass spectrometry (LCMS). In total, 344 metabolites were identified. Principal component analysis and orthogonal partial least-square discriminant analysis showed that the metabolic profiles at these two stages had distinct distribution patterns. Moreover, 91 significantly differential metabolites (62 upregulated and 29 downregulated) were identified at PD60 vs. PD14. N-acetylaspartylglutamic acid (NAAG), γ-aminobutyric acid (GABA), taurine, adenosine monophosphate (AMP), and choline were significantly upregulated at PD60. Pathway enrichment analysis suggested that alanine, aspartate, and glutamate metabolism; glycine, serine, and threonine metabolism; the mammalian target of rapamycin (mTOR) signaling pathway; and the AMP-activated protein kinase (AMPK) signaling pathway may be involved in key developmental events during maturation of the cochlear nucleus. Taken together, the metabolic profiles identified in this study could lead to the identification and understanding of specific key biomarkers and metabolic pathways involved in the maturation of hearing. Moreover, LC-MS-based metabolomics provides an alternative approach for the characterization of auditory maturation and auditory diseases.

Differential Expression of miRNAs and Their Predicted Target Pathways in Cochlear Nucleus Following Chronic Noise Exposure in Rats.

Several recent preclinical studies have reported that dynamic changes in miRNA expression contribute to hearing function. This study aims to investigate miRNA expression changes in the cochlear nuclei (CN) of rats following chronic noise exposure. Eight-week-old rats (n = 14) were exposed to noise for 4 weeks. The control rats (n = 14) were raised under identical conditions without noise. Two months after noise exposure, the auditory brainstem response (ABR) was examined, and the cochlea and CN were harvested. In the CN, the expression levels of arc, neurocan, and brevican were measured (n = 6 per group). Furthermore, the expression levels of miRNAs and their predicted target genes were measured in the CN (n = 8 per group). ABR thresholds were elevated after 4 weeks of noise exposure, which were maintained for 3 months. In CN, the protein expression of arc and brevican was higher in the noise-exposed group than in the control group (0.95 [standard deviation (SD) = 0.53] vs. 3.19 [SD = 1.00], p < 0.001 for arc and 1.02 [SD = 0.10] vs. 1.66 [SD = 0.24], p < 0.001 for brevican). The noise-exposed rats exhibited lower expression levels of miR-758-5p, miR-15b-5p, miR-212-3p, miR-199a-5p, and miR-134-3p than the control rats (all p < 0.001). The AMPK signaling pathway was predicted to be regulated by these miRNAs. The predicted target genes AKT3, SIRT1, and PRKAA1 were highly expressed in noise-exposed rats. In CN of noise-exposed rats, the miRNAs of miR-758-5p, miR-15b-5p, miR-212-3p, miR-199a-5p, and miR-134-3p were reduced and related to AMPK signaling including AKT3 and SIRT1 expression. These modulation of signaling pathways could mediate the increased expression of brevican in the CN of noise-exposed rats.

Development of Neuronal Excitability of the Bushy Cells in Anteroventral Cochlear Nucleus of Rats.

The ultrafast and precise single-onset action potential (AP) of the bushy cells (BCs) in the anteroventral cochlear nucleus (AVCN) plays an important role in precise processing of temporal auditory information for localizing sound sources and communication cues. The specialized properties of high conductance of the low-voltage-activated potassium (K+LVA) channel contribute to generate ultrafast and precise single-onset APs in BCs. However, the developmental changes of K+LVA distribution and their contributions to shape neuronal excitability of BCs remain unclear. Therefore, we investigated the developmental changes in neuronal excitability of BCs and K+LVA distribution at different developmental periods. Using electrophysiological recording, we first characterized the firing pattern of BCs in response to a sequence of current injections at different developmental periods. The expression of the K+LVA subunit Kv1.1 in AVCN was examined with Western blot. The results indicated that BCs showed single-onset AP firing patterns and paused multiple APs firing patterns at the postnatal time of day 7 (P7) and were then refined into single-onset firing patterns at P14 and P21. With development, the active membrane properties, including latency and half-width of AP, and passive membrane properties, including capacitance, input resistance, and time constant, were significantly decreased. Furthermore, the refinement of firing patterns in BCs was correlated with the upregulation of the Kv1.1 channel in AVCN. In summary, the present study indicated that BCs optimize precise and single-onset firing with development, possibly driven by the changes in membrane properties and upregulation of Kv1.1 in AVCN.

Effects of long-term Doppler ultrasound exposure on cochlea and cochlear nucleus in prenatal period in an experimental model.

BACKGROUND: New generation Doppler ultrasonography (DUSG) application effects on cochlea and cochlear nucleus (CN) are unclear. We aimed to investigate the effects of new generation DUSG application at different frequencies in prenatal period on cochlea and CN in rats. OBJECTIVE: Twenty-four pregnant female rats were divided into three groups (n = 8). Group 1 was the control group and was not subjected to any treatment. Group 2 was determined as the USG every day (USGED) treatment group. Group 2 has received DUSG application every day from the 4th to 18th day (20 min/15 per day). Group 3 has received DUSG application as "2 days/one dose as every other day application" (USG2D1) from the 4th to 18th day (20 min/8 every other day). Twenty-four female rats were sacrificed in 21 days. Also, 24 pups were sacrificed after two days. First day after born, the cochlear activities of the right ears of all pups were examined using DPOAEs. Second day, neural tissues from CN were evaluated histopathologically and immunohistochemically. RESULTS: There was no any statistical difference between the groups in respect of histopathologically. USGED group showed mild caspase-3 positive neurons and glial cells. However, there was no significant difference between the USGED and other groups (p>.05). Similarly, the rats applied with USG2D1 had mild caspase-3 expression, but no significant difference between the USG2D1 and other groups (p>.05). Differences in DPOAE amplitudes, and therefore in cochlear activity, between the groups were revealed. The decrease in cochlear activity between the groups involved frequencies at 2, 8, 16, and 32 kHz (p<.05). CONCLUSIONS: Multiple administration of new generation DUSG to pregnant rats has not shown harmful effects on the cochlear neural tissue. High frequencies are more sensitive in cochlea to apply DUSG.

Kv1 channels regulate variations in spike patterning and temporal reliability in the avian cochlear nucleus angularis.

Diverse physiological phenotypes in a neuronal population can broaden the range of computational capabilities within a brain region. The avian cochlear nucleus angularis (NA) contains a heterogeneous population of neurons whose variation in intrinsic properties results in electrophysiological phenotypes with a range of sensitivities to temporally modulated input. The low-threshold potassium conductance (GKLT) is a key feature of neurons involved in fine temporal structure coding for sound localization, but a role for these channels in intensity or spectrotemporal coding has not been established. To determine whether GKLT affects the phenotypical variation and temporal properties of NA neurons, we applied dendrotoxin-I (DTX), a potent antagonist of Kv1-type potassium channels, to chick brain stem slices in vitro during whole cell patch-clamp recordings. We found a cell-type specific subset of NA neurons that was sensitive to DTX: single-spiking NA neurons were most profoundly affected, as well as a subset of tonic-firing neurons. Both tonic I (phasic onset bursting) and tonic II (delayed firing) neurons showed DTX sensitivity in their firing rate and phenotypical firing pattern. Tonic III neurons were unaffected. Spike time reliability and fluctuation sensitivity measured in DTX-sensitive NA neurons was also reduced with DTX. Finally, DTX reduced spike threshold adaptation in these neurons, suggesting that GKLT contributes to the temporal properties that allow coding of rapid changes in the inputs to NA neurons. These results suggest that variation in Kv1 channel expression may be a key factor in functional diversity in the avian cochlear nucleus.NEW & NOTEWORTHY The dendrotoxin-sensitive voltage-gated potassium conductance typically associated with neuronal coincidence detection in the timing pathway for sound localization is demonstrated to affect spiking patterns and temporal input sensitivity in the intensity pathway in the avian auditory brain stem. The Kv1-family channels appear to be present in a subset of cochlear nucleus angularis neurons, regulate spike threshold dynamics underlying high-pass membrane filtering, and contribute to intrinsic firing diversity.

Spontaneous Calcium Oscillations through Differentiation: A Calcium Imaging Analysis of Rat Cochlear Nucleus Neural Stem Cells.

Causal therapies for the auditory-pathway and inner-ear diseases are still not yet available for clinical application. Regenerative medicine approaches are discussed and examined as possible therapy options. Neural stem cells could play a role in the regeneration of the auditory pathway. In recent years, neural stem and progenitor cells have been identified in the cochlear nucleus, the second nucleus of the auditory pathway. The current investigation aimed to analyze cell maturation concerning cellular calcium activity. Cochlear nuclei from PND9 CD rats were microscopically dissected and propagated as neurospheres in free-floating cultures in stem-cell medium (Neurobasal, B27, GlutaMAX, EGF, bFGF). After 30 days, the dissociation and plating of these cells took place under withdrawal of the growth factors and the addition of retinoic acid, which induces neural cell differentiation. Calcium imaging analysis with BAPTA-1/Oregon Green was carried out at different times during the differentiation phase. In addition, the influence of different voltage-dependent calcium channels was analyzed through the targeted application of inhibitors of the L-, N-, R- and T-type calcium channels. For this purpose, comparative examinations were performed on CN NSCs, and primary CN neurons. As the cells differentiated, a significant increase in spontaneous neuronal calcium activity was demonstrated. In the differentiation stage, specific frequencies of the spontaneous calcium oscillations were measured in different regions of the individual cells. Initially, the highest frequency of spontaneous calcium oscillations was ascertainable in the maturing somata. Over time, these were overtaken by calcium oscillations in the axons and dendrites. Additionally, in the area of the growth cones, an increasing activity was determined. By inhibiting voltage-dependent calcium channels, their expression and function in the differentiation process were confirmed. A comparable pattern of maturation of these channels was found in CN NSCs and primary CN neurons. The present results show that neural stem cells of the rat cochlear nucleus differentiated not only morphologically but also functionally. Spontaneous calcium activities are of great relevance in terms of neurogenesis and integration into existing neuronal structures. These functional aspects of neurogenesis within the auditory pathway could serve as future targets for the exogenous control of neuronal regeneration.

Developmental shift to mitochondrial respiration for energetic support of sustained transmission during maturation at the calyx of Held.

A considerable amount of energy is expended following presynaptic activity to regenerate electrical polarization and maintain efficient release and recycling of neurotransmitter. Mitochondria are the major suppliers of neuronal energy, generating ATP via oxidative phosphorylation. However, the specific utilization of energy from cytosolic glycolysis rather than mitochondrial respiration at the presynaptic terminal during synaptic activity remains unclear and controversial. We use a synapse specialized for high-frequency transmission in mice, the calyx of Held, to test the sources of energy used to maintain energy during short activity bursts (<1 s) and sustained neurotransmission (30-150 s). We dissect the role of presynaptic glycolysis versus mitochondrial respiration by acutely and selectively blocking these ATP-generating pathways in a synaptic preparation where mitochondria and synaptic vesicles are prolific, under near-physiological conditions. Surprisingly, if either glycolysis or mitochondrial ATP production is intact, transmission during repetitive short bursts of activity is not affected. In slices from young animals before the onset of hearing, where the synapse is not yet fully specialized, both glycolytic and mitochondrial ATP production are required to support sustained, high-frequency neurotransmission. In mature synapses, sustained transmission relies exclusively on mitochondrial ATP production supported by bath lactate, but not glycolysis. At both ages, we observe that action potential propagation begins to fail before defects in synaptic vesicle recycling. Our data describe a specific metabolic profile to support high-frequency information transmission at the mature calyx of Held, shifting during postnatal synaptic maturation from glycolysis to rely on monocarboxylates as a fuel source.NEW & NOTEWORTHY We dissect the role of presynaptic glycolysis versus mitochondrial respiration in supporting high-frequency neurotransmission, by acutely blocking these ATP-generating pathways at a synapse tuned for high-frequency transmission. We find that massive energy expenditure is required to generate failure when only one pathway is inhibited. Action potential propagation is lost before impaired synaptic vesicle recycling. Synaptic transmission is exclusively dependent on oxidative phosphorylation in mature synapses, indicating presynaptic glycolysis may be dispensable for ATP maintenance.

Immunohistochemical distribution of calcium binding proteins in the cochlear nuclear complex of circling mice / Distribución inmunohistoquímica de proteínas de unión a calcio en el complejo nuclear coclear de ratones circulantes

SUMMARY: The term "circling mouse" refers to an animal model of deafness, in which the mouse exhibits circling, head tossing, and hyperactivity, with pathological features including degenerated spiral ganglion cells in the cochlea, and the loss of the organ of Corti. The cochlear nuclear (CN) complex, a part of the auditory brain circuit, is essential to process both ascending and descending auditory information. Considering calcium's (Ca2+) importance in homeostasis of numerous biological processes, hearing loss by cochlear damage, either by ablation or genetic defect, could cause changes in the Ca2+ concentration that might trigger functional and structural alterations in the auditory circuit. However, little is known about the correlation of the central nervous system (CNS) pathology in circling mice, especially of the auditory pathway circuit and Ca2+ changes. This present study investigates the distribution of Ca2+- binding proteins (CaBPs), calbindin D-28k (CB), parvalbumin (PV), and calretinin (CR) by using a free floating immunohistochemical method inthe CN of the wild-type mouse (+/+), the heterozygous mouse (+/cir), and the homozygous (cir/cir) mouse. CaBPs are well known to be an important factor that regulates Ca2+ concentrations. Compared with the dorsal and ventral cochlear nuclei of +/+ and +/ cirmice, prominent decreases of CaBPs' immunoreactivity (IR) in cir/cirmice were observed in the somas, as well as in the neuropil. The present study reportson the overall distribution and changes in the immunoreactivity of CaBPs in the CN of cir/cirmice because ofa hearing defect. This data might be helpful to morphologically elucidate CNS disorders and their relation to CaBPs immunoreactivity related to hearing defects.

RESUMEN: El término "ratón circulante" se refiere a un modelo animal con sordera, en el que el ratón exhibe hiperactividad, movimientos circulares y movimientos de la cabeza, con características patológicas que incluyen células ganglionares espirales degeneradas en la cóclea, un canal de Rosenthal vacío y la pérdida del órgano de Corti. El complejo nuclear coclear (CN), una parte del circuito cerebral auditivo, es esencial para procesar la información auditiva tanto ascendente como descendente. Considerando la importancia del calcio (Ca2+) en la homeostasis de numerosos procesos biológicos, la hipoacusia por daño coclear, por ablación o por defecto genético, podría provocar cambios en la concentración de Ca2+que pueden desencadenar alteraciones funcionales y estructurales en el circuitoauditivo. Sin embargo, existe poca información de la correlación de la patología del sistema nervioso central (SNC) en ratones circulantes, especialmente del circuito de la víaauditiva y los cambios de Ca2+. Este estudio nvestiga la distribución de proteínas de unión a Ca2+ (CaBP), calbindina D-28k (CB), parvalbúmina (PV) y calretinina (CR) mediante el uso de un método inmunohistoquímico de flotaciónlibre en el CN del ratón de tiposalvaje (+/+), el ratón heterocigoto (+/cir) y el ratón homocigoto (cir/cir). Se sabe que los CaBP son un factor importante que regula las concentraciones de Ca2+. En comparación con los núcleos cocleares dorsal y ventral de los ratones +/+ y +/ cir, se observaron disminuciones prominentes de la inmunorreactividad (IR) de CaBPs en los ratonescir/cir en los somas, asícomo en el neuropilo. El presente estudio informa sobre la distribución general y los cambios en la inmunorreactividad de CaBP en el CN de ratones cir/cir debido a un defecto auditivo. Estos datos podrían ser útiles para dilucidar morfológicamente los trastornos del SNC y su relación con la inmunorreactividad de CaBP relacionada con los defectosauditivos.

The Lbx1 lineage differentially contributes to inhibitory cell types of the dorsal cochlear nucleus, a cerebellum-like structure, and the cerebellum.

The dorsal cochlear nucleus (DCN) is a mammalian-specific nucleus of the auditory system. Anatomically, it is classified as a cerebellum-like structure. These structures are proposed to share genetic programs with the cerebellum. Previous analyses demonstrated that inhibitory serial sister cell types (SCTs) of the DCN and cerebellum are derived from the pancreatic transcription factor 1a (Ptf1a) lineage. Postmitotic neurons of the Ptf1a lineage often express the transcription factor Ladybird homeobox protein homolog 1 (Lbx1) which is involved in neuronal cell fate determination. Lbx1 is therefore an attractive candidate for a further component of the genetic program shared between the DCN and cerebellum. Here, we used cell-type specific marker analysis in combination with an Lbx1 reporter mouse line to analyze in both tissues which cell types of the Ptf1a lineage express Lbx1. In the DCN, stellate cells and Purkinje-like cartwheel cells were part of the Lbx1 lineage and Golgi cells were not, as determined by cell counts. In contrast, in the cerebellum, stellate cells and Golgi cells were part of the Lbx1 lineage and Purkinje cells were not. Hence, two out of three phenotypically similar cell types differed with respect to their Lbx1 expression. Our study demonstrates that Lbx1 is differentially recruited to the developmental genetic program of inhibitory neurons both within a given tissue and between the DCN and cerebellum. The differential expression of Lbx1 within the DCN and the cerebellum might contribute to the genetic individuation of the inhibitory SCTs to adapt to circuit specific tasks.

Nrg1/ErbB2 regulates differentiation and apoptosis of neural stem cells in the cochlear nucleus through PI3K/Akt pathway.

Sensorineural hearing loss (SNHL) is a common causes of disability. Neural stem cells (NSCs) from the cochlear nuclei have been considered to be a potential direction for the treatment of SNHL. Neuregulin 1 (NRG1)/ErbB2 signaling displays an essential role in nervous system development. In this study, we aimed to explore the roles of NRG1/ErbB2 in differentiation and apoptosis of cochlear nuclei NSCs. The data showed that the expression of NGR1 and ErbB2 in cochlear nuclei NSCs isolated from rats were increased with the age of rats. NRG1 treatment reduced the nestin-positive cells number, increased the MAP2-positive and GFAP-positive cells number, decreased the expression of cleaved-caspase-3, and increased the activation of PI3K/AKT. ErbB2 knockdown by lentiviral-mediated ErbB2 shRNA infection reversed the effect of NRG1 on cochlear nuclei NSCs. LY294002 administration further enhanced the effect of ErbB2 silencing on the expression of nestin, MAP2, GFAP and cleaved-caspase-3. Taken together, NRG1/ErbB2 regulates differentiation and apoptosis of cochlear nucleus NSCs through PI3K/Akt pathway.

KATP and TRPM2-like channels couple metabolic status to resting membrane potential of octopus neurons in the mouse ventral cochlear nucleus.

ATP-sensitive potassium (KATP) channels and transient receptor potential melastatin 2 (TRPM2) channels are commonly expressed both pre- and postsynaptically in the central nervous system (CNS). We hypothesized that KATP and TRPM2 may couple metabolic status to the resting membrane potential of octopus neurons of the mouse ventral cochlear nucleus (VCN). Therefore, we studied the expression of KATP channels and TRPM2 channels in octopus cells by immunohistochemical techniques and their contribution to neuronal electrical properties by the electrophysiological patch clamp technique. In immunohistochemical staining of octopus cells, labelling with Kir6.2 and SUR1 antibodies was strong, and labelling with the SUR2 antibody was moderate, but labelling with Kir6.1 was very weak. Octopus cells had intense staining with TRPM2 antibodies. In patch clamp recordings, bath application of KATP channel agonists H2O2 (880 µM), ATZ (1 mM), cromakalim (50 µM), diazoxide (200 µM), NNC 55-0118 and NN 414 separately resulted in hyperpolarizations of resting potential to different extents. Application of 8-Bro-cADPR (50 µM), a specific antagonist of TRPM2 channels, in the presence of H2O2 (880 µM) resulted in further hyperpolarization by approximately 1 mV. The amplitudes of H2O2-induced outward KATP currents and ADPR-induced inward currents were 206.1 ± 31.5 pA (n = 4) and 136.8 ± 22.4 pA, respectively, at rest. Their respective reversal potentials were -77 ± 2.6 mV (n = 3) and -6.3 ± 2.9 (n = 3) and -6.3 ± 2.9 (n = 3). In conclusion, octopus cells appear to possess both KATP channels and TRPM2-like channels. KATP might largely be constituted by SUR1-Kir6.2 subunits and SUR2-Kir6.2 subunits. Both KATP and TRPM2-like channels might have a modulatory action in setting the membrane potential.

Outer Hair Cell Glutamate Signaling through Type II Spiral Ganglion Afferents Activates Neurons in the Cochlear Nucleus in Response to Nondamaging Sounds.

Cochlear outer hair cells (OHCs) are known to uniquely participate in auditory processing through their electromotility, and like inner hair cells, are also capable of releasing vesicular glutamate onto spiral ganglion (SG) neurons: in this case, onto the sparse Type II SG neurons. However, unlike glutamate signaling at the inner hair cell-Type I SG neuron synapse, which is robust across a wide spectrum of sound intensities, glutamate signaling at the OHC-Type II SG neuron synapse is weaker and has been hypothesized to occur only at intense, possibly damaging sound levels. Here, we tested the ability of the OHC-Type II SG pathway to signal to the brain in response to moderate, nondamaging sound (80 dB SPL) as well as to intense sound (115 dB SPL). First, we determined the VGluTs associated with OHC signaling and then confirmed the loss of glutamatergic synaptic transmission from OHCs to Type II SG neurons in KO mice using dendritic patch-clamp recordings. Next, we generated genetic mouse lines in which vesicular glutamate release occurs selectively from OHCs, and then assessed c-Fos expression in the cochlear nucleus in response to sound. From these analyses, we show, for the first time, that glutamatergic signaling at the OHC-Type II SG neuron synapse is capable of activating cochlear nucleus neurons, even at moderate sound levels.SIGNIFICANCE STATEMENT Evidence suggests that cochlear outer hair cells (OHCs) release glutamate onto Type II spiral ganglion neurons only when exposed to loud sound, and that Type II neurons are activated by tissue damage. Knowing whether moderate level sound, without tissue damage, activates this pathway has functional implications for this fundamental auditory pathway. We first determined that OHCs rely largely on VGluT3 for synaptic glutamate release. We then used a genetically modified mouse line in which OHCs, but not inner hair cells, release vesicular glutamate to demonstrate that moderate sound exposure activates cochlear nucleus neurons via the OHC-Type II spiral ganglion pathway. Together, these data indicate that glutamate signaling at the OHC-Type II afferent synapse participates in auditory function at moderate sound levels.

Expression pattern of cochlear microRNAs in the mammalian auditory hindbrain.

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.

Neuromodulation by mGluRs in Sound Localization Circuits in the Auditory Brainstem.

The ability of humans and animals to localize the source of a sound in a complex acoustic environment facilitates communication and survival. Two cues are used for sound localization at horizontal planes, interaural time and level differences (ITD and ILD), which are analyzed by distinct neural circuits in the brainstem. Here, we review the studies on metabotropic glutamate receptor (mGluR)-mediated neuromodulation of both intrinsic and synaptic properties of brainstem neurons in these circuits. Both mammalian and avian animal models have been used, with each having their advantages that are not present in the other. For the mammalian model, we discuss mGluR neuromodulation in the ILD circuit, with an emphasis on the recent discovery of differential modulation of synaptic transmission of different transmitter release modes. For the avian model, we focus on reviewing mGluR neuromodulation in the ITD pathway, with an emphasis on tonotopic distribution and synaptic plasticity of mGluR modulation in coincidence detector neurons. Future works are proposed to further investigate the functions and mechanisms of mGluRs in the sound localization circuits.