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.

Structural and Functional Refinement of the Axon Initial Segment in Avian Cochlear Nucleus during Development.

The axon initial segment (AIS) is involved in action potential initiation. Structural and biophysical characteristics of the AIS differ among cell types and/or brain regions, but the underlying mechanisms remain elusive. Using immunofluorescence and electrophysiological methods, combined with super-resolution imaging, we show in the developing nucleus magnocellularis of the chicken in both sexes that the AIS is refined in a tonotopic region-dependent manner. This process of AIS refinement differs among cells tuned to different frequencies. At hearing onset, the AIS was ∼50 µm long with few voltage-gated sodium channels regardless of tonotopic region. However, after hatching, the AIS matured and displayed an ∼20-µm-long structure with a significant enrichment of sodium channels responsible for an increase in sodium current and a decrease in spike threshold. Moreover, the shortening was more pronounced, while the accumulation of channels was not, in neurons tuned to higher frequency, creating tonotopic differences in the AIS. We conclude that AIS shortening is mediated by disassembly of the cytoskeleton at the distal end of the AIS, despite intact periodicity of the submembranous cytoskeleton across the AIS. Importantly, deprivation of afferent input diminished the shortening in neurons tuned to a higher frequency to a larger extent in posthatch animals, with little effect on the accumulation of sodium channels. Thus, cytoskeletal reorganization and sodium channel enrichment at the AIS are differentially regulated depending on tonotopic region, but work synergistically to optimize neuronal output in the auditory nucleus.SIGNIFICANCE STATEMENT The axon initial segment (AIS) plays fundamental roles in determining neuronal output. The AIS varies structurally and molecularly across tonotopic regions in avian cochlear nucleus. However, the mechanism underlying these variations remains unclear. The AIS is immature around hearing onset, but becomes shorter and accumulates more sodium channels during maturation, with a pronounced shortening and a moderate channel accumulation at higher tonotopic regions. Afferent input adjusts sodium conductance at the AIS by augmenting AIS shortening (via disassembly of cytoskeletons at its distal end) specifically at higher-frequency regions. However, this had little effect on channel accumulation. Thus, cytoskeletal structure and sodium channel accumulation at the AIS are regulated differentially but work synergistically to optimize the neuronal output.

Morphological development of the human cochlear nucleus.

Morphological studies in developing brain determine critical periods of proliferation, neurogenesis, gliogenesis, and apoptosis. During these periods both intrinsic and extrinsic pathological factors can hamper development. These time points are not available for the human cochlear nucleus (CN). We have used design-based stereology and determined that 18-22 weeks of gestation (WG) are critical in the development of the human CN. Twenty-three fetuses and seven postnatal brainstems were processed for cresyl violet (CV) staining and immunoexpression of NeuN (neurons), GFAP (astrocytes), Ki-67 (proliferation) and TUNEL (apoptosis) and 3-D reconstruction. The volume of CN, total number of neurons selected profiles and the volume of neurons and their nuclei were estimated. Data were grouped (G) into: G1:18-20 WG, G2: 21-24 WG, G3: 25-28 WG and G4 >29 WG. The dimensions of morphologically identified neurons were also measured. The CN primordium was first identifiable at 10WG. Definitive DCN (Dorsal cochlear nucleus) and VCN (ventral cochlear nucleus) were identifiable at 16 WG. There was a sudden growth spurt in total volume of CN, number of neurons and astrocytes from 18 WG. We also observed an increase in proliferation and apoptosis after 22 WG. The number of neurons identifiable by CV was significantly lower than that by NeuN-immunostaining till 25 WG (p = 0.020), after which, both methods were equivalent. Eight morphological types of neurons were identifiable by 26 WG and could be resolved into four clusters by volume and diameter. The CN changed orientation from small, flat and horizontal at 10-16 WG to larger and oblique from 18WG onwards. Prevention of exposure to noxious factors at 18-22 WG may be important in preventing congenital deafness.

Relationships between neuronal birthdates and tonotopic positions in the mouse cochlear nucleus.

Tonotopy is a key anatomical feature of the vertebrate auditory system, but little is known about the mechanisms underlying its development. Since date of birth of a neuron correlates with tonotopic position in the cochlea, we investigated if it also correlates with tonotopic position in the cochlear nucleus (CN). In the cochlea, spiral ganglion neurons are organized in a basal to apical progression along the length of the cochlea based on birthdates, with neurons in the base (responding to high-frequency sounds) born early around mouse embryonic day (E) 9.5-10.5, and those in the apex (responding to low-frequency sounds) born late around E12.5-13.5. Using a low-dose thymidine analog incorporation assay, we examine whether CN neurons are arranged in a spatial gradient according to their birthdates. Most CN neurons are born between E10.5 and E13.5, with a peak at E12.5. A second wave of neuron birth was observed in the dorsal cochlear nucleus (DCN) beginning on E14.5 and lasts until E18.5. Large excitatory neurons were born in the first wave, and small local circuit neurons were born in the second. No spatial gradient of cell birth was observed in the DCN. In contrast, neurons in the anteroventral cochlear nucleus (AVCN) were found to be arranged in a dorsal to ventral progression according to their birthdates, which are aligned with the tonotopic axis. Most of these AVCN neurons are endbulb-innervated bushy cells. The correlation between birthdate and tonotopic position suggests testable mechanisms for specification of tonotopic position.

Cholinergic innervation of principal neurons in the cochlear nucleus of the Mongolian gerbil.

Principal neurons in the ventral cochlear nucleus (VCN) receive powerful ascending excitation and pass on the auditory information with exquisite temporal fidelity. Despite being dominated by ascending inputs, the VCN also receives descending cholinergic connections from olivocochlear neurons and from higher regions in the pontomesencephalic tegmentum. In Mongolian gerbils, acetylcholine acts as an excitatory and modulatory neurotransmitter on VCN neurons, but the anatomical structure of cholinergic innervation of gerbil VCN is not well described. We applied fluorescent immunohistochemical staining to elucidate the development and the cellular localization of presynaptic and postsynaptic components of the cholinergic system in the VCN of the Mongolian gerbil. We found that cholinergic fibers (stained with antibodies against the vesicular acetylcholine transporter) were present before hearing onset at P5, but innervation density increased in animals after P10. Early in development cholinergic fibers invaded the VCN from the medial side, spread along the perimeter and finally innervated all parts of the nucleus only after the onset of hearing. Cholinergic fibers ran in a rostro-caudal direction within the nucleus and formed en-passant swellings in the neuropil between principal neurons. Nicotinic and muscarinic receptors were expressed differentially in the VCN, with nicotinic receptors being mostly expressed in dendritic areas while muscarinic receptors were located predominantly in somatic membranes. These anatomical data support physiological indications that cholinergic innervation plays a role in modulating information processing in the cochlear nucleus.

Auditory Input Shapes Tonotopic Differentiation of Kv1.1 Expression in Avian Cochlear Nucleus during Late Development.

Tonotopic differentiation is fundamental for signal processing in the auditory system. However, when and how this differentiation arises remain elusive. We addressed this issue using electrophysiology and immunohistochemistry in nucleus magnocellularis of chickens of both sexes, which is known to differ in the expression of Kv1.1 channels depending on characteristic frequency (CF). Just after hearing onset (embryonic day 12-14), Kv1 current gradually increased to a slightly larger extent in neurons with higher CF, causing a tonotopic difference of Kv1 current before hatch. However, after hatch, a much larger increase of Kv1 current occurred, particularly in higher-CF neurons, due to an augmentation of Kv1.1 expression at the plasma membrane. This later change in expression led to the large tonotopic difference of Kv1 current characteristic of mature animals. Attenuation of auditory input by inducing conductive or sensorineural hearing loss around hatch suppressed the differentiation in a level-dependent manner. Moreover, elevation of auditory input during embryonic periods could not reproduce the differentiation, suggesting that the capacity of neurons to drive Kv1.1 expression via auditory input develops in a cell-specific manner, thus underlying the frequency-specific expression of the channel within the nucleus. The results indicated that the tonotopic differentiation of Kv1.1 in nucleus magnocellularis is partially determined before hatch, but largely driven by afferent input after hatch. Our results highlight the importance of neuronal capacity for sound to drive ion channel expression as well as the level of auditory experience in the frequency tuning of brainstem auditory circuits.SIGNIFICANCE STATEMENT Tuning-frequency-specific expression of ion channels is a prerequisite for auditory system function, but its underlying mechanisms remain unclear. Here, we revealed in avian cochlear nucleus that the expression of Kv1.1 became more dependent on auditory input at a late period of maturation in neurons tuned to higher-frequency sound, leading to frequency-specific Kv1.1 expression. Attenuation of auditory input during this period suppressed the differentiation in a level-dependent manner, whereas elevation of input in earlier periods could not reproduce the differentiation. Thus, the capacity of neurons to drive Kv1.1 expression via auditory input develops in a cell-specific manner and directs differentiation, highlighting the importance of neuronal character as well as the level of input in the frequency tuning of auditory circuits.

miR-96 is required for normal development of the auditory hindbrain.

The peripheral deafness gene Mir96 is expressed in both the cochlea and central auditory circuits. To investigate whether it plays a role in the auditory system beyond the cochlea, we characterized homozygous Dmdo/Dmdo mice with a point mutation in miR-96. Anatomical analysis demonstrated a significant decrease in volume of auditory nuclei in Dmdo/Dmdo mice. This decrease resulted from decreased cell size. Non-auditory structures in the brainstem of Dmdo/Dmdo mice or auditory nuclei of the congenital deaf Cldn14-/- mice revealed no such differences. Electrophysiological analysis in the medial nucleus of the trapezoid body (MNTB) showed that principal neurons fired preferentially multiple action potentials upon depolarization, in contrast to the single firing pattern prevalent in controls and Cldn14-/- mice. Immunohistochemistry identified significantly reduced expression of two predicted targets of the mutated miR-96, Kv1.6 and BK channel proteins, possibly contributing to the electrophysiological phenotype. Microscopic analysis of the Dmdo/Dmdo calyx of Held revealed a largely absent compartmentalized morphology, as judged by SV2-labeling. Furthermore, MNTB neurons from Dmdo/Dmdo mice displayed larger synaptic short-term depression, slower AMPA-receptor decay kinetics and a larger NMDA-receptor component, reflecting a less matured stage. Again, these synaptic differences were not present between controls and Cldn14-/- mice. Thus, deafness genes differentially affect the auditory brainstem. Furthermore, our study identifies miR-96 as an essential gene regulatory network element of the auditory system which is required for functional maturation in the peripheral and central auditory system alike.

Distinct Neural Properties in the Low-Frequency Region of the Chicken Cochlear Nucleus Magnocellularis.

Topography in the avian cochlear nucleus magnocellularis (NM) is represented as gradually increasing characteristic frequency (CF) along the caudolateral-to-rostromedial axis. In this study, we characterized the organization and cell biophysics of the caudolateral NM (NMc) in chickens (Gallus gallus). Examination of cellular and dendritic architecture first revealed that NMc contains small neurons and extensive dendritic processes, in contrast to adendritic, large neurons located more rostromedially. Individual dye-filling study further demonstrated that NMc is divided into two subregions, with NMc2 neurons having larger and more complex dendritic fields than NMc1. Axonal tract tracing studies confirmed that NMc1 and NMc2 neurons receive afferent inputs from the auditory nerve and the superior olivary nucleus, similar to the adendritic NM. However, the auditory axons synapse with NMc neurons via small bouton-like terminals, unlike the large end bulb synapses on adendritic NM neurons. Immunocytochemistry demonstrated that most NMc2 neurons express cholecystokinin but not calretinin, distinct from NMc1 and adendritic NM neurons that are cholecystokinin negative and mostly calretinin positive. Finally, whole-cell current clamp recordings revealed that NMc neurons require significantly lower threshold current for action potential generation than adendritic NM neurons. Moreover, in contrast to adendritic NM neurons that generate a single-onset action potential, NMc neurons generate multiple action potentials to suprathreshold sustained depolarization. Taken together, our data indicate that NMc contains multiple neuron types that are structurally, connectively, molecularly, and physiologically different from traditionally defined NM neurons, emphasizing specialized neural properties for processing low-frequency sounds.

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.

Tonotopic action potential tuning of maturing auditory neurons through endogenous ATP.

KEY POINTS: Following the genetically controlled formation of neuronal circuits, early firing activity guides the development of sensory maps in the auditory, visual and somatosensory system. However, it is not clear whether the activity of central auditory neurons is specifically regulated depending on the position within the sensory map. In the ventral cochlear nucleus, the first central station along the auditory pathway, we describe a mechanism through which paracrine ATP signalling enhances firing in a cell-specific and tonotopically-determined manner. Developmental down-regulation of P2X2/3R currents along the tonotopic axis occurs simultaneously with an increase in AMPA receptor currents, suggesting a high-to-low frequency maturation pattern. Facilitated action potential (AP) generation, measured as higher firing rate, shorter EPSP-AP delay in vivo and shorter AP latency in slice experiments, is consistent with increased synaptic efficacy caused by ATP. The long lasting change in intrinsic neuronal excitability is mediated by the heteromeric P2X2/3 receptors. ABSTRACT: Synaptic refinement and strengthening are activity-dependent processes that establish orderly arranged cochleotopic maps throughout the central auditory system. The maturation of auditory brainstem circuits is guided by action potentials (APs) arising from the inner hair cells in the developing cochlea. The AP firing of developing central auditory neurons can be modulated by paracrine ATP signalling, as shown for the cochlear nucleus bushy cells and principal neurons in the medial nucleus of the trapezoid body. However, it is not clear whether neuronal activity may be specifically regulated with respect to the nuclear tonotopic position (i.e. sound frequency selectivity). Using slice recordings before hearing onset and in vivo recordings with iontophoretic drug applications after hearing onset, we show that cell-specific purinergic modulation follows a precise tonotopic pattern in the ventral cochlear nucleus of developing gerbils. In high-frequency regions, ATP responsiveness diminished before hearing onset. In low-to-mid frequency regions, ATP modulation persisted after hearing onset in a subset of low-frequency bushy cells (characteristic frequency< 10 kHz). Down-regulation of P2X2/3R currents along the tonotopic axis occurs simultaneously with an increase in AMPA receptor currents, thus suggesting a high-to-low frequency maturation pattern. Facilitated AP generation, measured as higher firing frequency, shorter EPSP-AP delay in vivo, and shorter AP latency in slice experiments, is consistent with increased synaptic efficacy caused by ATP. Finally, by combining recordings and pharmacology in vivo, in slices, and in human embryonic kidney 293 cells, it was shown that the long lasting change in intrinsic neuronal excitability is mediated by the P2X2/3R.

Effects of brain-derived neurotrophic factor (BDNF) on the cochlear nucleus in cats deafened as neonates.

Many previous studies have shown significant neurotrophic effects of intracochlear delivery of BDNF in preventing degeneration of cochlear spiral ganglion (SG) neurons after deafness in rodents and our laboratory has shown similar results in developing cats deafened prior to hearing onset. This study examined the morphology of the cochlear nucleus (CN) in a group of neonatally deafened cats from a previous study in which infusion of BDNF elicited a significant improvement in survival of the SG neurons. Five cats were deafened by systemic injections of neomycin sulfate (60 mg/kg, SQ, SID) starting one day after birth, and continuing for 16-18 days until auditory brainstem response (ABR) testing demonstrated profound bilateral hearing loss. The animals were implanted unilaterally at about 1 month of age using custom-designed electrodes with a drug-delivery cannula connected to an osmotic pump. BDNF (94 µg/ml; 0.25 µl/hr) was delivered for 10 weeks. The animals were euthanized and studied at 14-23 weeks of age. Consistent with the neurotrophic effects of BDNF on SG survival, the total CN volume in these animals was significantly larger on the BDNF-treated side than on the contralateral side. However, total CN volume, both ipsi- and contralateral to the implants in these deafened juvenile animals, was markedly smaller than the CN in normal adult animals, reflecting the severe effects of deafness on the central auditory system during development. Data from the individual major CN subdivisions (DCN, Dorsal Cochlear Nucleus; PVCN, Posteroventral Cochlear Nucleus; AVCN, Anteroventral Cochlear Nucleus) also were analyzed. A significant difference was observed between the BDNF-treated and control sides only in the AVCN. Measurements of the cross-sectional areas of spherical cells showed that cells were significantly larger in the AVCN ipsilateral to the implant than on the contralateral side. Further, the numerical density of spherical cells was significantly lower in the AVCN ipsilateral to the implant than on the contralateral side, consistent with the larger AVCN volume observed with BDNF treatment. Together, findings indicate significant neurotrophic effects of intracochlear BDNF infusion on the developing CN.

Tonotopic Optimization for Temporal Processing in the Cochlear Nucleus.

UNLABELLED: In the auditory system, sounds are processed in parallel frequency-tuned circuits, beginning in the cochlea. Auditory nerve fibers reflect this tonotopy and encode temporal properties of acoustic stimuli by "locking" discharges to a particular stimulus phase. However, physiological constraints on phase-locking depend on stimulus frequency. Interestingly, low characteristic frequency (LCF) neurons in the cochlear nucleus improve phase-locking precision relative to their auditory nerve inputs. This is proposed to arise through synaptic integration, but the postsynaptic membrane's selectivity for varying levels of synaptic convergence is poorly understood. The chick cochlear nucleus, nucleus magnocellularis (NM), exhibits tonotopic distribution of both input and membrane properties. LCF neurons receive many small inputs and have low input thresholds, whereas high characteristic frequency (HCF) neurons receive few, large synapses and require larger currents to spike. NM therefore presents an opportunity to study how small membrane variations interact with a systematic topographic gradient of synaptic inputs. We investigated membrane input selectivity and observed that HCF neurons preferentially select faster input than their LCF counterparts, and that this preference is tolerant of changes to membrane voltage. We then used computational models to probe which properties are crucial to phase-locking. The model predicted that the optimal arrangement of synaptic and membrane properties for phase-locking is specific to stimulus frequency and that the tonotopic distribution of input number and membrane excitability in NM closely tracks a stimulus-defined optimum. These findings were then confirmed physiologically with dynamic-clamp simulations of inputs to NM neurons. SIGNIFICANCE STATEMENT: One way that neurons represent temporal information is by phase-locking, which is discharging in response to a particular phase of the stimulus waveform. In the auditory system, central neurons are optimized to retain or improve phase-locking precision compared with input from the auditory nerve. However, the difficulty of this computation varies systematically with stimulus frequency. We examined properties that contribute to temporal processing both physiologically and in a computational model. Neurons processing low-frequency input benefit from integration of many weak inputs, whereas those processing higher frequencies progressively lose precision by integration of multiple inputs. Here, we reveal general features of input-output optimization that apply to all neurons that process time varying input.

Bhlhb5::flpo allele uncovers a requirement for Bhlhb5 for the development of the dorsal cochlear nucleus.

Auditory information is initially processed in the cochlear nuclei before being relayed to the brain. The cochlear nuclei are subdivided into dorsal, anterior ventral, and posterior ventral domains, each containing several subtypes of neurons that are thought to play discrete roles in the processing of sound. However, the ontogeny of these neurons is poorly understood, and this gap in knowledge hampers efforts to understand the basic neural circuitry of this nucleus. Here, we reveal that Bhlhb5 is expressed in both excitatory (unipolar brush cells) and inhibitory neurons (cartwheel cells) of the DCN during development. To gain genetic access to Bhlhb5-expressing neurons in the DCN, we generated a Bhlhb5::flpo knockin allele. Using an intersectional genetic strategy, we labeled cartwheel cells, thereby providing proof of concept that subpopulations of Bhlhb5-expressing neurons can be genetically targeted. Moreover, fate-mapping experiments using this allele revealed that Bhlhb5 is required for the proper development of the DCN, since mice lacking Bhlhb5 showed a dramatically diminished number of neurons, including unipolar brush and cartwheel cells. Intriguingly, the Bhlhb5::flpo allele also genetically labels numerous other regions of the nervous system that process sensory input, including the dorsal horn, the retina, and the nucleus of the lateral olfactory tract, hinting at a more general role for Bhlhb5 in the development of neurons that mediate sensory integration.

Induction of single-sided deafness in the newborn rat and its consequence for cochlear nucleus volume development.

Aim of this study was to induce a single-sided deafness (SSD) in rats before hearing onset. Rats were operated at postnatal day 10 by approaching the tympanic cavity along a retroauricular path without manipulating ossicles or tympanic membrane. The ototoxic aminoglycoside neomycin was injected intracochlearly through the round window membrane on one side. When the animals have reached young adult stages, their hearing threshold was determined by their auditory brainstem response (ABR). Monaural deafening was considered successful when the hearing threshold was at least 95 dB above the threshold of the normal hearing ear. Growing up with one non-functional ear, rats developed a striking anatomical asymmetry of their cochlear nuclei (CN). The CN from age-matched normal hearing brains and from both sides of single-sided deaf brains were cut into series of frontal sections and their volumes calculated. No difference was detected between the volume of the normal hearing CN and the contralateral CN in SSD rats. By contrast, growth retardation was found for the ventral CN on the deaf side to result in a volume of only 57% compared to the normal hearing side. Marginal growth retardation was also observed for the dorsal CN on the deaf side. Thus, loss of sensory activation leads mainly, but not exclusively, to a reduction of tissue volume in the ventral CN of the deaf side, leaving the contralateral side apparently unaffected.

Selective deletion of cochlear hair cells causes rapid age-dependent changes in spiral ganglion and cochlear nucleus neurons.

During nervous system development, critical periods are usually defined as early periods during which manipulations dramatically change neuronal structure or function, whereas the same manipulations in mature animals have little or no effect on the same property. Neurons in the ventral cochlear nucleus (CN) are dependent on excitatory afferent input for survival during a critical period of development. Cochlear removal in young mammals and birds results in rapid death of target neurons in the CN. Cochlear removal in older animals results in little or no neuron death. However, the extent to which hair-cell-specific afferent activity prevents neuronal death in the neonatal brain is unknown. We further explore this phenomenon using a new mouse model that allows temporal control of cochlear hair cell deletion. Hair cells express the human diphtheria toxin (DT) receptor behind the Pou4f3 promoter. Injections of DT resulted in nearly complete loss of organ of Corti hair cells within 1 week of injection regardless of the age of injection. Injection of DT did not influence surrounding supporting cells directly in the sensory epithelium or spiral ganglion neurons (SGNs). Loss of hair cells in neonates resulted in rapid and profound neuronal loss in the ventral CN, but not when hair cells were eliminated at a more mature age. In addition, normal survival of SGNs was dependent on hair cell integrity early in development and less so in mature animals. This defines a previously undocumented critical period for SGN survival.

The Structural Development of the Mouse Dorsal Cochlear Nucleus.

The dorsal cochlear nucleus (DCN) is a major subdivision of the mammalian cochlear nucleus (CN) that is thought to be involved in sound localization in the vertical plane and in feature extraction of sound stimuli. The main principal cell type (pyramidal cells) integrates auditory and non-auditory inputs, which are considered to be important in performing sound localization tasks. This study aimed to investigate the histological development of the CD-1 mouse DCN, focussing on the postnatal period spanning the onset of hearing (P12). Fluorescent Nissl staining revealed that the three layers of the DCN were identifiable as early as P6 with subsequent expansion of all layers with age. Significant increases in the size of pyramidal and cartwheel cells were observed between birth and P12. Immunohistochemistry showed substantial changes in synaptic distribution during the first two postnatal weeks with subsequent maturation of the presumed mossy fibre terminals. In addition, GFAP immunolabelling identified several glial cell types in the DCN including the observation of putative tanycytes for the first time. Each glial cell type had specific spatial and temporal patterns of maturation with apparent rapid development during the first two postnatal weeks but little change thereafter. The rapid maturation of the structural organization and DCN components prior to the onset of hearing possibly reflects an influence from spontaneous activity originating in the cochlea/auditory nerve. Further refinement of these connections and development of the non-auditory connections may result from the arrival of acoustic input and experience dependent mechanisms.

Postnatal expression of neurotrophic factors accessible to spiral ganglion neurons in the auditory system of adult hearing and deafened rats.

Spiral ganglion neurons (SGNs) receive input from cochlear hair cells and project from the cochlea to the cochlear nucleus. After destruction of hair cells with aminoglycoside antibiotics or noise, SGNs gradually die. It has been assumed that SGN death is attributable to loss of neurotrophic factors (NTFs) derived from hair cells or supporting cells in the organ of Corti (OC). We used quantitative PCR (qPCR) to assay NTF expression-neurotrophin-3 (NT-3), BDNF, GDNF, neurturin, artemin, and CNTF-in the OC and cochlear nucleus at various ages from postnatal day 0 (P0) to P90 in control hearing and neonatally deafened rats. NT-3, neurturin, and CNTF were most abundant in the postnatal hearing OC; CNTF and neurturin most abundant in the cochlear nucleus. In the OC, NT-3 and CNTF showed a postnatal increase in expression approximately concomitant with hearing onset. In rats deafened by daily kanamycin injections (from P8 to P16), surviving inner hair cells were evident at P16 but absent by P19, with most postsynaptic boutons lost before P16. NT-3 and CNTF, which normally increase postnatally, had significantly reduced expression in the OC of deafened rats, although CNTF was expressed throughout the time that SGNs were dying. In contrast, neurturin expression was constant, unaffected by deafening or by age. CNTF and neurturin expression in the cochlear nucleus was unaffected by deafening or age. Thus, NTFs other than NT-3 are available to SGNs even as they are dying after deafening, apparently conflicting with the hypothesis that SGN death is attributable to lack of NTFs.

Distribution of glial cells in the auditory brainstem: normal development and effects of unilateral lesion.

Auditory brainstem networks facilitate sound source localization through binaural integration. A key component of this circuitry is the projection from the ventral cochlear nucleus (VCN) to the medial nucleus of the trapezoid body (MNTB), a relay nucleus that provides inhibition to the superior olivary complex. This strictly contralateral projection terminates in the large calyx of Held synapse. The formation of this pathway requires spatiotemporal coordination of cues that promote cell maturation, axon growth, and synaptogenesis. Here we have examined the emergence of distinct classes of glial cells, which are known to function in development and in response to injury. Immunofluorescence for several astrocyte markers revealed unique expression patterns. Aldehyde dehydrogenase 1 family member L1 (ALDH1L1) was expressed earliest in both nuclei, followed by S100ß, during the first postnatal week. Glial fibrillary acidic protein (GFAP) expression was seen in the second postnatal week. GFAP-positive cell bodies remained outside the boundaries of VCN and MNTB, with a limited number of labeled fibers penetrating into the margins of the nuclei. Oligodendrocyte transcription factor 2 (OLIG2) expression revealed the presence of oligodendrocytes in VCN and MNTB from birth until after hearing onset. In addition, ionized calcium binding adaptor molecule 1 (IBA1)-positive microglia were observed after the first postnatal week. Following hearing onset, all glial populations were found in MNTB. We then determined the distribution of glial cells following early (P2) unilateral cochlear removal, which results in formation of ectopic projections from the intact VCN to ipsilateral MNTB. We found that following perturbation, astrocytic markers showed expression near the ectopic ipsilateral calyx. Taken together, the developmental expression patterns are consistent with a role for glial cells in the maturation of the calyx of Held and suggest that these cells may have a similar role in maturation of lesion-induced connections.

Activity-dependent and activity-independent development of the axon initial segment.

The axon initial segment (AIS) is the site of spike initiation in neurons. Previous studies revealed that spatial distribution of the AIS varies greatly among neurons to meet their specific needs. However, when and how this differentiation arises is unknown. Neurons in the avian nucleus laminaris (NL) are binaural coincidence detectors for sound localization and show differentiation in the distribution of the AIS, with shorter length and a more distal position from the soma with an increase in tuning frequency. We studied these characteristics of the AIS in NL neurons of the chicken during development and found that the AIS differentiates in its distribution after initial formation, and this is driven by activity-dependent and activity-independent mechanisms that differentially regulate distal and proximal boundaries of the AIS. Before hearing onset, the ankyrinG-positive AIS existed at a wide stretch of proximal axon regardless of tuning frequency, but Na+ channels were only partially distributed within the AIS. Shortly after hearing onset, Na+ channels accumulated along the entire AIS, which started shortening and relocating distally to a larger extent in neurons with higher tuning frequencies. Ablation of inner ears abolished the shortening of the AIS without affecting the position of its proximal boundary, indicating that both distal and proximal AIS boundaries are disassembled during development, and the former is dependent on afferent activity. Thus, interaction of these activity-dependent and activity-independent mechanisms determines the cell-specific distribution of the AIS in NL neurons and plays a critical role in establishing the function of sound localization circuit.

Depolarizing chloride gradient in developing cochlear nucleus neurons: underlying mechanism and implication for calcium signaling.

Precise regulation of the chloride homeostasis crucially determines the action of inhibitory transmitters GABA and glycine and thereby endows neurons or even discrete neuronal compartments with distinct physiological responses to the same transmitters. In mammals, the signaling mediated by GABAA/glycine receptors shifts during early postnatal life from depolarization to hyperpolarization, due to delayed maturation of the chloride homeostasis system. While the activity of the secondary active, K(+)-Cl(-)-extruding cotransporter KCC2, renders GABA/glycine hyperpolarizing in auditory brainstem nuclei of altricial rodents, the mechanisms contributing to the initially depolarizing transmembrane gradient for Cl(-) in respective neurons remained unknown. Here we used gramicidin-perforated patch recordings, non-invasive Cl(-) and Ca(2+) imaging, and immunohistochemistry to identify the Cl(-)-loading transporter that renders depolarizing effects of GABA/glycine in early postnatal life of spherical bushy cells in the cochlear nucleus of gerbil. Our data identify the 1Na(+):1K(+):2Cl(-) cotransporter 1 (NKCC1) as the major Cl(-)-loader responsible for depolarizing action of GABA/glycine at postnatal days 3-5 (P3-5). Extracellular GABA/muscimol elicited calcium signaling through R-, L-, and T-type channels, which was dependent on bumetanide- and [Na(+)]e-sensitive Cl(-) accumulation. The "adult like", low intracellular Cl(-) concentration is established during the second postnatal week, through a mechanism engaging the NKCC1-down regulation between P5 and P15 and ongoing KCC2-mediated Cl(-)-extrusion.