Resurgent sodium current promotes action potential firing in the avian auditory brainstem.

KEY POINTS: Auditory brainstem neurons of all vertebrates fire phase-locked action potentials (APs) at high rates with remarkable fidelity, a process controlled by specialized anatomical and biophysical properties. This is especially true in the avian nucleus magnocellularis (NM) - the analogue of the mammalian anteroventral cochlear nucleus. In addition to high voltage-activated potassium (KHVA ) channels, we report, using whole cell physiology and modelling, that resurgent sodium current (INaR ) of sodium channels (NaV ) is equally important and operates synergistically with KHVA channels to enable rapid AP firing in NM. Anatomically, we detected strong NaV 1.6 expression near hearing maturation, which was less distinct during hearing development despite functional evidence of INaR , suggesting that multiple NaV channel subtypes may contribute to INaR . We conclude that INaR plays an important role in regulating rapid AP firing for NM neurons, a property that may be evolutionarily conserved for functions related to similar avian and mammalian hearing. ABSTRACT: Auditory brainstem neurons are functionally primed to fire action potentials (APs) at markedly high rates in order to rapidly encode the acoustic information of sound. This specialization is critical for survival and the comprehension of behaviourally relevant communication functions, including sound localization and distinguishing speech from noise. Here, we investigated underlying ion channel mechanisms essential for high-rate AP firing in neurons of the chicken nucleus magnocellularis (NM) - the avian analogue of bushy cells of the mammalian anteroventral cochlear nucleus. In addition to the established function of high voltage-activated potassium channels, we found that resurgent sodium current (INaR ) plays a role in regulating rapid firing activity of late-developing (embryonic (E) days 19-21) NM neurons. INaR of late-developing NM neurons showed similar properties to mammalian neurons in that its unique mechanism of an 'open channel block state' facilitated the recovery and increased the availability of sodium (NaV ) channels after depolarization. Using a computational model of NM neurons, we demonstrated that removal of INaR reduced high-rate AP firing. We found weak INaR during a prehearing period (E11-12), which transformed to resemble late-developing INaR properties around hearing onset (E14-16). Anatomically, we detected strong NaV 1.6 expression near maturation, which became increasingly less distinct at hearing onset and prehearing periods, suggesting that multiple NaV channel subtypes may contribute to INaR during development. We conclude that INaR plays an important role in regulating rapid AP firing for NM neurons, a property that may be evolutionarily conserved for functions related to similar avian and mammalian hearing.

Effects of Temporal Processing on Speech-in-Noise Perception in Middle-Aged Adults.

Auditory temporal processing is a vital component of auditory stream segregation, or the process in which complex sounds are separated and organized into perceptually meaningful objects. Temporal processing can degrade prior to hearing loss, and is suggested to be a contributing factor to difficulties with speech-in-noise perception in normal-hearing listeners. The current study tested this hypothesis in middle-aged adults-an under-investigated cohort, despite being the age group where speech-in-noise difficulties are first reported. In 76 participants, three mechanisms of temporal processing were measured: peripheral auditory nerve function using electrocochleography, subcortical encoding of periodic speech cues (i.e., fundamental frequency; F0) using the frequency following response, and binaural sensitivity to temporal fine structure (TFS) using a dichotic frequency modulation detection task. Two measures of speech-in-noise perception were administered to explore how contributions of temporal processing may be mediated by different sensory demands present in the speech perception task. This study supported the hypothesis that temporal coding deficits contribute to speech-in-noise difficulties in middle-aged listeners. Poorer speech-in-noise perception was associated with weaker subcortical F0 encoding and binaural TFS sensitivity, but in different contexts, highlighting that diverse aspects of temporal processing are differentially utilized based on speech-in-noise task characteristics.

Revisiting the Chicken Auditory Brainstem Response: Frequency Specificity, Threshold Sensitivity, and Cross Species Comparison.

The auditory brainstem response (ABR) is important for both clinical and basic auditory research. It is a non-invasive measure of hearing function with millisecond-level precision. The ABR can not only measure the synchrony, speed, and efficacy of auditory physiology but also detect different modalities of hearing pathology and hearing loss. ABRs are easily acquired in vertebrate animal models like reptiles, birds, and mammals, and complement existing molecular, developmental, and systems-level research. One such model system is the chicken; an excellent animal for studying auditory development, structure, and function. However, the ABR for chickens was last reported nearly 4 decades ago. The current study examines how decades of ABR characterization in other animal species support findings from the chicken ABR. We replicated and expanded on previous research using 43 chicken hatchlings 1- and 2-day post-hatch. We report that click-evoked chicken ABRs presented with a peak waveform morphology, amplitude, and latency like previous avian studies. Tone-evoked ABRs were found for frequencies from 250 to 4000 Hertz (Hz) and exhibited a range of best sensitivity between 750 and 2000 Hz. Objective click-evoked and tone-evoked ABR thresholds were comparable to subjective thresholds. With these revisited measurements, the chicken ABR still proves to be an excellent example of precocious avian development that complements decades of molecular, neuronal, and systems-level research in the same model organism.

TrkB downregulation is required for dendrite retraction in developing neurons of chicken nucleus magnocellularis.

The chick embryo (Gallus domesticus) is one of the most important model systems in vertebrate developmental biology. The development and function of its auditory brainstem circuitry is exceptionally well studied. These circuits represent an excellent system for genetic manipulation to investigate mechanisms controlling neural circuit formation, synaptogenesis, neuronal polarity, and dendritic arborization. The present study investigates the auditory nucleus, nucleus magnocellularis (NM). The neurotrophin receptor TrkB regulates dendritic structure in CNS neurons. TrkB is expressed in NM neurons at E7-E8 when these neurons have dendritic arbors. Downregulation of TrkB occurs after E8 followed by retraction of dendrites and by E18 most NM cells are adendritic. Is cessation of TrkB expression in NM necessary for dendritic retraction? To answer this question we combined focal in ovo electroporation with transposon mediated gene transfer to obtain stable expression of Doxycycline (Dox) regulated transgenes, specifically TrkB coexpressed with EGFP in a temporally controlled manner. Electroporation was performed at E2 and Dox added onto the chorioallointoic membrane from E7.5 to E16. Expression of EGFP had no effect on development of the embryo, or cell morphology and organization of auditory brainstem nuclei. NM cells expressing EGFP and TrkB at E17-E18 had dendrites and biophysical properties uncharacteristic for normal NM cells, indicating that cessation of TrkB expression is essential for dendrite retraction and functional maturation of these neurons. These studies indicate that expression of transposon based plasmids is an effective method to genetically manipulate events in mid to late embryonic brain development in chick.

Exploring Electrode Placements to Optimize the Identification and Measurement of Early Auditory Evoked Potentials.

Cochlear synaptic loss (termed cochlear synaptopathy) has been suggested to contribute to suprathreshold hearing difficulties. However, its existence and putative effects in humans remain inconclusive, largely due to the heterogeneous methods used across studies to indirectly evaluate the health of cochlear synapses. There is a need to standardize proxies of cochlear synaptopathy to appropriately compare and interpret findings across studies. Early auditory evoked potentials (AEPs), including the compound action potential (AP)/Wave I of the auditory brainstem response are a popular proxy, yet remain variable based on technical considerations. This study evaluated one such consideration-electrode array (i.e., montage)-to optimize the use of early AEP waveforms. In 35 young adults, electrocochleography (ECochG) responses were collected using vertical and horizontal montages. Standard ECochG measures and AP/Wave I and Wave II peak-to-trough amplitudes and latencies were compared between montages. Vertical montage recordings consistently produced significantly larger AP/Wave I peak-to-trough amplitudes compared to horizontal recordings. These findings support the use of a vertical electrode montage for optimal recordings of peripheral cochlear nerve activity. As cochlear synaptopathy continues to be explored in humans, the methods highlighted here should be considered in the development of a standardized assessment.

Control of neuronal excitability by NMDA-type glutamate receptors in early developing binaural auditory neurons.

Precise control of neuronal excitability in the auditory brainstem is fundamental for processing timing cues used for sound localization and signal discrimination in complex acoustic environments. In mature nucleus laminaris (NL), the first nucleus responsible for binaural processing in chickens, neuronal excitability is governed primarily by voltage-activated potassium conductances (K(VA)). High levels of K(VA) expression in NL neurons result in one or two initial action potentials (APs) in response to high-frequency synaptic activity or sustained depolarization. Here we show that during a period of synaptogenesis and circuit refinement, before hearing onset, K(VA) conductances are relatively small, in particular low-voltage-activated K(+) conductances (K(LVA)). In spite of this, neuronal output is filtered and repetitive synaptic activity generates only one or two initial APs during a train of stimuli. During this early developmental time period, synaptic NMDA-type glutamate receptors (NMDA-Rs) contain primarily the GluN2B subunit. We show that the slow decay kinetics of GluN2B-containing NMDA-Rs allows synaptic responses to summate, filtering the output of NL neurons before intrinsic properties are fully developed. Weaker Mg(2+) blockade of NMDA-Rs and ambient glutamate early in development generate a tonic NMDA-R-mediated current that sets the membrane potential at more depolarized values. Small KLVA conductances, localized in dendrites, prevent excessive depolarization caused by tonic activation of NMDA-Rs. Thus, before intrinsic properties are fully developed, NMDA-Rs control the output of NL neurons during evoked synaptic transmission.

Evaluation of Auditory Brainstem Response in Chicken Hatchlings.

The auditory brainstem response (ABR) is an invaluable assay in clinical audiology, non-human animals, and human research. Despite the widespread use of ABRs in measuring auditory neural synchrony and estimating hearing sensitivity in other vertebrate model systems, methods for recording ABRs in the chicken have not been reported in nearly four decades. Chickens provide a robust animal research model because their auditory system is near functional maturation during late embryonic and early hatchling stages. We have demonstrated methods used to elicit one or two-channel ABR recordings using subdermal needle electrode arrays in chicken hatchlings. Regardless of electrode recording configuration (i.e., montage), ABR recordings included 3-4 positive-going peak waveforms within the first 6 ms of a suprathreshold click stimulus. Peak-to-trough waveform amplitudes ranged from 2-11 µV at high-intensity levels, with positive peaks exhibiting expected latency-intensity functions (i.e., increase in latency as a function of decreased intensity). Standardized earphone position was critical for optimal recordings as loose skin can occlude the ear canal, and animal movement can dislodge the stimulus transducer. Peak amplitudes were smaller, and latencies were longer as animal body temperature lowered, supporting the need for maintaining physiological body temperature. For young hatchlings (<3 h post-hatch day 1), thresholds were elevated by ~5 dB, peak latencies increased ~1-2 ms, and peak to trough amplitudes were decreased ~1 µV compared to older hatchlings. This suggests a potential conductive-related issue (i.e., fluid in the middle ear cavity) and should be considered for young hatchlings. Overall, the ABR methods outlined here permit accurate and reproducible recording of in-vivo auditory function in chicken hatchlings that could be applied to different stages of development. Such findings are easily compared to human and mammalian models of hearing loss, aging, or other auditory-related manipulations.

Slicing the Embryonic Chicken Auditory Brainstem to Evaluate Tonotopic Gradients and Microcircuits.

The chicken embryo is a widely accepted animal model to study the auditory brainstem, composed of highly specialized microcircuitry and neuronal topology differentially oriented along a tonotopic (i.e., frequency) axis. The tonotopic axis permits the segregated encoding of high-frequency sounds in the rostral-medial plane and low-frequency encoding in caudo-lateral regions. Traditionally, coronal brainstem slices of embryonic tissue permit the study of relative individual iso-frequency lamina. Although sufficient to investigate anatomical and physiological questions pertaining to individual iso-frequency regions, the study of tonotopic variation and its development across larger auditory brainstem areas is somewhat limited. This protocol reports brainstem slicing techniques from chicken embryos that encompass larger gradients of frequency regions in the lower auditory brainstem. The utilization of different slicing methods for chicken auditory brainstem tissue permits electrophysiological and anatomical experiments within one brainstem slice, where larger gradients of tonotopic properties and developmental trajectories are better preserved than coronal sections. Multiple slicing techniques allow for improved investigation of the diverse anatomical, biophysical, and tonotopic properties of auditory brainstem microcircuits.

Development of glutamatergic synaptic transmission in binaural auditory neurons.

Glutamatergic synaptic transmission is essential for binaural auditory processing in birds and mammals. Using whole cell voltage clamp recordings, we characterized the development of synaptic ionotropic glutamate receptor (iGluR) function from auditory neurons in the chick nucleus laminaris (NL), the first nucleus responsible for binaural processing. We show that synaptic transmission is mediated by AMPA- and N-methyl-d-aspartate (NMDA)-type glutamate receptors (AMPA-R and NMDA-R, respectively) when hearing is first emerging and dendritic morphology is being established across different sound frequency regions. Puff application of glutamate agonists at embryonic day 9 (E9) revealed that both iGluRs are functionally present prior to synapse formation (E10). Between E11 and E19, the amplitude of isolated AMPA-R currents from high-frequency (HF) neurons increased 14-fold. A significant increase in the frequency of spontaneous events is also observed. Additionally, AMPA-R currents become faster and more rectifying, suggesting developmental changes in subunit composition. These developmental changes were similar in all tonotopic regions examined. However, mid- and low-frequency neurons exhibit fewer spontaneous events and evoked AMPA-R currents are smaller, slower, and less rectifying than currents from age-matched HF neurons. The amplitude of isolated NMDA-R currents from HF neurons also increased, reaching a peak at E17 and declining sharply by E19, a trend consistent across tonotopic regions. With age, NMDA-R kinetics become significantly faster, indicating a developmental switch in receptor subunit composition. Dramatic increases in the amplitude and speed of glutamatergic synaptic transmission occurs in NL during embryonic development. These changes are first seen in HF neurons suggesting regulation by peripheral inputs and may be necessary to enhance coincidence detection of binaural auditory information.

Effects of Neurotrophin-3 on Intrinsic Neuronal Properties at a Central Auditory Structure.

Neurotrophins, a class of growth factor proteins that control neuronal proliferation, morphology, and apoptosis, are found ubiquitously throughout the nervous system. One particular neurotrophin (NT-3) and its cognate tyrosine receptor kinase (TrkC) have recently received attention as a possible therapeutic target for synaptopathic sensorineural hearing loss. Additionally, research shows that NT-3-TrkC signaling plays a role in establishing the sensory organization of frequency topology (ie, tonotopic order) in the cochlea of the peripheral inner ear. However, the neurotrophic effects of NT-3 on central auditory properties are unclear. In this study we examined whether NT-3-TrkC signaling affects the intrinsic electrophysiological properties at a first-order central auditory structure in chicken, known as nucleus magnocellularis (NM). Here, the expression pattern of specific neurotrophins is well known and tightly regulated. By using whole-cell patch-clamp electrophysiology, we show that NT-3 application to brainstem slices does not affect intrinsic properties of high-frequency neuronal regions but had robust effects for low-frequency neurons, altering voltage-dependent potassium functions, action potential repolarization kinetics, and passive membrane properties. We suggest that NT-3 may contribute to the precise establishment and organization of tonotopy in the central auditory pathway by playing a specialized role in regulating the development of intrinsic neuronal properties of low-frequency NM neurons.

Glycinergic "inhibition" mediates selective excitatory responses to combinations of sounds.

In the mustached bat's inferior colliculus (IC), combination-sensitive neurons display time-sensitive facilitatory interactions between inputs tuned to distinct spectral elements in sonar or social vocalizations. Here we compare roles of ionotropic receptors to glutamate (iGluRs), glycine (GlyRs), and GABA (GABA(A)Rs) in facilitatory combination-sensitive interactions. Facilitatory responses to 36 single IC neurons were recorded before, during, and after local application of antagonists to these receptors. The NMDA receptor antagonist CPP [(+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid], alone (n = 14) or combined with AMPA receptor antagonist NBQX (n = 22), significantly reduced or eliminated responses to best frequency (BF) sounds across a broad range of sound levels, but did not eliminate combination-sensitive facilitation. In a subset of neurons, GABA(A)R blockers bicuculline or gabazine were applied in addition to iGluR blockers. GABA(A)R blockers did not "uncover" residual iGluR-mediated excitation, and only rarely eliminated facilitation. In nearly all neurons for which the GlyR antagonist strychnine was applied in addition to iGluR blockade (22 of 23 neurons, with or without GABA(A)R blockade), facilitatory interactions were eliminated. Thus, neither glutamate nor GABA neurotransmission are required for facilitatory combination-sensitive interactions in IC. Instead, facilitation may depend entirely on glycinergic inputs that are presumed to be inhibitory. We propose that glycinergic inputs tuned to two distinct spectral elements in vocal signals each activate postinhibitory rebound excitation. When rebound excitations from two spectral elements coincide, the neuron discharges. Excitation from glutamatergic inputs, tuned to the BF of the neuron, is superimposed onto this facilitatory interaction, presumably mediating responses to a broader range of acoustic signals.

Contribution of NMDA and AMPA receptors to temporal patterning of auditory responses in the inferior colliculus.

Although NMDA receptors (NMDARs) are associated with synaptic plasticity, they form an essential part of responses to sensory stimuli. We compared contributions of glutamatergic NMDARs and AMPA receptors (AMPARs) to auditory responses in the inferior colliculus (IC) of awake, adult mustached bats. We examined the magnitude and temporal pattern of responses to tonal signals in single units before, during, and after local micro-iontophoretic application of selective antagonists to AMPARs [NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide)] and NMDARs [CPP ((+/-)3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid)]. Combined blockade of AMPARs and NMDARs eliminated excitatory responses in nearly all neurons, whereas separate blockade of each receptor was quantitatively similar, causing substantial (> 50%) spike reductions in approximately 75% of units. The major result was that effects of receptor blockade were most closely related to the first-spike latency of a unit. Thus, AMPAR blockade substantially reduced spikes in all short-latency units (< 12 ms) but never in long-latency units (> or = 12 ms). NMDAR blockade had variable effects on short-latency units but reduced spikes substantially for all long-latency units. There were no distinct contributions of AMPARs and NMDARs to early and late elements of responses. Thus, AMPAR blockade reduced early (onset) spikes somewhat more effectively than NMDAR blockade in short-latency units, but NMDAR blockade reduced onset spikes more effectively in long-latency units. AMPAR and NMDAR blockade were equally effective in reducing later elements of sustained responses in short-latency units, whereas NMDAR blockade was much more effective in long-latency units. These results indicate that NMDARs play multiple roles for signal processing in adult IC neurons.

Need for Speed and Precision: Structural and Functional Specialization in the Cochlear Nucleus of the Avian Auditory System.

Birds such as the barn owl and zebra finch are known for their remarkable hearing abilities that are critical for survival, communication, and vocal learning functions. A key to achieving these hearing abilities is the speed and precision required for the temporal coding of sound-a process heavily dependent on the structural, synaptic, and intrinsic specializations in the avian auditory brainstem. Here, we review recent work from us and others focusing on the specialization of neurons in the chicken cochlear nucleus magnocellularis (NM)-a first-order auditory brainstem structure analogous to bushy cells in the mammalian anteroventral cochlear nucleus. Similar to their mammalian counterpart, NM neurons are mostly adendritic and receive auditory nerve input through large axosomatic endbulb of Held synapses. Axonal projections from NM neurons to their downstream auditory targets are sophisticatedly programmed regarding their length, caliber, myelination, and conduction velocity. Specialized voltage-dependent potassium and sodium channel properties also play important and unique roles in shaping the functional phenotype of NM neurons. Working synergistically with potassium channels, an atypical current known as resurgent sodium current promotes rapid and precise action potential firing for NM neurons. Interestingly, these structural and functional specializations vary dramatically along the tonotopic axis and suggest a plethora of encoding strategies for sounds of different acoustic frequencies, mechanisms likely shared across species.

Diverse Intrinsic Properties Shape Functional Phenotype of Low-Frequency Neurons in the Auditory Brainstem.

In the auditory system, tonotopy is the spatial arrangement of where sounds of different frequencies are processed. Defined by the organization of neurons and their inputs, tonotopy emphasizes distinctions in neuronal structure and function across topographic gradients and is a common feature shared among vertebrates. In this study we characterized action potential firing patterns and ion channel properties from neurons located in the extremely low-frequency region of the chicken nucleus magnocellularis (NM), an auditory brainstem structure. We found that NM neurons responsible for encoding the lowest sound frequencies (termed NMc neurons) have enhanced excitability and fired bursts of action potentials to sinusoidal inputs ≤10 Hz; a distinct firing pattern compared to higher-frequency neurons. This response property was due to lower amounts of voltage dependent potassium (KV) conductances, unique combination of KV subunits and specialized sodium (NaV) channel properties. Particularly, NMc neurons had significantly lower KV1 and KV3 currents, but higher KV2 current. NMc neurons also showed larger and faster transient NaV current (INaT) with different voltage dependence of inactivation from higher-frequency neurons. In contrast, significantly smaller resurgent sodium current (INaR) was present in NMc with kinetics and voltage dependence that differed from higher-frequency neurons. Immunohistochemistry showed expression of NaV1.6 channel subtypes across the tonotopic axis. However, various immunoreactive patterns were observed between regions, likely underlying some tonotopic differences in INaT and INaR. Finally, using pharmacology and computational modeling, we concluded that KV3, KV2 channels and INaR work synergistically to regulate burst firing in NMc.

A Comparison of Commercially Available Auditory Brainstem Response Stimuli at a Neurodiagnostic Intensity Level.

iChirp-evoked auditory brainstem responses (ABRs) yield a larger wave V amplitude at low intensity levels than traditional broadband click stimuli, providing a reliable estimation of hearing sensitivity. However, advantages of iChirp stimulation at high intensity levels are unknown. We tested to see if high-intensity (i.e., 85 dBnHL) iChirp stimulation results in larger and more reliable ABR waveforms than click. Using the commercially available Intelligent Hearing System SmartEP platform, we recorded ABRs from 43 normal hearing young adults. We report that absolute peak latencies were more variable for iChirp and were ~3 ms longer: the latter of which is simply due to the temporal duration of the signal. Interpeak latencies were slightly shorter for iChirp and were most evident between waves I-V. Interestingly, click responses were easier to identify and peak-to-trough amplitudes for waves I, III and V were significantly larger than iChirp. These differences were not due to residual noise levels. We speculate that high intensity iChirp stimulation reduces neural synchrony and conclude that for retrocochlear evaluations, click stimuli should be used as the standard for ABR neurodiagnostic testing.

Ion channel mechanisms underlying frequency-firing patterns of the avian nucleus magnocellularis: A computational model.

We have previously shown that late-developing avian nucleus magnocellularis (NM) neurons (embryonic [E] days 19-21) fire action potentials (APs) that resembles a band-pass filter in response to sinusoidal current injections of varying frequencies. NM neurons located in the mid- to high-frequency regions of the nucleus fire preferentially at 75 Hz, but only fire a single onset AP to frequency inputs greater than 200 Hz. Surprisingly, NM neurons do not fire APs to sinusoidal inputs less than 20 Hz regardless of the strength of the current injection. In the present study we evaluated intrinsic mechanisms that prevent AP generation to low frequency inputs. We constructed a computational model to simulate the frequency-firing patterns of NM neurons based on experimental data at both room and near physiologic temperatures. The results from our model confirm that the interaction among low- and high-voltage activated potassium channels (KLVA and KHVA, respectively) and voltage dependent sodium channels (NaV) give rise to the frequency-firing patterns observed in vitro. In particular, we evaluated the regulatory role of KLVA during low frequency sinusoidal stimulation. The model shows that, in response to low frequency stimuli, activation of large KLVA current counterbalances the slow-depolarizing current injection, likely permitting NaV closed-state inactivation and preventing the generation of APs. When the KLVA current density was reduced, the model neuron fired multiple APs per sinusoidal cycle, indicating that KLVA channels regulate low frequency AP firing of NM neurons. This intrinsic property of NM neurons may assist in optimizing response to different rates of synaptic inputs.

In Ovo Electroporation in the Chicken Auditory Brainstem.

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that the chicken embryo is an effective research model for studying basic biological functions of auditory system development. More recently, the chicken embryo has become particularly valuable in studying gene expression, regulation and function associated with hearing. In ovo electroporation can be used to target auditory brainstem regions responsible for highly specialized auditory functions. These regions include the chicken nucleus magnocellularis (NM) and nucleus laminaris (NL). NM and NL neurons arise from distinct precursors of rhombomeres 5 and 6 (R5/R6). Here, we present in ovo electroporation of plasmid-encoded genes to study gene-related properties in these regions. We show a method for spatial and temporal control of gene expression that promote either gain or loss of functional phenotypes. By targeting auditory neural progenitor regions associated with R5/R6, we show plasmid transfection in NM and NL. Temporal regulation of gene expression can be achieved by adopting a tet-on vector system. This is a drug inducible procedure that expresses the genes of interest in the presence of doxycycline (Dox). The in ovo electroporation technique - together with either biochemical, pharmacological, and or in vivo functional assays - provides an innovative approach to study auditory neuron development and associated pathophysiological phenomena.

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.

Effects of artifact rejection and bayesian weighting on the auditory brainstem response during quiet and active behavioral conditions.

PURPOSE: To evaluate the effects of 2 noise reduction techniques on the auditory brainstem response (ABR). METHOD: ABRs of 20 normal hearing adults were recorded during quiet and active behavioral conditions using 2 stimulus intensity levels. Wave V amplitudes and residual noise root-mean-square values were measured following the offline application of artifact rejection and Bayesian weighting. Repeated measures analysis of variance and Bonferroni adjusted pairwise t tests were utilized to evaluate significant main effects and interactions between the 2 noise reduction techniques. RESULTS: ABRs recorded during the quiet behavioral condition resulted in minimal differences in wave V amplitude and noise reduction improvement, suggesting that the 2 techniques were equally effective under ideal recording situations. During the active behavioral condition, however, the techniques differed significantly in the ability to preserve the evoked potential and reduce noise. Consequently, strict artifact rejection levels resulted in an inherent underestimation of wave V amplitudes when compared with the Bayesian approach. CONCLUSION: Artifact rejection had a detrimental effect on waveform morphology of the ABR. This could lead to difficulty in ABR interpretation when patients are active and ultimately result in diagnostic errors.

Developmental Profile of Ion Channel Specializations in the Avian Nucleus Magnocellularis.

Ultrafast and temporally precise action potentials (APs) are biophysical specializations of auditory brainstem neurons; properties necessary for encoding sound localization and communication cues. Fundamental to these specializations are voltage dependent potassium (KV) and sodium (NaV) ion channels. Here, we characterized the functional development of these ion channels and quantified how they shape AP properties in the avian cochlear nucleus magnocellularis (NM). We report that late developing NM neurons (embryonic [E] days 19-21) generate fast APs that reliably phase lock to sinusoidal inputs at 75 Hz. In contrast, early developing neurons (E19) contained NaV channels that inactivate at more negative voltages, suggesting alterations in NaV channel subtypes. Taken together, our results indicate that the refinement of passive and active ion channel properties operate differentially in order to develop fast and reliable APs in the avian NM.