Nucleus Magnocellularis Neurons Research Articles

Tonotopic Specializations in Number, Size, and Reversal Potential of GABAergic Inputs Fine-Tune Temporal Coding at Avian Cochlear Nucleus.

GABAergic inhibition in neurons plays a critical role in determining the output of neural circuits. Neurons in avian nucleus magnocellularis (NM) use several tonotopic-region-dependent specializations to relay the timing information of sound in the auditory nerve to higher auditory nuclei. Previously, we showed that feedforward GABAergic inhibition in NM has a different dependence on the level of auditory nerve activity, with the low-frequency region having a low-threshold and linear relationship, while the high-frequency region has a high-threshold and step-like relationship. However, it remains unclear how the GABAergic synapses are tonotopically regulated and interact with other specializations of NM neurons. In this study, we examined GABAergic transmission in the NM of chickens of both sexes and explored its contributions to the temporal coding of sound at each tonotopic region. We found that the number and size of unitary GABAergic currents and their reversal potential were finely tuned at each tonotopic region in the NM. At the lower-frequency region, unitary GABAergic currents were larger in number but smaller in size. In addition, their reversal potential was close to the resting potential of neurons, which enabled reliable inhibition despite the smaller potassium conductance. At the higher-frequency region, on the other hand, unitary GABAergic currents were fewer, larger, and highly depolarizing, which enabled powerful inhibition via activating the large potassium conductance. Thus, we propose that GABAergic synapses are coordinated with the characteristics of excitatory synapses and postsynaptic neurons, ensuring the temporal coding for wide frequency and intensity ranges.SIGNIFICANCE STATEMENT We found in avian cochlear nucleus that the number and size of unitary GABAergic inputs differed among tonotopic regions and correlated to respective excitatory inputs; it was larger in number but smaller in size for neurons tuned to lower-frequency sound. Furthermore, GABAergic reversal potential also differed among the regions in accordance with the size of Kv1 current; it was less depolarized in the lower-frequency neurons with smaller Kv1 current. These differentiations of GABAergic transmission maximized the effects of inhibition at each tonotopic region, ensuring precise and reliable temporal coding across frequencies and intensities. Our results emphasize the importance of optimizing characteristics of GABAergic transmission within individual neurons for proper neural circuit function.

Dynamics of the fragile X mental retardation protein correlates with cellular and synaptic properties in primary auditory neurons following afferent deprivation.

Afferent activity dynamically regulates neuronal properties and connectivity in the central nervous system. The Fragile X mental retardation protein (FMRP) is an RNA-binding protein that regulates cellular and synaptic properties in an activity-dependent manner. Whether and how FMRP level and localization are regulated by afferent input remains sparsely examined and how such regulation is associated with neuronal response to changes in sensory input is unknown. We characterized changes in FMRP level and localization in the chicken nucleus magnocellularis (NM), a primary cochlear nucleus, following afferent deprivation by unilateral cochlea removal. We observed rapid (within 2 hr) aggregation of FMRP immunoreactivity into large granular structures in a subset of deafferented NM neurons. Neurons that exhibited persistent FMRP aggregation at 12-24 hr eventually lost cytoplasmic Nissl substance, indicating cell death. A week later, FMRP expression in surviving neurons regained its homeostasis, with a slightly reduced immunostaining intensity and enhanced heterogeneity. Correlation analyses under the homeostatic status (7-14 days) revealed that neurons expressing relatively more FMRP had a higher capability of maintaining cell body size and ribosomal activity, as well as a better ability to detach inactive presynaptic terminals. Additionally, the intensity of an inhibitory postsynaptic protein, gephyrin, was reduced following deafferentation and was positively correlated with FMRP intensity, implicating an involvement of FMRP in synaptic dynamics in response to reduced afferent inputs. Collectively, this study demonstrates that afferent input regulates FMRP expression and localization in ways associated with multiple types of neuronal responses and synaptic rearrangements.

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.

Excitatory-Inhibitory Synaptic Coupling in Avian Nucleus Magnocellularis.

The activity of neurons is determined by the balance between their excitatory and inhibitory synaptic inputs. Neurons in the avian nucleus magnocellularis (NM) integrate monosynaptic excitatory and polysynaptic inhibitory inputs from the auditory nerve, and transmit phase-locked output to higher auditory centers. The excitatory input is graded tonotopically, such that neurons tuned to higher frequency receive fewer, but larger, axon terminals. However, it remains unknown how the balance between excitatory and inhibitory inputs is determined in NM. We here examined synaptic and spike responses of NM neurons during stimulation of the auditory nerve in thick brain slices of chicken of both sexes, and found that the excitatory-inhibitory balance varied according to tonotopic region, ensuring reliable spike output across frequencies. Auditory nerve stimulation elicited IPSCs in NM neurons regardless of tonotopic region, but the dependence of IPSCs on intensity varied in a systematic way. In neurons tuned to low frequency, IPSCs appeared and increased in parallel with EPSCs with elevation of intensity, which expanded dynamic range by preventing saturation of spike generation. On the other hand, in neurons tuned to higher frequency, IPSCs were smaller than EPSCs and had higher thresholds for activation, thus facilitating high-fidelity transmission. Computer simulation confirmed that these differences in inhibitory input were optimally matched to the patterns of excitatory input, and enabled appropriate level of neuronal output for wide intensity and frequency ranges of sound in the auditory system.SIGNIFICANCE STATEMENT Neurons in nucleus magnocellularis encode timing information of sound across wide intensity ranges by integrating excitatory and inhibitory synaptic inputs from the auditory nerve, but underlying synaptic mechanisms of this integration are not fully understood. We here show that the excitatory-inhibitory relationship was expressed differentially at each tonotopic region; the relationship was linear in neurons tuned to low-frequency, expanding dynamic range by preventing saturation of spike generation; by contrast inhibitory input remained much smaller than excitatory input in neurons tuned to higher frequency, thus ensuring high-fidelity transmission. The tonotopic regulation of excitatory and inhibitory input optimized the output across frequencies and intensities, playing a fundamental role in the timing coding pathway in the auditory system.

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.

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 (I NaR) of sodium channels (NaV) is equally important and operates synergistically with KHVA channels to enable rapid AP firing in NM.Anatomically, we detected strong NaV1.6 expression near hearing maturation, which was less distinct during hearing development despite functional evidence of I NaR, suggesting that multiple NaV channel subtypes may contribute to I NaR.We conclude that I NaR 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. 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 (I NaR) plays a role in regulating rapid firing activity of late‐developing (embryonic (E) days 19–21) NM neurons. I NaR 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 I NaR reduced high‐rate AP firing. We found weak I NaR during a prehearing period (E11–12), which transformed to resemble late‐developing I NaR properties around hearing onset (E14–16). Anatomically, we detected strong NaV1.6 expression near maturation, which became increasingly less distinct at hearing onset and prehearing periods, suggesting that multiple NaV channel subtypes may contribute to I NaR during development. We conclude that I NaR 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.

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.

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.

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

ABSTRACTWe 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.

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.

Tonotopic Optimization for Temporal Processing in the Cochlear Nucleus.

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. 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.

A role for inhibition in deafness-induced plasticity of the avian auditory brainstem

To better understand the effects of deafness on the brain, these experiments examine how disrupted balance between excitatory and inhibitory neurotransmission following the loss of excitatory input from the auditory nerve alters the central auditory system. In the avian cochlear nucleus, nucleus magnocellularis (NM), deprivation of excitatory input induced by deafness triggers neuronal death. While this neuronal death was previously accredited to the loss of excitatory drive, the present experiments examine an alternative hypothesis: that inhibitory input to NM, which may also be affected by deafness, contributes to neuronal death in NM. Using an in vitro slice preparation in which excitatory input from the auditory nerve is absent, we pharmacologically altered GABA receptor activation in NM, and assayed an early marker of neuronal health, antigenicity for the ribosomal antibody Y10B (Y10B-ir). We found that GABA decreases Y10B-ir, and that GABAA activation is necessary for the GABA-induced effect. We further found that endogenous GABAA activation similarly decreases Y10B-ir and this decrease requires extracellular Ca2+. Our results suggest that, in the absence of excitatory input, endogenous activation of ionotropic GABAA receptors is detrimental to NM neurons.

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 (<E12) have slower and less reliable APs that preferentially fire to lower frequencies (5–10 Hz). With development, the membrane time constant of NM neurons became faster, while input resistance and capacitance decreased. Change in input resistance was due to a 2-fold increase in KV current from E10 to E21 and when high-voltage activated potassium (K+HVA) channels were blocked, APs for all ages became significantly slower. This was most evident for early developing neurons where the ratio of K+HVA current accounted for ~85% of the total KV response. This ratio dropped to ~50% for late developing neurons, suggesting a developmental upregulation of low-voltage activated potassium (K+LVA) channels. Indeed, blockade of K+LVA eliminated remaining current and increased neural excitability for late developing neurons. We also report developmental changes in the amplitude, kinetics and voltage dependence of NaV currents. For early developing neurons, increase in NaV current amplitude was due to channel density while channel conductance dominated for late developing neurons. From E10 to E21, NaV channel currents became faster but differed in their voltage dependence; early developing neurons (<E16) had similar NaV channel inactivation voltages while late developing NM 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.

Activity-dependent regulation of calcium and ribosomes in the chick cochlear nucleus

Cochlea removal results in the death of 20–30% of neurons in the chick cochlear nucleus, nucleus magnocellularis (NM). Two potentially cytotoxic events, a dramatic rise in intracellular calcium concentration ([Ca2+]i) and a decline in the integrity of ribosomes are observed within 1h of deafferentation. Glutamatergic input from the auditory nerve has been shown to preserve NM neuron health by activating metabotropic glutamate receptors (mGluRs), maintaining both normal [Ca2+]i and ribosomal integrity. One interpretation of these results is that a common mGluR-activated signaling cascade is required for the maintenance of both [Ca2+]i and ribosomal integrity. This could happen if both responses are influenced directly by a common messenger, or if the loss of mGluR activation causes changes in one component that secondarily causes changes in the other. The present studies tested this common-mediator hypothesis in slice preparations by examining activity-dependent regulation of [Ca2+]i and ribosomes in the same tissue after selectively blocking group I mGluRs (1-Aminoindan-1,5-dicarboxylic acid (AIDA)) or group II mGluRs (LY 341495) during unilateral auditory nerve stimulation. Changes in [Ca2+]i of NM neurons were measured using fura-2 ratiometric calcium imaging and the tissue was subsequently processed for Y10B immunoreactivity (Y10B-ir), an antibody that recognizes a ribosomal epitope. The group I mGluR antagonist blocked the activity-dependent regulation of both [Ca2+]i and Y10B-ir, but the group II antagonist blocked only the activity-dependent regulation of Y10B-ir. That is, even when group II receptors were blocked, stimulation continued to maintain low [Ca2+]i, but it did not maintain Y10B-ir. These results suggest a dissociation in how calcium and ribosomes are regulated in NM neurons and that ribosomes can be regulated through a mechanism that is independent of calcium regulation.

Short-term synaptic depression is topographically distributed in the cochlear nucleus of the chicken.

In the auditory system, sounds are processed in parallel frequency-tuned circuits, beginning in the cochlea. Activity of auditory nerve fibers reflects this frequency-specific topographic pattern, known as tonotopy, and imparts frequency tuning onto their postsynaptic target neurons in the cochlear nucleus. In birds, cochlear nucleus magnocellularis (NM) neurons encode the temporal properties of acoustic stimuli by "locking" discharges to a particular phase of the input signal. Physiological specializations exist in gradients corresponding to the tonotopic axis in NM that reflect the characteristic frequency (CF) of their auditory nerve fiber inputs. One feature of NM neurons that has not been investigated across the tonotopic axis is short-term synaptic plasticity. NM offers a rather homogeneous population of neurons with a distinct topographical distribution of synaptic properties that is ideal for the investigation of specialized synaptic plasticity. Here we demonstrate for the first time that short-term synaptic depression (STD) is expressed topographically, where unitary high CF synapses are more robust with repeated stimulation. Correspondingly, high CF synapses drive spiking more reliably than their low CF counterparts. We show that postsynaptic AMPA receptor desensitization does not contribute to the observed difference in STD. Further, rate of recovery from depression, a presynaptic property, does not differ tonotopically. Rather, we show that another presynaptic feature, readily releasable pool (RRP) size, is tonotopically distributed and inversely correlated with vesicle release probability. Mathematical model results demonstrate that these properties of vesicle dynamics are sufficient to explain the observed tonotopic distribution of STD.

Alteration of CaBP Expression Pattern in the Nucleus Magnocellularis following Unilateral Cochlear Ablation in Adult Zebra Finches

Songbirds have the rare ability of auditory-vocal learning and maintenance. Up to now, the organization and function of the nucleus magnocellularis (NM), the first relay of the avian ascending auditory pathway is largely based on studies in non-vocal learning species, such as chickens and owls. To investigate whether NM exhibits different histochemical properties associated with auditory processing in songbirds, we examined the expression patterns of three calcium-binding proteins (CaBPs), including calretinin (CR), parvalbumin (PV) and calbindin-D28k (CB), and their relations to auditory inputs in NM in adult zebra finches. We found enriched and co-localized immunostaining of CR, PV and CB in the majority of NM neurons, without neuronal population preference. Furthermore, they were sensitive to adult deafferentation with differential plasticity patterns. After unilateral cochlear removal, CR staining in the ipsilateral NM decreased appreciably at 3 days after surgery, and continued to decline thereafter. PV staining showed down-regulation first at 3 days, but subsequently recovered slightly. CB staining did not significantly decrease until 7 days after surgery. Our findings suggest that the three CaBPs might play distinct roles in association with auditory processing in zebra finches. These results are in contrast to the findings in the NM of chickens where CR is the predominant CaBP and deafferentation had no apparent effect on its expression. Further extended studies in other avian species are required to establish whether the difference in CaBP patterns in NM is functionally related to the different auditory-vocal behaviors.

Slowly emerging glycinergic transmission enhances inhibition in the sound localization pathway of the avian auditory system

Localization of low-frequency acoustic stimuli is processed in dedicated neural pathways where coincidence-detecting neurons compare the arrival time of sound stimuli at the two ears, or interaural time disparity (ITD). ITDs occur in the submillisecond range, and vertebrates have evolved specialized excitatory and inhibitory circuitry to compute these differences. Glycinergic inhibition is a computationally significant and prominent component of the mammalian ITD pathway. However, evidence for glycinergic transmission is limited in birds, where GABAergic inhibition has been thought to be the dominant or exclusive inhibitory transmitter. Indeed, previous work showed that GABA antagonists completely eliminate inhibition in avian nuclei specialized for processing temporal features of sound, nucleus magnocellularis (NM) and nucleus laminaris (NL). However, more recent work shows that glycine is coexpressed with GABA in synaptic terminals apposed to neurons in both nuclei (Coleman WL, Fischl MJ, Weimann SR, Burger RM. J Neurophysiol 105: 2405-2420, 2011; Kuo SP, Bradley LA, Trussell LO. J Neurosci 29: 9625-9634, 2009). Here we show complementary evidence of functional glycine receptor (GlyR) expression in NM and NL. Additionally, we show that glycinergic input can be evoked under particular stimulus conditions. Stimulation at high but physiologically relevant rates evokes a slowly emerging glycinergic response in NM and NL that builds over the course of the stimulus. Glycinergic response magnitude was stimulus rate dependent, representing 18% and 7% of the total inhibitory current in NM and NL, respectively, at the end of the 50-pulse, 200-Hz stimulus. Finally, we show that the glycinergic component is functionally relevant, as its elimination reduced inhibition of discharges evoked by current injection into NM neurons.

Expression of calcium-binding proteins in the chick auditory nuclei following prenatal auditory stimulation

Aim: Auditory stimulation during development influences the morphological and neurochemical substrate of chick brainstem auditory nuclei, nucleus magnocellularis (NM) and nucleus laminaris (NL). Calcium-binding proteins (CaBPs) - calretinin (CR), calbindin (CALB), and parvalbumin (PV) - are known to buffer cytosolic calcium transients that occur with activation of neurons. In the present study, we examined the expression of three CaBPs - CR, CALB, and PV - in the NM and NL at embryonic days E12, E16, E20, and posthatch dayl (PH1), following prenatal sound enrichment protocol. Materials and methods: The incubating eggs were exposed to species-specific sound or music (sitar) at 65 dB for 15 min/h over a day/night cycle from E10 to E14 (low frequency) and E15 till hatching (high frequency). Results: Calretinin and parvalbumin were present in the developing normal and stimulated auditory nuclei, while CALB was absent. Calretinin-immunoreactivity (CR-IR) was present from E12 onward in NM and NL neurons of all the groups. The auditory stimulated groups showed no change in the expression of CR-IR in NM and NL. During normal development, PV was restricted to the cochlear nerve fibers at E16, and appeared in their terminals on the NM somata at PH1. In both stimulated groups, however, PV appeared earlier at E12 in the cochlear fibers and was prominently visualized from E16 in the NM and E20 in the NL neurons. Conclusions: Thus, CR and PV but not CALB are present in chick brainstem auditory nuclei for mediating calcium signaling and homeostasis. Prenatal sound caused an early activity-dependent maturation of PV but not CR which is a constitutive protein.

Pre‐target axon sorting in the avian auditory brainstem

Topographic organization of neurons is a hallmark of brain structure. The establishment of the connections between topographically organized brain regions has attracted much experimental attention, and it is widely accepted that molecular cues guide outgrowing axons to their targets in order to construct topographic maps. In a number of systems afferent axons are organized topographically along their trajectory as well, and it has been suggested that this pre-target sorting contributes to map formation. Neurons in auditory regions of the brain are arranged according to their best frequency (BF), the sound frequency they respond to optimally. This BF changes predictably with position along the so-called tonotopic axis. In the avian auditory brainstem, the tonotopic organization of the second- and third-order auditory neurons in nucleus magnocellularis (NM) and nucleus laminaris (NL) has been well described. In this study we examine whether the decussating NM axons forming the crossed dorsal cochlear tract (XDCT) and innervating the contralateral NL are arranged in a systematic manner. We electroporated dye into cells in different frequency regions of NM to anterogradely label their axons in XDCT. The placement of dye in NM was compared to the location of labeled axons in XDCT. Our results show that NM axons in XDCT are organized in a precise tonotopic manner along the rostrocaudal axis, spanning the entire rostrocaudal extent of both the origin and target nuclei. We propose that in the avian auditory brainstem, this pretarget axon sorting contributes to tonotopic map formation in NL.

Afferent regulation of chicken auditory brainstem neurons: Rapid changes in phosphorylation of elongation factor 2

The relationships between protein synthesis and neuronal survival are poorly understood. In chicken nucleus magnocellularis (NM), significant alterations in overall protein synthesis precede neuronal death induced by deprivation of excitatory afferent activity. Previously we demonstrated an initial reduction in the overall rate of protein synthesis in all deprived NM neurons, followed by quick recovery (starting at 6 hours) in some, but not all, neurons. Neurons with recovered protein synthesis ultimately survive, whereas others become "ghost" cells (no detectable Nissl substance) at 12-24 hours and die within 48 hours. To explore the mechanisms underlying this differential influence of afferent input on protein synthesis and cell survival, the current study investigates the involvement of eukaryotic translation elongation factor 2 (eEF2), the phosphorylation of which reduces overall protein synthesis. Using immunocytochemistry for either total or phosphorylated eEF2 (p-eEF2), we found significant reductions in the level of phosphorylated, but not total, eEF2 in NM neurons as early as 0.5-1 hour following cochlea removal. Unexpectedly, neurons with low levels of p-eEF2 show reduced protein synthesis at 6 hours, indicated by a marker for active ribosomes. At 12 hours, all "ghost" cells exhibited little or no p-eEF2 staining, although not every neuron with a comparable low level of p-eEF2 was a "ghost" cell. These observations demonstrate that a reduced level of p-eEF2 is not responsible for immediate responses (including reduced overall protein synthesis) of a neuron to compromised afferent input but may impair the neuron's ability to initiate recovery signaling for survival and make the neuron more vulnerable to death.