Search

The avian nucleus laminaris (NL) is a brainstem nucleus necessary for binaural processing, analogous in structure and function to the mammalian medial superior olive. In chickens (Gallus gallus), NL is a well-studied model system for activity-dependent neural plasticity. Its neurons have bipolar extension of dendrites, which receive segregated inputs from two ears and display rapid and compartment-specific reorganization in response to unilateral changes in auditory input. More recently, fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates local protein translation, has been shown to be enriched in NL dendrites, suggesting its potential role in the structural dynamics of these dendrites. To explore the molecular role of FMRP in this nucleus, we performed proteomic analysis of NL, using micro laser capture and liquid chromatography tandem mass spectrometry. We identified 657 proteins, greatly represented in pathways involved in mitochondria, translation and metabolism, consistent with high levels of activity of NL neurons. Of these, 94 are potential FMRP targets, by comparative analysis with previously proposed FMRP targets in mammals. These proteins are enriched in pathways involved in cellular growth, cellular trafficking and transmembrane transport. Immunocytochemistry verified the dendritic localization of several proteins in NL. Furthermore, we confirmed the direct interaction of FMRP with one candidate, RhoC, by in vitro RNA binding assays. In summary, we provide a database of highly expressed proteins in NL and in particular a list of potential FMRP targets, with the goal of facilitating molecular characterization of FMRP signaling in future studies., (© 2017 Wiley Periodicals, Inc.)

Glial cells, previously thought to have generally supporting roles in the central nervous system, are emerging as essential contributors to multiple aspects of neuronal circuit function and development. This review focuses on the contributions of glial cells to the development of auditory pathways in the brainstem. These pathways display specialized synapses and an unusually high degree of precision in circuitry that enables sound source localization. The development of these pathways thus requires highly coordinated molecular and cellular mechanisms. Several classes of glial cells, including astrocytes, oligodendrocytes and microglia, have now been explored in these circuits in both avian and mammalian brainstems. Distinct populations of astrocytes are found over the course of auditory brainstem maturation. Early appearing astrocytes are associated with spatial compartments in the avian auditory brainstem. Factors from late appearing astrocytes promote synaptogenesis and dendritic maturation, and astrocytes remain integral parts of specialized auditory synapses. Oligodendrocytes play a unique role in both birds and mammals in highly regulated myelination essential for proper timing to decipher interaural cues. Microglia arise early in brainstem development and may contribute to maturation of auditory pathways. Together these studies demonstrate the importance of non-neuronal cells in the assembly of specialized auditory brainstem circuits.

A brainstem circuit for encoding the spatial location of sounds involves neurons in the cochlear nucleus that project to medial superior olivary (MSO) neurons on both sides of the brain via a single bifurcating axon. Neurons in MSO act as coincidence detectors, responding optimally when signals from the two ears arrive within a few microseconds. To achieve this, transmission of signals along the contralateral collateral must be faster than transmission of the same signals along the ipsilateral collateral. We demonstrate that this is achieved by differential regulation of myelination and axon caliber along the ipsilateral and contralateral branches of single axons; ipsilateral axon branches have shorter internode lengths and smaller caliber than contralateral branches. The myelination difference is established prior to the onset of hearing. We conclude that this differential myelination and axon caliber requires local interactions between axon collaterals and surrounding oligodendrocytes on the two sides of the brainstem., (© 2015 Wiley Periodicals, Inc.)

Neuronal dendrites are structurally and functionally dynamic in response to changes in afferent activity. The fragile X mental retardation protein (FMRP) is an mRNA binding protein that regulates activity-dependent protein synthesis and morphological dynamics of dendrites. Loss and abnormal expression of FMRP occur in fragile X syndrome (FXS) and some forms of autism spectrum disorders. To provide further understanding of how FMRP signaling regulates dendritic dynamics, we examined dendritic expression and localization of FMRP in the reptilian and avian nucleus laminaris (NL) and its mammalian analogue, the medial superior olive (MSO), in rodents and humans. NL/MSO neurons are specialized for temporal processing of low-frequency sounds for binaural hearing, which is impaired in FXS. Protein BLAST analyses first demonstrate that the FMRP amino acid sequences in the alligator and chicken are highly similar to human FMRP with identical mRNA-binding and phosphorylation sites, suggesting that FMRP functions similarly across vertebrates. Immunocytochemistry further reveals that NL/MSO neurons have very high levels of dendritic FMRP in low-frequency hearing vertebrates including alligator, chicken, gerbil, and human. Remarkably, dendritic FMRP in NL/MSO neurons often accumulates at branch points and enlarged distal tips, loci known to be critical for branch-specific dendritic arbor dynamics. These observations support an important role for FMRP in regulating dendritic properties of binaural neurons that are essential for low-frequency sound localization and auditory scene segregation, and support the relevance of studying this regulation in nonhuman vertebrates that use low frequencies in order to further understand human auditory processing disorders., (Copyright © 2013 Wiley Periodicals, Inc.)

Information processing in the brain relies on precise timing of signal propagation. The highly conserved neuronal network for computing spatial representations of acoustic signals resolves microsecond timing of sounds processed by the two ears. As such, it provides an excellent model for understanding how precise temporal regulation of neuronal signals is achieved and maintained. The well described avian and mammalian brainstem circuit for computation of interaural time differences is composed of monaural cells in the cochlear nucleus (CN; nucleus magnocellularis in birds) projecting to binaurally innervated coincidence detection neurons in the medial superior olivary nucleus (MSO) in mammals or nucleus laminaris (NL) in birds. Individual axons from CN neurons issue a single axon that bifurcates into an ipsilateral branch and a contralateral branch that innervate segregated dendritic regions of the MSO/NL coincidence detector neurons. We measured conduction velocities of the ipsilateral and contralateral branches of these bifurcating axon collaterals in the chicken by antidromic stimulation of two sites along each branch and whole-cell recordings in the parent neurons. At the end of each experiment, the individual CN neuron and its axon collaterals were filled with dye. We show that the two collaterals of a single axon adjust the conduction velocities individually to achieve the specific conduction velocities essential for precise temporal integration of information from the two ears, as required for sound localization. More generally, these results suggest that individual axonal segments in the CNS interact locally with surrounding neural structures to determine conduction velocity.

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., (Copyright © 2012 Wiley Periodicals, Inc.)

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., (Copyright © 2012 Wiley Periodicals, Inc.)

Research performed on transgenic animals has led to numerous advances in biological research. However, using traditional retroviral methods to generate transgenic avian research models has proved problematic. As a result, experiments aimed at genetic manipulations on birds have remained difficult for this popular research tool. Recently, lentiviral methods have allowed the production of transgenic birds, including a transgenic Japanese quail (Coturnix coturnix japonica) line showing neuronal specificity and stable expression of enhanced green fluorescent protein (eGFP) across generations (termed here GFP quail). To test whether the GFP quail may serve as a viable alternative to the popular chicken model system, with the additional benefit of genetic manipulation, we compared the development, organization, structure, and function of a specific neuronal circuit in chicken (Gallus gallus domesticus) with that of the GFP quail. This study focuses on a well-defined avian brain region, the principal nuclei of the sound localization circuit in the auditory brainstem, nucleus magnocellularis (NM), and nucleus laminaris (NL). Our results demonstrate that structural and functional properties of NM and NL neurons in the GFP quail, as well as their dynamic properties in response to changes in the environment, are nearly identical to those in chickens. These similarities demonstrate that the GFP quail, as well as other transgenic quail lines, can serve as an attractive avian model system, with the advantage of being able to build on the wealth of information already available from the chicken., (Copyright © 2012 Wiley Periodicals, Inc.)

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.

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.

Afferent input regulates neuronal dendritic patterning locally and globally through distinct mechanisms. To begin to understand these mechanisms, we differentially manipulate afferent input in vivo and assess effects on dendritic patterning of individual neurons in chicken nucleus laminaris (NL). Dendrites of NL neurons segregate into dorsal and ventral domains, receiving excitatory input from the ipsilateral and contralateral ears, respectively, via nucleus magnocellularis (NM). Blocking action potentials from one ear, by either cochlea removal or temporary treatment with tetrodotoxin (TTX), leads to rapid and significant retraction of affected NL dendrites (dorsal ipsilaterally and ventral contralaterally) within 8 h compared with the other dendrites of the same neurons. The degree of retraction is comparable with that induced by direct deafferentation resulting from transection of NM axons. Importantly, when inner ear activity is allowed to recover from TTX treatments, retracted NL dendrites regrow to their normal length within 48 h. The retraction and growth involve elimination of terminal branches and addition of new branches, respectively. Examination of changes in NL dendrites at 96 h after unilateral cochlea removal, a manipulation that induces cell loss in NM and persistent blockage of afferent excitatory action potentials, reveals a significant correlation between cell death in the ipsilateral NM and the degree of dendritic retraction in NL. These results demonstrate that presynaptic action potentials rapidly and reversibly regulate dendritic patterning of postsynaptic neurons in a compartment specific manner, whereas long-term dendritic maintenance may be regulated in a way that is correlated with the presence of silent presynaptic appositions.

Competition between presynaptic inputs has been suggested to shape dendritic form. This hypothesis can be directly tested on bitufted, auditory neurons in chicken nucleus laminaris (NL). Each NL neuron contains two relatively symmetrical dendritic arbors; the dorsal dendrites receive excitatory glutamatergic input from the ipsilateral ear, and the ventral dendrites receive corresponding input from the contralateral ear. To assess the effect of relative synaptic strength on NL dendrites, we used single-cell electroporation; electrophysiology; and live, two-photon laser scanning microscopy to manipulate both the amount and the balance of synaptic input to the two matching sets of dendrites. With simultaneous activation, both sets of dendrites changed together, either growing or retracting over the imaging period. In contrast, stimulation of only one set of dendrites (either dorsal or ventral) resulted in the unstimulated dendrites losing total dendritic branch length, whereas the stimulated dendrites exhibited a tendency to grow. In this system, balanced input leads to balanced changes in the two sets of dendrites, but imbalanced input results in differential changes. Time-lapse imaging revealed that NL dendrites respond to differential stimulation by first decreasing the size of their unstimulated dendrites and then increasing the size of their stimulated dendrites. This result suggests that the relative activity of presynaptic neurons dynamically controls dendritic structure in NL and that dendritic real estate can rapidly be shifted from inactive inputs to active inputs., (Copyright © 2011 Wiley-Liss, Inc.)

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at discreet periods of development as well as mechanisms for temporal coding in the central nervous system. Here we present a method to prepare chicken auditory brainstem slices that can be used for acute experimental procedures or to culture organotypic slices for long-term experimental manipulations. The chicken auditory brainstem is composed of nucleus angularis, magnocellularis, laminaris and superior olive. These nuclei are responsible for binaural sound processing and single coronal slice preparations preserve the entire circuitry. Ultimately, organotypic slice cultures can provide the opportunity to manipulate several developmental parameters such as synaptic activity, expression of pre and postsynaptic components, expression of aspects controlling excitability and differential gene expression This approach can be used to broaden general knowledge about neural circuit development, refinement and maturation.

The avian nucleus laminaris (NL) is involved in computation of interaural time differences (ITDs) that encode the azimuthal position of a sound source. Neurons in NL are bipolar, with dorsal and ventral dendritic arbors receiving input from separate ears. NL neurons act as coincidence detectors that respond maximally when input from each ear arrives at the two dendritic arbors simultaneously. Computational and physiological studies demonstrated that the sensitivity of NL neurons to coincident inputs is modulated by an inhibitory feedback circuit via the superior olivary nucleus (SON). To understand the mechanism of this modulation, the topography of the projection from SON to NL was mapped, and the morphology of the axon terminals of SON neurons in NL was examined in chickens (Gallus gallus). In vivo injection of AlexaFluor 568 dextran amine into SON demonstrated a coarse topographic projection from SON to NL. Retrogradely labeled neurons in NL were located within the zone of anterogradely labeled terminals, suggesting a reciprocal projection between SON to NL. In vivo extracellular physiological recording further demonstrated that this topography is consistent with tonotopic maps in SON and NL. In addition, three-dimensional reconstruction of single SON axon branches within NL revealed that individual SON neurons innervate a large area of NL and terminate on both dorsal and ventral dendritic arbors of NL neurons. The organization of the projection from SON to NL supports its proposed functions of controlling the overall activity level of NL and enhancing the specificity of frequency mapping and ITD detection., (© 2010 Wiley-Liss, Inc.)

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.

The organotypic slice culture (Stoppini et al. A simple method for organotypic cultures of nervous tissue. 1991;37:173-182) has become the method of choice to answer a variety of questions in neuroscience. For many experiments, however, it would be beneficial to image or manipulate a slice culture repeatedly, for example, over the course of many days. We prepared organotypic slice cultures of the auditory brainstem of P3 and P4 mice and kept them in vitro for up to 4 weeks. Single cells in the auditory brainstem were transfected with plasmids expressing fluorescent proteins by way of electroporation (Haas et al. Single-cell electroporation for gene transfer in vivo. 2001;29:583-591). The culture was then placed in a chamber perfused with oxygenated ACSF and the labeled cell imaged with an inverted wide-field microscope repeatedly for multiple days, recording several time-points per day, before returning the slice to the incubator. We describe a simple method to image a slice culture preparation during the course of multiple days and over many continuous hours, without noticeable damage to the tissue or photobleaching. Our method uses a simple, inexpensive custom-built insulator constructed around the microscope to maintain controlled temperature and uses a perfusion chamber as used for in vitro slice recordings., ((c) 2009 Wiley-Liss, Inc.)

Calcium signaling plays a role in synaptic regulation of dendritic structure, usually on the time scale of hours or days. Here we use immunocytochemistry to examine changes in expression of plasma membrane calcium ATPase type 2 (PMCA2), a high-affinity calcium efflux protein, in the chick nucleus laminaris (NL) following manipulations of synaptic inputs. Dendrites of NL neurons segregate into dorsal and ventral domains, receiving excitatory input from the ipsilateral and contralateral ears, respectively, via nucleus magnocellularis (NM). Deprivation of the contralateral projection from NM to NL leads to rapid retraction of ventral, but not the dorsal, dendrites of NL neurons. Immunocytochemistry revealed symmetric distribution of PMCA2 in two neuropil regions of normally innervated NL. Electron microscopy confirmed that PMCA2 localizes in both NM terminals and NL dendrites. As early as 30 minutes after transection of the contralateral projection from NM to NL or unilateral cochlea removal, significant decreases in PMCA2 immunoreactivity were seen in the deprived neuropil of NL compared with the other neuropil that continued to receive normal input. The rapid decrease correlated with reductions in the immunoreactivity for microtubule-associated protein 2, which affects cytoskeleton stabilization. These results suggest that PMCA2 is regulated independently in ventral and dorsal NL dendrites and/or their inputs from NM in a way that is correlated with presynaptic activity. This provides a potential mechanism by which deprivation can change calcium transport that, in turn, may be important for rapid, compartment-specific dendritic remodeling.

Mature nucleus magnocellularis (NM) neurons, the avian homolog of bushy cells of the mammalian anteroventral cochlear nucleus, maintain high [Cl-]i and depolarize in response to GABA. Depolarizing GABAergic postsynaptic potentials (GPSPs) activate both the synaptic conductance and large outward currents, which, when coupled together, inhibit spikes via shunting and spike threshold accommodation. We studied the maturation of the synaptic and voltage-dependent components of inhibition in embryonic NM neurons using whole-cell and gramicidin-perforated patch-clamp techniques to measure Cl- reversal potential, GABAergic synaptic responses, and voltage-dependent outward currents. We found that GABA enhanced excitability in immature NM neurons, undergoing a switch to inhibitory between embryonic day 14 (E14) and E18. Low-voltage-activated Kv1-type (dendrotoxin-I sensitive) K+ currents increased in amplitude between E14 and E18, whereas Cl- reversal potential and synaptic conductances remained relatively stable during this period. GABA was rendered inhibitory because of this increase in low-voltage activated outward currents. GPSPs summed with other inputs to increase spike probability at E14. GPSPs shunted spikes at E18, but blocking Kv1 channels transformed this inhibition to excitation, similar to E14 neurons. Subthreshold depolarizing current steps, designed to activate outward currents similar to depolarizing GPSPs, enhanced excitability at E14 but inhibited spiking in E18 neurons. Blocking Kv1 channels reversed this effect, rendering current steps excitatory. We present the novel finding that the developmental transition of GABAergic processing from increasing neuronal excitability to inhibiting spiking can depend on changes in the expression of voltage-gated channels rather than on a change in Cl- reversal potential.

Nucleus magnocellularis (NM), nucleus angularis (NA), and nucleus laminaris (NL), second- and third-order auditory neurons in the avian brainstem, receive GABAergic input primarily from the superior olivary nucleus (SON). Previous studies have demonstrated that both GABA(A) and GABA(B) receptors (GABA(B)Rs) influence physiological properties of NM neurons. We characterized the distribution of GABA(B)R expression in these nuclei during development and after deafferentation of the excitatory auditory nerve (nVIII) inputs. We used a polyclonal antibody raised against rat GABA(B)Rs in the auditory brainstem during developmental periods that are thought to precede and include synaptogenesis of GABAergic inputs. As early as embryonic day (E)14, dense labeling is observed in NA, NM, NL, and SON. At earlier ages immunoreactivity is present in somas as diffuse staining with few puncta. By E21, when the structure and function of the auditory nuclei are known to be mature, GABA(B) immunoreactivity is characterized by dense punctate labeling in NM, NL, and a subset of NA neurons, but label is sparse in the SON. Removal of the cochlea and nVIII neurons in posthatch chicks resulted in only a small decrease in immunoreactivity after survival times of 14 or 28 days, suggesting that a major proportion of GABA(B)Rs may be expressed postsynaptically or on GABAergic terminals. We confirmed this interpretation with immunogold TEM, where expression at postsynaptic membrane sites is clearly observed. The characterization of GABA(B)R distribution enriches our understanding of the full complement of inhibitory influences on central auditory processing in this well-studied neuronal circuit., (Copyright (c) 2005 Wiley-Liss, Inc.)

Afferent activity, especially in young animals, can have profound influences on postsynaptic neuronal structure, function and metabolic processes. Most studies evaluating activity regulation of cellular components have examined the expression of ubiquitous cellular proteins as opposed to molecules that are specialized in the neurons of interest. Here we consider the regulation of two proteins (voltage-gated potassium channel subunits Kv1.1 and Kv3.1) that auditory brainstem neurons in birds and mammals express at uniquely high levels. Unilateral removal of the avian cochlea leads to rapid and dramatic reduction in the expression of both proteins in the nucleus magnocellularis (NM; a division of the avian cochlear nucleus) neurons as detected by immunocytochemistry. Uniform downregulation of Kv1.1 was reliable by 3 hours after cochlea removal, was sustained through 96 hours, and returned to control levels in the surviving neurons by 2 weeks. The activity-dependent changes in Kv3.1 appear to be bimodal and are more transient, being observed at 3 hours after cochlea removal and recovering to control levels within 24 hours. We also explored the functional properties of Kv1.1 in NM neurons deprived of auditory input for 24 hours by whole-cell recordings. Low-threshold potassium currents in deprived NM neurons were not significantly different from control neurons in their amplitude or sensitivity to dendrotoxin-I, a selective K+ channel antagonist. We conclude that the highly specialized abundant expression of Kv1.1 and 3.1 channel subunits is not permanently regulated by synaptic activity and that changes in overall protein levels do not predict membrane pools., (Copyright 2004 Wiley-Liss, Inc.)

Deprivation of afferent input in young animals results in transneuronal degeneration of postsynaptic sensory neurons in a variety of species and sensory pathways. Transneuronal degeneration is generally not seen in adult animals. The cellular and molecular basis for this dramatic developmental change in susceptibility is not understood. One possibility is that genes involved in the apoptotic process are involved in determining cell death or survival after afferent deprivation. To further investigate this possibility, we performed unilateral cochlear ablation on wild-type and bcl-2-overexpressing mice at a variety of ages. In postnatal day 5 (P5) or P8 wild-type mice, cochlea removal resulted in a 54% or 31% neuronal loss in the anteroventral cochlear nucleus (AVCN), respectively. When the same manipulation is performed on a P30 mouse, no loss of AVCN neurons occurs. This confirmed a rather abrupt change in the sensitivity to disruption of afferent input, a critical period. However, in littermates expressing bcl-2 under a neuron-specific enolase promoter, no significant loss of AVCN neurons was observed at any age after unilateral cochlear ablation. Furthermore, wild-type mice demonstrate rapid expression of activated caspase-3 in AVCN neurons within hours of deafferentation, whereas bcl-2-overexpressing mice do not. This suggests that bcl-2 can influence cell survival after removal of afferent input during the critical period and is consistent with the hypothesis that caspase-3 is one effector of cell death under these circumstances. These data are the first to indicate that known apoptotic mediators can play a role in central neuronal plasticity in models of afferent deprivation.

Neurons in the avian cochlear nucleus are depolarized by GABAergic synaptic input. We recorded GABAergic synaptic currents using the gramicidin-perforated-patch method and found their reversal potential (V(rev)) to be depolarized relative to spike threshold, which is surprising given that these inputs are inhibitory. Depolarizing IPSPs (dIPSPs) are kept below spike generation threshold by the activation of a dendrotoxin-I-sensitive, voltage-gated K(+) conductance. We show experimentally that the polarity of IPSPs contributes to their efficacy; dIPSPs induce accommodation, the positive shift in spike threshold, and are therefore more strongly inhibitory than conventional, hyperpolarizing IPSPs in the same neurons. A similar inhibitory mechanism has been described in invertebrate sensory fibers and axons of dorsal root ganglion cells and may be a general means of amplifying the strength of inhibition in cases where the size of excitatory conductances greatly exceeds that of inhibitory conductances.

The avian auditory brainstem nuclei nucleus magnocellularis (NM) and nucleus laminaris (NL) display highly precise patterns of neuronal connectivity. NM projects tonotopically to the dorsal dendrites of ipsilateral NL neurons and to the ventral dendrites of contralateral NL neurons. The precision of this binaural segregation is evident at the earliest developmental stage at which connections can be observed. We have begun to examine the possibility that Eph receptor tyrosine kinase signaling is involved in establishing these spatially segregated connections. The expression of the EphA4 tyrosine kinase was examined at several developmental stages. EphA4 is expressed in rhombomere 5, which contains progenitors for both NM and NL. In this rhombomere, the labeling becomes striped during the time that precursor cells migrate to the auditory anlage. At the precise time when NM-NL projections are forming, EphA4 expression in NL is asymmetric, with markedly higher expression in the dorsal NL neuropil than in the ventral neuropil, suggesting a possible role in guiding growing axons to the appropriate region. At later embryonic ages EphA4 expression is symmetric around NL, and is absent in NM. As auditory function matures, EphA4 expression decreases so that by 4 days after hatch no EphA4 antibody labeling is evident in the auditory brainstem nuclei., (Copyright 2000 Wiley-Liss, Inc.)

Proliferation of astrocytes is a dramatic response of the central nervous system (CNS) to injury and disease. Such proliferation results in the formation of the neural/glial scar and the reconstitution of the glial limitans. However, not all astrocytes enter the proliferative cycle following injury, and for those that do, the period of cell division is limited. Little attention has focused on the events that regulate the duration and extent of astrocyte proliferation following damage, but clearly control mechanisms are in place as CNS injury does not result in the continuous astrocyte proliferation seen in glial tumorigenesis. Protein tyrosine phosphorylation has been implicated in both astrocyte proliferation and differentiation and plays an important role in the regulation of the cell cycle in a number of different systems. We have found a small subset of astrocytes in the chick auditory brainstem that are immunopositive for the protein tyrosine phosphatase SHP-1. SHP-1 appears to negatively regulate cellular division in the hematopoietic system and is involved in the mitogenic response to various growth factors. Following cochlea removal, there is a marked increase within the auditory brainstem nucleus, nucleus magnocellularis (NM), in both in the number of SHP-1-positive astrocytes and the length of their immunopositive fibers. Significantly, those animals showing the greatest increases in SHP-1 immunoreactivity do not exhibit large amounts of astrocyte proliferation. We hypothesize that the expression of SHP-1 plays a role in negatively regulating the mitotic behavior of astrocytes following deafferentation., (Copyright 2000 Wiley-Liss, Inc.)

Neurotrophins and their cognate receptors are critical to normal nervous system development. Trk receptors are high-affinity receptors for nerve-growth factor (trkA), brain-derived neurotrophic factor and neurotrophin-4/5 (trkB), and neurotrophin-3 (trkC). We examine the expression of these three neurotrophin tyrosine kinase receptors in the chick auditory system throughout most of development. Trks were localized in the auditory brainstem, the cochlear ganglion, and the basilar papilla of chicks from embryonic (E) day 5 to E21, by using antibodies and standard immunocytochemical methods. TrkB mRNA was localized in brainstem nuclei by in situ hybridization. TrkB and trkC are highly expressed in the embryonic auditory brainstem, and their patterns of expression are both spatially and temporally dynamic. During early brainstem development, trkB and trkC are localized in the neuronal cell bodies and in the surrounding neuropil of nucleus magnocellularis (NM) and nucleus laminaris (NL). During later development, trkC is expressed in the cell bodies of NM and NL, whereas trkB is expressed in the nerve calyces surrounding NM neurons and in the ventral, but not the dorsal, dendrites of NL. In the periphery, trkB and trkC are located in the cochlear ganglion neurons and in peripheral fibers innervating the basilar papilla and synapsing at the base of hair cells. The protracted expression of trks seen in our materials is consistent with the hypothesis that the neurotrophins/tyrosine kinase receptors play one or several roles in the development of auditory circuitry. In particular, the polarized expression of trkB in NL is coincident with refinement of NM terminal arborizations on NL.

Located in the ventrolateral region of the avian brainstem, the superior olivary nucleus (SON) receives inputs from nucleus angularis (NA) and nucleus laminaris (NL) and projects back to NA, NL, and nucleus magnocellularis (NM). The reciprocal connections between the SON and NL are of particular interest because they constitute a feedback circuit for coincidence detection. In the present study, the chick SON was investigated. In vivo tracing studies show that the SON projects predominantly to the ipsilateral NM, NL, and NA. In vitro whole-cell recording reveals single-cell morphology, firing properties, and postsynaptic responses. SON neurons are morphologically and physiologically suited for temporal integration; their firing patterns do not reflect the temporal structure of their excitatory inputs. Of most interest, direct stimulation of the SON evokes long-lasting inhibition in NL neurons. The inhibition blocks both intrinsic spike generation and orthodromically evoked activity in NL neurons and can be eliminated by bicuculline methiodide, a potent antagonist for GABAA receptor-mediated neurotransmission. These results strongly suggest that the SON provides GABAergic inhibitory feedback to laminaris neurons. We discuss a mechanism whereby SON-evoked GABAergic inhibition can influence the coding of interaural time differences for sound localization in the avian auditory brainstem.

The developing brainstem auditory system has been studied in detail by using anatomical and physiological techniques. However, it is not known whether immature auditory neurons exhibit different molecular characteristics than those of physiologically mature neurons. To address this issue, we examined the distribution of Cat-301 immunoreactivity in the developing auditory brainstem of gerbils. Cat-301 is a monoclonal antibody that recognizes a 680-kD chondroitin sulfate proteoglycan similar to aggrecan, a high-molecular-weight chondroitin sulfate proteoglycan found in cartilage. In the central nervous system, Cat-301 immunoreactivity is localized to the extrasynaptic surface of neurons. It has been hypothesized by Hockfield and co-workers (Hockfield et al. [1990a]Cold Spring Harbor Symp. Quart. Biol. 55:504-514) that the Cat-301 proteoglycan is a molecular marker indicating that a neuron has acquired mature neuronal properties. In the current study, Cat-301 staining is first seen at 7 days after birth in the anterior ventral cochlear nucleus (AVCN), the posterior VCN (PVCN), and the medial nucleus of the trapezoid body (MNTB) shortly before the onset of sound-evoked activity. By 21 days after birth, neurons in the AVCN, the PVCN, and the lateral and medial superior olive have attained adult-like distributions of Cat-301 staining concomitant with the physiological maturation of these neurons. Neurons in MNTB attain adult-like distributions of Cat-301 immunoreactivity at 1 year. The maturation of Cat-301 immunoreactivity parallels the physiological maturation of gerbil auditory neurons, and the Cat-301 proteoglycan may play a role in the formation and/or stabilization of auditory synapses.

1. Fura-2 imaging was used to measure the effects of glutamate on caffeine-sensitive Ca2+ stores in neurons of the avian cochlear nucleus, n. magnocellularis (NM). 2. On average, 100-mM caffeine stimulated a 250-nM increase in intracellular calcium ion concentration {[Ca2+]i} in Ca(2+)-free media; 1-mM glutamate significantly attenuated caffeine-stimulated Ca2+ responses. 3. The metabotropic glutamate receptor agonist, ACPD, also inhibited the caffeine-stimulated rise in [Ca2+]i. 4. Glutamate has an important role in regulating Ca2+ stores in NM neurons. Glutamate-deprivation (viz. cochlear removal) results in a rise in [Ca2+]i that may, in part, be the result of release from Ca2+ stores. We hypothesize that Ca(2+)-induced Ca2+ release stores (CICRs) may be involved in deprivation-induced cell death.

Neuronal survival in the cochlear nucleus of young animals is regulated by afferent activity. Removal or blockade of nerve VIII input results in the death of 20-40% of neurons in the cochlear nucleus, nucleus magnocellularis (NM), of the 10-14 days posthatch chick. Neuronal death in NM is preceded by complete failure of protein synthesis and degradation of ribosomes. In addition, there is a biphasic change in the immunoreactivity of ribosomes for a monoclonal antiribosomal RNA antibody, Y10B. Initially, the entire population of afferent-deprived NM neurons loses Y10B immunoreactivity, but, after 6 or 12 hours of afferent deprivation, lack of Y10B immunoreactivity specifically marks dying NM neurons. Whether RNA synthesis is also altered in afferent-deprived NM neurons has not previously been studied. To determine whether RNA synthesis in NM neurons is regulated by loss of afferent activity, we injected chicks with 3H-uridine following unilateral cochlea removal and measured the incorporation of RNA precursor with tissue autoradiography. As early as 1 hour after cochlea removal, there was a significant decrease in 3H-uridine incorporation by afferent-deprived NM neurons. After longer periods of afferent deprivation (6 or 12 hours), the majority of dying NM neurons (marked by loss of Y10B immunoreactivity) fail to incorporate RNA precursor. Six or 12 hours following cochlea removal, the subpopulation of surviving NM neurons incorporates 3H-uridine at increased levels over those observed 1 or 3 hours after cochlea removal. These findings suggest that nuclear function is regulated by afferent synaptic activity and that failure of RNA synthesis occurs early in the cell death process.

Neurons in the brainstem auditory nuclei, n. magnocellularis and n. laminaris, of the chick are contacted by terminals containing the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). In this report we describe the physiological response of these neurons to GABA using an in vitro slice preparation. In brainstem auditory neurons, GABA produced a depolarization of up to 20 mV and an associated decrease in input resistance. This depolarization was inhibitory; action potentials generated by orthodromic synaptic drive, antidromic stimulation and intracellular current injection were prevented by GABA application. The GABA response still occurred when synaptic transmission was prevented by perfusing the slice with a medium containing low Ca2+ and high Mg2+ concentrations. Thus, the effects of GABA were directly on the postsynaptic neuron and not via an interneuron. Whole-cell voltage clamp of neurons revealed that the reversal potential of the inward current was approximately -45 mV, suggesting that the channel responsible for this response is not selective for Cl- or K+. Pharmacological analyses suggest that this GABA receptor has properties distinct from those typical of either GABAa or GABAb receptors. Although a similar response was observed with the GABAa agonist, muscimol, it was not blocked by the GABAa antagonist, bicuculline. The response was not evoked by the GABAb agonist, baclofen, and was not blocked by the GABAb antagonist phaclofen. This unusual depolarizing response is not a common feature of all brainstem neurons. Neurons located in the neighboring medial vestibular nucleus show a more traditional response to GABA application. At resting potential, these neurons show a hyperpolarizing or biphasic response associated with a decrease in input resistance and inhibition of their spontaneous activity. GABA-induced responses in the medial vestibular nucleus are blocked by bicuculline. These results suggest that an unusual form of the GABA receptor is present in the brainstem auditory system of the chick. It is possible that this form of GABA receptor provides an efficient mechanism for inhibiting the relatively powerful EPSPs received by brainstem auditory neurons, or it may play a trophic role in the afferent regulation of neuronal integrity in this system.

The identification, purification, and biochemical characterization of specific markers for neuroglial cells in the central nervous system is an essential step toward a better understanding of the function of glial cells. This manuscript reports the identification and purification of a neuroglia-associated protein (NAP-185) with an apparent molecular mass of 185 kDa. While its expression is not restricted to the brain, it was first identified in a specific subpopulation of glial cells when chick brain stem sections were analyzed with an affinity-purified rabbit antiserum raised against the catalytic domain of the T-cell protein tyrosine phosphatase. This 185-kDa antigen was purified to apparent homogeneity and confirmed to be responsible for the neuroglial staining observed. In spite of its immunological relation to T-cell protein tyrosine phosphatase, purified NAP-185 failed to display tyrosine phosphatase activity. The primary sequence of five NAP-185-derived peptides shows that this protein has not yet been characterized and that it is possibly related to AP180, a clathrin-associated protein.

Astrocytes in the central nervous system (CNS) respond to injury and disease by proliferating and extending processes. The intermediate filament protein of astrocytes, glial fibrillary acidic protein (GFAP) also increases in astrocytes. These cells are called "reactive astrocytes" and are thought to play a role in CNS repair. We have previously demonstrated rapid increases (< 6 hours) in GFAP-immunoreactive and silver-impregnated glial processes in the chick cochlear nucleus, nucleus magnocellularis (NM), following cochlea removal or activity blockade of the eighth nerve. It was not known whether these changes were the result of glial proliferation, glial hypertrophy, or both. The present study examined the time course of astrocyte proliferation in NM following cochlea removal. Postnatal chicks received unilateral cochlea removal and survived for 6, 12, 18, 24, 36, 48, and 72 hours. Bromodeoxyuridine was used to label proliferating cells. The volume and number of labeled cells in NM was calculated for both the experimental and control sides of the brains for experimental animals was well as for unoperated control animals. A subset of astrocytes continuously divide in the normal posthatch chick brainstem. The percentage of labeled nuclei increases within NM 36 hours following cochlea removal and is robust by 48 hours. This increase is due to astrocyte proliferation within, rather than migration to, NM. These results indicate that rapid increases in GFAP following reduced activity are independent of cell proliferation. The time course of astrocyte proliferation suggests that cellular degeneration within the nucleus may play a role in upregulating astrocyte proliferation.

Third-order auditory neurons in the avian nucleus laminaris (NL) are the first to receive binaural input. In the chick, NL consists of a monolayer of neurons with polarized dendritic arbors oriented dorsally and ventrally. Afferents from second-order neurons in the ipsilateral nucleus magnocellularis (NM) innervate the dorsal dendrites of NL neurons, distributing processes of approximately equal length to NL neurons along an isofrequency band (roughly caudomedial to rostrolateral). Afferents from the contralateral NM innervate the ventral dendrites of NL neurons, distributing collateral branches sequentially as they proceed from caudomedial to rostrolateral along the isofrequency band of neurons. This innervation pattern could be the basis of a "delay line" circuit, as postulated in models of neural networks mediating sound localization. We examined this circuit by analyzing evoked field potentials using a brain slice preparation containing both NL and NM. The results were consistent with the previous anatomical findings. When the ipsilateral auditory nerve or ipsilateral NM was stimulated, there was no consistent variation in the latency of postsynaptic field potentials across the medial-to-lateral extent of NL. In contrast, when the contralateral NM or NM axons in the crossed dorsal cochlear tract were stimulated, a linear increase in the latency of postsynaptic potentials was observed from medial to lateral positions in NL. When stimulation amplitudes for both the ipsilateral and contralateral inputs were adjusted so as to produce little or no postsynaptic field potential, simultaneous bilateral stimulation evoked a pronounced response. Thus, NL neurons can act as "coincidence detectors." The amplitude of the postsynaptic response was dependent on the relative timing of stimulation of the two inputs. The optimal time difference changed systematically across the medial-to-lateral extent of NL. This system of delay lines and coincidence detectors could provide a mechanism for converting interaural time differences into a "place map" within NL.

We describe here rapid proliferation of astrocytic processes immunoreactive for glial fibrillary acidic protein (GFAP) in the chick cochlear nucleus following blockade of action potentials in the afferent nerve. Unilateral eighth nerve activity blockade was achieved through intralabyrinthine injection of TTX. Within 1 hr of activity blockade, a 56% increase in area density of GFAP-immunoreactive processes was found in the ipsilateral cochlear nucleus as compared to the contralateral side of the brain. This increase reached 152% by 3 hr. When eighth nerve activity was allowed to recover and animals were studied 1 week after TTX injection, no difference was found in GFAP immunoreactivity between the ipsilateral and contralateral cochlear nuclei. This is the first report of a glial reaction to documented neuronal inactivity in the absence of neuropathology. These results indicate that neuronal activity may regulate the structure of astrocytic processes.

Elimination of presynaptic elements often results in marked changes, such as atrophy and death, in postsynaptic neurons in the central nervous system. These transneuronal changes are particularly rapid and profound in young animals. In order to understand the cellular events underlying transneuronal regulation it is necessary to explore changes in the local environment of neurons following manipulations of their afferents. In previous investigations we have documented a variety of rapid and marked cellular changes in neurons of the cochlear nucleus of neonatal chicks (n. magnocellularis) following cochlea removal. In adult chickens, however, these transneuronal changes are either absent or minor. The goals of the studies presented here were to examine changes in the electrical activity of nucleus magnocellularis cells and their afferents following removal of the cochlea and to determine if these changes were similar in adult and neonatal animals. Two measures of electrical activity were used; multiunit recording with microelectrodes and incorporation of radiolabeled 2-deoxyglucose (2-DG). Microelectrode recordings revealed high levels of spontaneous activity in n. magnocellularis and n. laminaris, the binaural target of n. magnocellularis neurons. Neither puncturing of the tympanic membrane nor removal of the columella causes significant changes in spontaneous activity, although the latter results in a profound hearing loss (40-50 dB). Removal of the cochlea, on the other hand, results in immediate cessation of all extracellular electrical activity in the ipsilateral n. magnocellularis. Recordings from the same location for up to 6 h failed to reveal any return of spontaneous activity. When the electrode tip was placed in n. laminaris, unilateral cochlea removal had no discernible effect on extracellularly recorded spontaneous activity, probably due to the high levels of excitatory input from the intact ear. Bilateral cochlea removal, however, completely eliminated activity in n. laminaris. 2-DG studies conducted 1 h to 8 days following unilateral cochlea removal revealed marked decreases in 2-DG incorporation in the ipsilateral n. magnocellularis and bilaterally in the n. laminaris target of the ablated cochlea. No compensatory return of 2-DG incorporation was observed for up to 8 days. Comparisons of adult and neonatal chicks failed to reveal significant differences in the effects of cochlea removal on multiunit activity or 2-DG incorporation, suggesting that age differences in transneuronal regulation are due to intrinsic biochemical differences in young and adult neurons rather than differences in the proportion of synaptic input that has been abolished.

The ABR wave forms of 16-day-old and adult Mongolian gerbils were evoked by click stimuli presented at rates ranging from 1 to 80/sec. Wave I and wave IV thresholds were determined for each of 5 click rates. Amplitudes and latencies of waves I and IV were measured at each of 7 click rates and 3 intensity levels (15, 40 and 65 dB above threshold). Thresholds for waves I and IV in the adult gerbil and wave I in the 16 day gerbil were unaffected by changes in stimulus repetition rate. Neonatal wave IV thresholds were unaffected by click rate for rates below 25/sec but increased approximately 7 dB/decade increase in click rate when rate exceeded 25/sec. Increasing click rate produced greater reductions in ABR amplitude among neonates than adults for both waves I and IV. Decreases in amplitude due to increasing rate were independent of intensity level in both neonatal and adult subjects. Increasing rate produced similar increases in wave I latency among 16 day and adult subjects, but produced much greater increases in wave IV latency among neonates. Stimulus intensity level and click rate acted independently on wave I and wave IV latency in adult subjects and wave I latency in neonates. However, an interaction between rate and intensity was observed with respect to neonatal wave IV latency.

We have reviewed a series of experiments which begin to examine the cellular events underlying afferent regulation of neuronal structure. Our initial interest in such experiments stemmed from a desire to understand the cellular nature of experiential influences on brain development. While this remains a long-range goal, it's elusive nature has become increasingly apparent; how will we know when such a goal is achieved? On the other hand, it has become increasingly clear that by approaching this question as a subset of the larger problem of tissue interactions regulating nervous system structure and function, some progress is possible. In this respect, understanding afferent regulation is part and parcel of understanding "competition." Both exemplify the fact that we are dealing with a dynamic system, where changes in the balance of extracellular factors result in a cascade of events defining a new "steady state." Unfortunately, most of our methods are limited to taking "snap-shots" of a few parameters and attempting to reconstruct an epic. Our analyses of the postsynaptic events following cochlea removal have only scratched the surface. They are beginning to reveal myriad cellular processes that are dramatically altered by changing the balance of synaptic activity, or "synaptic drive," in a neuronal system. We have been continually struck by the rapidity of these postsynaptic changes when the manipulations are performed on immature animals. While the kinetics of metabolic and structural events we have studied do not yet match those of ionic events involved in information transmission, the two classes of intercellular communication are coming much closer. Some neuromodulators can alter synaptic currents for up to many seconds, and we have shown that altering afferent activity can cause changes in protein synthesis within a few minutes. The merging of these two classes of phenomena should come as no surprise since our studies and many others have definitively linked a variety of metabolic and structural events to changes in the synaptic drive between two neurons. On the other hand, this progress does highlight the need for increased attention to the short-term changes following manipulations of afferent activity. Hopefully such studies will lead to an understanding of the intracellular chain of events responsible for the regulation of neuronal form. A second area of interest has been the age restrictions on the events we have studied.(ABSTRACT TRUNCATED AT 400 WORDS)

The tonotopic organization of brainstem auditory nuclei was compared in embryonic and hatchling chickens. In embryos, neurons at any given position in these nuclei were maximally sensitive to lower frequency sounds than the best frequency after hatching. This finding indicates that neurons are maximally stimulated by sounds of different frequencies as development proceeds and supports the hypothesis that during development there is a change in the spatial encoding of frequency along the cochlea.

Studies of the avian auditory system indicate that neurons in nucleus magnocellularis (NM) and nucleus laminaris of young animals are dramatically altered by changes in the auditory receptor. We examined the role of presynaptic activity on these transneuronal regulatory events. TTX was used to block action potentials in the auditory nerve. TTX injections into the perilymph reliably blocked all neuronal activity in the cochlear nerve and NM. Far-field recordings of sound-evoked potentials revealed that responses returned within 6-12 hr after a single TTX injection. Changes in protein synthesis by NM neurons were measured by determining the incorporation of 3H-leucine using autoradiography. NM neurons on the side of the brain ipsilateral to the TTX injection were compared to normally active cells on the other side of the same tissue section. Grain counts over individual neurons revealed that a single injection of TTX produced a 40% decrease in grain density in ipsilateral NM neurons within 1.5 hr after the TTX injection. However, by 24 hr after a single TTX injection, grain densities were not different on the 2 sides of the brain. Continuous activity blockade for 6 hr caused the cessation of amino acid incorporation in a portion of NM neurons and a 15-20% decrease in the remaining neurons. These changes in amino acid incorporation are comparable to those following complete removal of the cochlea (Steward and Rubel, 1985). We also examined NM for neuron loss and soma shrinkage after blocking eighth nerve action potentials. TTX injected every 12 hr for 48 hr caused a 20% neuron loss and an 8% shrinkage of the remaining neurons. Similar reductions were found following cochlea removal (Born and Rubel, 1985). It is concluded that neuronal activity plays a major role in the maintenance of normal NM neurons. Furthermore, these results suggest that transneuronal morphological changes seen in neurons following deafferentation or alterations of sensory experience are a result of changes in the level of presynaptic activity.

Synaptic potentials were examined in the second- and third-order auditory neurons of nucleus magnocellularis and nucleus laminaris in the chick. Brain stems of mature chick embryos were explanted and maintained in vitro for 4 to 8 h. Field potentials, extracellular spike potentials and intracellular potentials evoked by 8th-nerve stimulation were examined. Eighth-nerve stimulation reliability elicited four identifiable field potentials which could be attributed to: (i) the afferent volley of the 8th-nerve axons, (ii) postsynaptic responses of n. magnocellularis neurons, and (iii) ipsilaterally and, (iv) contralaterally-evoked n. laminaris postsynaptic responses. Intracellular-recorded postsynaptic potentials were characterized by a rapid rise time and short duration. They were apparently monosynaptic with a synaptic delay of 0.4 ms. In each n. magnocellularis neuron the 'fast' excitatory postsynaptic potentials were composed of 1 to 3 all-or-none components. 'Slow' excitatory postsynaptic potentials were characterized by a longer latency, a longer duration and graded amplitude variation in proportion to the intensity of 8th-nerve stimulation. Both 'fast' and 'slow' excitatory postsynaptic potentials had similar reversal potentials. Since the 8th nerve makes monosynaptic connection with n. magnocellularis neurons, it is likely that at this synapse the 'fast' excitatory postsynaptic potentials were produced, while the 'slow' potential may be attributable to the convergence of many boutonal synapses of unknown origin. Intracellular injections of horseradish peroxidase into n. magnocellularis revealed that its efferents bifurcate below the nucleus and send one axon to the contralateral n. laminaris while the other axon forms a highly divergent projection to the ipsilateral laminar nucleus. The intracellular records obtained from n. laminaris are consistent with this anatomical finding in that graded excitatory postsynaptic potentials were elicited by 8th-nerve stimulation.

This study examined the development of individual axon terminal fields in n. laminaris (NL) of the chicken brainstem. In their mature form axons from the nucleus magnocellularis (NM), second-order auditory neurons in the chicken brainstem, project bilaterally onto the NL. Axons from the ipsilateral and contralateral NM neurons form spatially segregated, elongated arbors in the dorsal and ventral neuropil of NL, respectively. The long axes of these arbors correspond to physiologically defined isofrequency bands. To assess the development of this stereotyped arborization pattern, 6-17-day embryonic chicken brain stems were maintained in vitro while injecting horseradish peroxidase into small groups of axons. Three-dimensional reconstructions were made from serial sections and projected onto a cartesian plane for quantitative analyses. At embryonic day 6 (E6), the ventral axons already course beneath the recently migrated NL neurons. The arrival of the dorsal NM axon branches is delayed and their paths are indirect. They first loop dorsally into the the ventricular layer, where they seem to make specific connections with migrating NL neurons and use these as guides to their appropriate positions in the NL. During the period from E9 to E17 the dorsal and ventral terminal fields become similar, each adopting properties of the other's initial pattern. The dorsal terminal fields extend to form bands similar to the early ventral terminal fields, while the ventral terminal fields narrow and appear to shift position in order to achieve the tonotopic specificity characteristic of the early dorsal terminal fields. The results show that a complex, mature pattern of neuronal connections can be formed during development by the combination and reorganization of two simple patterns--each shaped, in turn, by its respective axonal trajectory.

The tonotopic and topographic organization of the bilateral projection from second-order auditory neurons of nucleus magnocellularis (NM) to nucleus laminaris (NL) was examined in young chickens. In one group of birds, the NM axons which innvervate the contralateral NL were severed by cutting the crossed dorsal cochlear tract at the midline. Heavy terminal degeneration in NL was confined to the neuropil area immediately ventral to the perikaryl lamina. Very little degeneration was seen in the dorsal neuropil region. In a second series of animals, the charactertistic frequency (CF) of cells in an area of NM was first determined by microelectrode recording techniques and then a small electrolytic lesion was made through the recording electrode. Following survival periods of 24-48 hours, the distribution of projections from the lesioned area to the ipsilateral and contralateral NL was examined using the Fink-Heimer method. As previously described in the pigeon, projections from NM terminate densely in the neuropil region immediately dorsal to the ipsilateral NL cell bodies and ventral to the perikaryl layer on the contralateral side, providing each NL neuron with segregated binaural innervation. Lesions in any area of the NM produced degeneration confined to a limited caudo-rostral and medio-lateral portion of both laminar nuclei. To investigate this topographic relationship, the cuado-rostral extents of the lesion in NM and of the resulting degeneration in both NL were determined. Linear regression and correlation analyses then related these positional values to each other and to the CF found at the center of each lesion. All correlations were highly significant and ranged from 0.78 between the position of the lesion in NM and CF to 0.91 between the caudo-rostral position of degeneration in the NL ipsilateral and contralateral to the lesion. It is concluded that neurons in NM project in a very discrete topographic, tonotopic and symmetrical fashion to NL on both sides of the brain, contributing to the binaural response properties and tonotopic organization of neurons in NL. The results also suggest that the organization of projections from NM to NL could provide a mechanism for the differential transmission delay required by a "place" model of low-frequency sound localization.

The brainstem auditory pathway of the chicken were used to examine the relationship between the maintenance of dendrites following denervation and the successful reinnervation (sprouting) by surviving afferents. In the system the third-order cells in n. laminaris receive spatially segregated binaural innervation from n. magnocellularis. Afferents from the ipsilateral n. magnocellularis innervate the dendrites on the dorsal aspect of n. laminaris cells, while afferents from contralateral magnocellular neurons innervate ventral dendrites via the crossed dorsal cochlear tract. Denervation of the ventral dendrites of n. laminaris cells by transection at the midline results in rapid and severe atrophy of the denervated dendrite. Unilateral cochlea removal induces transneuronal degeneration of 30-45% of the ipsilateral magnocellular cells, thereby partially denervating one dendrite of the n. laminaris cells on each side of the brain. In animals with long-standing transections of the crossed dorsal cochlear tract there is no evidence of sprouting the fibers from the ipsilateral n. magnocellularis when the projections of the surviving magnocellular neurons are traced with degeneration methods after a secondary cochlea removal. However, when dendrites of n. laminaris are partially denervated dendrites do not disappear. Furthermore, secondary lesions of the crossed dorsal cochlear tract or secondary cochlea removal reveal that these denervated dendrites are reinnervated by the afferents from the opposite n. magnocellularis which are normally restricted to the opposite dendrite of the n. laminaris cells.

Glycine-immunoreactivity (Gly-I) was studied in the auditory brain stem of chick using a polyclonal antiserum to glycine conjugated to bovine serum albumin (Wenthold et al., 1987). During embryonic development, little Gly-I is present in nucleus magnocellularis (NM), nucleus laminaris (NL), or nucleus angularis (NA). In posthatch chicks, a few Gly-I terminals are found on nerve cell bodies in NM. Gly-labeled terminals are rare in NL and NA. When present in NL, they appear to be apposed to neuronal somata and not to dendrites. Occasionally, a Gly-labeled cell can be found in NM. After unilateral cochlea removal, Gly-labeled terminals are still present in NM. Thus, the cochlea does not appear to be the source of the glycinergic afferents to NM. The pattern of staining of Gly-I contrasts sharply with that of gamma-aminobutyric acid-immunoreactivity (GABA-I). The number of Gly-I terminals in NM, NL and NA appears to be much less than that of GABA-I. In addition, GABA-I terminals are very abundant around both the cell bodies and dendrites of neurons in NM and NL, whereas Gly-I terminals are found only on neuronal somata. Gly-I in NM also appears to be much less than that in its mammalian homologue, the anteroventral cochlear nucleus.

The influence of monaural acoustic deprivation on the size of dendrites in n. laminaris in the chick was examined. Chicks were raised in a controlled acoustic environment with one ear occluded from 2 days prior to hatching until 25 days after hatching by an earplug which provided a conductive hearing loss of approximately 40 dB across the audible frequency range. Each n. laminaris cell receives spatially segregated binaural excitatory innervation; one dendritic field received input from the plugged ear while the other received input from the normal ear. This arrangement allowed comparison of the size (length) of the "deprived" dendrites and the "nondeprived" dendrites for each cell. The tonotopic organization of n. laminaris allowed these comparisons to be made as a function of the frequency organization of the nucleus. We observed systematic changes in dendritic size which differed as a function of the tonotopic position of the neurons. In high-frequency regions the dendrites receiving information from the deprived ear were shorter than those receiving input from the normal ear. Unexpectedly, cells responsive to low frequencies showed the opposite result; the dendrites innervated from the deprived ear were longer than those responsive to the nondeprived ear. These results suggests that a relatively flat conductive hearing loss may cause nonuniform changes in activity impinging on high- and low-frequency areas of the auditory system.

The effect of unilateral basilar papilla removal on glucose uptake in the 2nd and 3rd order auditory nuclei in the chick's brain stem, nucleus magnocellularis and nucleus laminaris, respectively, was examined with [3H]2-deoxy-D-glucose (2-DG) autoradiography. The tissue was processed according to a thaw-mount technique, and the number of grains in the resulting autoradiographs was counted to assess changes in glucose uptake. It was observed that there is a greater density of grains over the neuropil regions of nucleus laminaris which receive input from the normal ear than over the corresponding regions which receive input from the operated ear. Similarly, differences in grain density are found between the normally innervated and deafferented magnocellular nuclei although these differences are not as great as those in nucleus laminaris. Differences in grain density were also apparent between the glial/fiber regions which bound the neuropil areas of nucleus laminaris; there is a greater density of grains overlying those glial/fiber regions through which fibers receiving input from the normal ear course than over those regions through which fibers which normally carry input from the operated ear travel. It is likely that this difference mainly reflects glucose uptake in the fibers although a possible contribution of glial tissue cannot be excluded. All these effects of basilar papilla removal are seen with survival times as short as 70 min and thus likely reflect the reduction of neural activity rather than the degeneration of pre- or postsynaptic elements. Finally, the same pattern of results as described above was found when using the more common [14C]2-DG procedure or when using [3H]2-DG but processing the tissue using the freeze-dried technique. The present results thus show the neuropil regions of nucleus laminaris and the adjacent glial/fiber areas to be areas of high glucose utilization. Unilateral basilar papilla removal results in the removal of an excitatory input to these regions, and this results in a reduction of glucose utilization that is specific to those neuropil regions and glial/fiber areas that receive input from the operated ear. These findings are contrasted with another study in which removal of a major excitatory input to the dentate gyrus of the rat results in a reduced glucose utilization which is not specific to the deafferented region and which largely reflects post- rather than presynatic events.

The pattern of primary auditory projections to the brain stem of young chickens was investigated using terminal degeneration methods and orthograde transport of horseradish peroxidase (HRP) or tritiated amino acid. Of particular interest was the question of whether nucleus laminaris (NL) receives primary afferents. A study of silver-stained degeneration pattersn in nucleus magnocellularis (NM) and NL at three intervals following unilateral interruption of the cochlear nerve revealed that by 48 hours after the lesion, degenerating terminals were found only in the ipsilateral nucleus angularis (NA), NM and lagenar projection areas but not in NL. Five- and eight-day survival times, however, also revealed degeneration bilaterally in NL. The appearance of terminal degeneration in NL at the longer survival times is attributed to the previously-reported severe and rapid transneuronal degeneration of neurons in NM following deafferentation and not to the presence of cochlear nerve terminals in NL. Injection of HRP or tritiated proline into the basilar papilla produced patterns of labeling similar to that seen in the 2-day degeneration material; HRP reaction product or autoradiographic label were seen only in the ipsilateral NA and NM and in the ipsilateral projection areas of the macula lagena but not in either NL. The patterns of primary auditory projections revealed by the three methods were quite similar to each other and to that previously reported for the pigeon and confirm the conslucion that the laminar nucleus of chickens does not receive primary afferents.

Previous studies of the avian auditory system have revealed that removal of the peripheral receptor (the cochlea) leads to a transneuronal degeneration of auditory relay neurons in nucleus magnocellularis (NM) of the brain stem. An early manifestation of the degeneration which can be observed within 12 hours is a decrease of histochemical staining for RNA (Nissl staining); such a decrease could reflect an alteration in protein synthetic activity within the NM neurons. The present study evaluates this possibility by determining whether the cochlea removal led to an alteration incorporation of protein precursors in the target neurons which exhibit transneuronal degeneration and if so, how early the changes appeared. The cochlea was removed unilaterally in seventeen 10-day-old chicks and two 66-week-old mature chickens, and incorporation of protein precursors was evaluated in the neurons of NM at 0.5, 1.5, 3, 6, 12, and 24 hours following the cochlea removal. Each chick received an intravenous injection of 3H leucine, and was allowed to survive for 30 minutes after the injection of precursor. The brains were then prepared for autoradiography. The extent of incorporation by neurons in NM was determined by counting grains overlying each cell body and determining grain density/micrometers2 of neuron cross-sectional area. We found that auditory relay neurons whose synaptic inputs have been silenced exhibit dramatic decreases in protein synthesis within 30 minutes after removal of the cochlea; leucine incorporation was reduced by about 50%. In chicks sacrificed 3 to 24 hours after removal of the cochlea, some neurons (about 1/3) were entirely unlabeled despite heavy labeling of their neighbors and heavy labeling of all NM neurons on the opposite side of the brain. The remaining neurons exhibited about a 15% reduction in incorporation in comparison with the cells in the contralateral (control) NM. While the decreases in incorporation were apparent at all survival intervals, there was no consistent decrease in Nissl staining until 6 hours after cochlea removal. There were no changes in protein precursor incorporation following removal of the cochlea in adult birds, a result which is in keeping with the relative absence of transneuronal degeneration following removal of the cochlea at maturity. The results suggest a very rapid transneuronal regulation of protein metabolism within target neurons in young animals, perhaps by activity-related events.

The consequences of cochlea removal on neuron number and soma cross-sectional area were examined in the second order auditory nucleus (n. magnocellularis) of chickens. Both the age of the subjects at the time of cochlea (basilar papilla) removal (1-66 weeks) and the survival period (1-45 days) were varied. Neuron number and soma cross-sectional area were determined from Nissl stained sections. Additional material was processed to examine the relationship of ganglion cell loss to changes in n. magnocellularis. Neuron number decreased by 25-30% and soma cross-sectional area decreased by 10-20% ipsilateral to the cochlea removal in chickens operated on during the first 6 weeks after hatching. In contrast, in chickens operated on at 66 weeks posthatch neuron number decreased less than 10% and there was no change in soma area. The changes were rapid, being nearly complete 2 days after cochlea removal. An initial change (1 and 2 days after surgery) observed in animals operated on up to 6 weeks posthatch was the presence of a large number of neurons in which no Nissl substance could be detected. These results demonstrate an age-dependent change in the susceptibility of NM neurons to deafferentation. This change is not temporally related to other measures of functional maturation of the auditory system.

4- to 8-day-old chicks were used to examine the ultrastructure of end bulbs and neurons of nucleus magnocellularis by freeze-fracture and thin section techniques. Both normal animals and animals deafferented by removal of the basilar papilla were used to describe the pre- and postsynaptic membranes of these neurons. Postsynaptic membrane specializations similar to those described in rodents were found in normal and deafferented chicks. In the presynaptic end bulb there was an immediate cessation of endocytotic vesicle formation after deafferentation. There was also a shift in the preferential fracture plane from the presynaptic to postsynaptic membranes 48-72 h after basilar papilla removal. These results indicate that the ultrastructure of this synaptic complex is comparable to that seen in mammals.