Rapid regulation of microtubule-associated protein 2 in dendrites of nucleus laminaris of the chick following deprivation of afferent activity.

Differential innervation of segregated dendritic domains in the chick nucleus laminaris (NL), composed of third-order auditory neurons, provides a unique model to study synaptic regulation of dendritic structure. Altering the synaptic input to one dendritic domain affects the structure and length of the manipulated dendrites while leaving the other set of unmanipulated dendrites largely unchanged. Little is known about the effects of neuronal input on the cytoskeletal structure of NL dendrites and whether changes in the cytoskeleton are responsible for dendritic remodeling following manipulations of synaptic input. In this study, we investigate changes in the immunoreactivity of high-molecular weight microtubule associated protein 2 (MAP2) in NL dendrites following two different manipulations of their afferent input. Unilateral cochlea removal eliminates excitatory synaptic input to the ventral dendrites of the contralateral NL and the dorsal dendrites of the ipsilateral NL. This manipulation produced a dramatic decrease in MAP2 immunoreactivity in the deafferented dendrites. This decrease was detected as early as 3 h following the surgery, well before any degeneration of afferent axons. A similar decrease in MAP2 immunoreactivity in deafferented NL dendrites was detected following a midline transection that silences the excitatory synaptic input to the ventral dendrites on both sides of the brain. These changes were most distinct in the caudal portion of the nucleus where individual deafferented dendritic branches contained less immunoreactivity than intact dendrites. Our results suggest that the cytoskeletal protein MAP2, which is distributed in dendrites, perikarya, and postsynaptic densities, may play a role in deafferentation-induced dendritic remodeling.

Accommodation enhances depolarizing inhibition in central neurons.

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.

Tonotopic map of potassium currents in chick auditory hair cells using an intact basilar papilla.

The avian basilar papilla is tonotopically organized such that hair cells along the sensory epithelium respond best to acoustic stimulation at differing frequencies. This specificity arises due to the mechanics of the cochlea itself and intrinsic electrical properties of the hair cells. Tall hair cells show membrane voltage oscillations in response to step current injection that may allow cells to act as electrical resonators, boosting the response at the resonant frequency. These oscillations and the underlying currents have been studied in enzymatically isolated cells. This study uses a whole chick (Gallus domesticus) basilar papilla preparation where the entire epithelium and its afferent connections are intact. With this preparation, a map of changes in potassium currents of tall hair cells was produced. All cells recorded from expressed two K+ currents, a calcium-activated K+ current, I(K(Ca)), and a voltage-activated K+ current, I(K). Also, apical cells expressed an inward rectifier K+ current, I(IR). The amplitude of total outward current increases in a gradient along the tonotopic axis. Pharmacological blockers were used to separate the outward K+ currents. These experiments showed that both currents individually increase in magnitude along a gradient from apex to base. Finally, measurements of oscillation frequency in response to current steps suggest a discontinuous change in the electrical resonances at about 33% from the apex. This study demonstrates a new preparation to study the electrical properties of hair cells in more detail along the tonotopic axis of the chick basilar papilla.

Visual influences on the development and recovery of the vestibuloocular reflex in the chicken.

Whenever the head turns, the vestibuloocular reflex (VOR) produces compensatory eye movements to help stabilize the image of the visual world on the retina. Uncompensated slip of the visual world across the retina results in a gradual change in VOR gain to minimize the image motion. VOR gain changes naturally during normal development and during recovery from neuronal damage. We ask here whether visual slip is necessary for the development of the chicken VOR (as in other species) and whether it is required for the recovery of the VOR after hair cell loss and regeneration. In the first experiment, chickens were reared under stroboscopic illumination, which eliminated visual slip. The horizontal and vertical VORs (h- and vVORs) were measured at different ages and compared with those of chickens reared in normal light. Strobe-rearing prevented the normal development of both h- and vVORs. After 8 wk of strobe-rearing, 3 days of exposure to normal light caused the VORs to recover partially but not to normal values. In the second experiment, 1-wk-old chicks were treated with streptomycin, which destroys most vestibular hair cells and reduces hVOR gain to zero. In birds, vestibular hair cells regenerate so that after 8 wk in normal illumination they appear normal and hVOR gain returns to values that are normal for birds of that age. The treated birds in this study recovered in either normal or stroboscopic illumination. Their hVOR and vVOR and vestibulocollic reflexes (VCR) were measured and compared with those of untreated, age-matched controls at 8 wk posthatch, when hair cell regeneration is known to be complete. As in previous studies, the gain of the VOR decreased immediately to zero after streptomycin treatment. After 8 wk of recovery under normal light, the hVOR was normal, but vVOR gain was less than normal. After 8 wk of recovery under stroboscopic illumination, hVOR gain was less than normal at all frequencies. VCR recovery was not affected by the strobe environment. When streptomycin-treated, strobe-recovered birds were then placed in normal light for 2 days, hVOR gain returned to normal. Taken together, the results of these experiments suggest that continuous visual feedback can adjust VOR gain. In the absence of appropriate visual stimuli, however, there is a default VOR gain and phase to which birds recover or revert, regardless of age. Thus an 8-wk-old chicken raised in a strobe environment from hatch would have the same gain as a streptomycin-treated chicken that recovers in a strobe environment.

Hair cell regeneration and recovery of auditory thresholds following aminoglycoside ototoxicity in Bengalese finches.

Birds regenerate auditory hair cells when original hair cells are lost. Regenerated hair cells become innervated and restore hearing function. Functional recovery during hair cell regeneration is particularly interesting in animals that depend on hearing for vocal communication. Bengalese finches are songbirds that depend on auditory feedback for normal song learning and maintenance. We examined the structural and functional recovery of the Bengalese finch basilar papilla after aminoglycoside ototoxicity. Birds were treated with the ototoxic aminoglycoside, amikacin, daily for 1 week. Treatment resulted in hair cell loss across the basal half of the basilar papilla and corresponding high frequency hearing loss. Hair cell regeneration and recovery of auditory brainstem responses were compared in the same animals. Survival times following treatment were between 1 day and 12 weeks. Analysis of structural recovery at weekly intervals indicated that hair cells in the Bengalese finch papilla require a maximum of 1 week to regenerate and appear with immature morphology at the epithelial surface. An additional 6 days are required for adult-like morphology to develop. Repopulation of the damaged region was complete by 8 weeks. Recovery of auditory thresholds began 1 week after treatment and reached asymptote by 4 weeks. Slight residual threshold shifts at 2.0 kHz and above were observed up to 12 weeks after treatment. Direct comparison of structural and functional recovery indicates that auditory thresholds recover maximally before a full complement of hair cells has regenerated.

FGFR3 expression during development and regeneration of the chick inner ear sensory epithelia.

Several studies suggest fibroblast growth factor receptor 3 (FGFR3) plays a role in the development of the auditory epithelium in mammals. We undertook a study of FGFR3 in the developing and mature chicken inner ear and during regeneration of this epithelium to determine whether FGFR3 shows a similar pattern of expression in birds. FGFR3 mRNA is highly expressed in most support cells in the mature chick basilar papilla but not in vestibular organs of the chick. The gene is expressed early in the development of the basilar papilla. Gentamicin treatment sufficient to destroy hair cells in the basilar papilla causes a rapid, transient downregulation of FGFR3 mRNA in the region of damage. In the initial stages of hair cell regeneration, the support cells that reenter the mitotic cycle in the basilar papilla do not express detectable levels of FGFR3 mRNA. However, once the hair cells have regenerated in this region, the levels of FGFR3 mRNA and protein expression rapidly return to approximate those in the undamaged epithelium. These results indicate that FGFR3 expression changes after drug-induced hair cell damage to the basilar papilla in an opposite way to that found in the mammalian cochlea and may be involved in regulating the proliferation of support cells.

Patterns of cell death in mouse anteroventral cochlear nucleus neurons after unilateral cochlea removal.

Developmental changes that influence the results of removal of afferent input on the survival of neurons of the anteroventral cochlear nucleus (AVCN) of mice were examined with the hope of providing a suitable model for understanding the cellular and molecular basis for these developmental changes in susceptibility. We performed unilateral cochlear ablation on wild-type mice at a variety of ages around the time of hearing onset to determine developmental changes in the sensitivity of AVCN neurons to afferent deprivation. In postnatal day 5 (P5) mice, cochlea removal resulted in 61% neuronal loss in the AVCN. By age P14, fewer than 1% of AVCN neurons were lost after this manipulation. This reveals a rather abrupt change in the sensitivity to disruption of afferent input, a critical period. We next investigated the temporal events associated with neuron loss after cochlea removal in susceptible animals. We demonstrate that significant cell loss occurs within 48 hours of cochlea removal in P7 animals. Furthermore, evidence of apoptosis was observed within 12 hours of cochlea removal, suggesting that the molecular events leading to cell loss after afferent deprivation begin to occur within hours of cochlea removal. Finally, we began to examine the role of the bcl-2 gene family in regulating afferent deprivation-induced cell death in the mouse AVCN. AVCN neurons in mature bcl-2 knockout mice demonstrate susceptibility to removal of afferent input comparable to neonatal sensitivity of wild-type controls. These data suggest that bcl-2 is one effector of cell survival as these cells switch from afferent-dependent to -independent survival mechanisms.

Cellular studies of auditory hair cell regeneration in birds.

A decade ago it was discovered that mature birds are able to regenerate hair cells, the receptors for auditory perception. This surprising finding generated hope in the field of auditory neuroscience that new hair cells someday may be coaxed to form in another class of warm-blooded vertebrates, mammals. We have made considerable progress toward understanding some cellular and molecular events that lead to hair cell regeneration in birds. This review discusses our current understanding of avian hair cell regeneration, with some comparisons to other vertebrate classes and other regenerative systems.

Developmental regulation of EphA4 expression in the chick auditory brainstem.

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.

Embryonic origins of auditory brain-stem nuclei in the chick hindbrain.

The auditory nuclei of the chick brain stem have distinct morphologies and highly specific synaptic connectivity. Nucleus magnocellularis (NM) and nucleus angularis receive tonotopically ordered cochlear input. NM in turn projects tonotopically to nucleus laminaris (NL), maintaining binaural specificity with projections to either dorsal or ventral NL dendrites. NM and NL arise from a common anlage, which differentiates as the cells migrate and acquire their mature morphologies. NM and NL cells are closely associated during embryogenesis and synapse formation. However, the morphologies of the nuclei and of the cells within the nuclei differ greatly between NM and NL. While later maturation of these nuclei has been described in considerable detail, relatively little is known about the early embryonic events that lead to the formation of these nuclei. We examined the embryonic origins of cells in brain-stem auditory nuclei with particular emphasis on NM and NL. Lipophilic dyes were injected into small regions of the embryonic hindbrain prior to the birth and migration of cells that contribute to these nuclei. We found that NM arises from rhombomeres r5, r6, and r7, and NL arises mostly from r5 with a few cells arising from r6. NM and NL thus have partially overlapping rhombomeres of origin. However, we found that the precursors for NM and NL are found in distinct regions within rhombomere 5, with NM precursors in medial regions and NL precursors in lateral regions. Our results do not support a lineage relationship between NM and NL cells and they suggest that NM and NL are specified prior to migration of precursors to the auditory anlage.

Tyrosine phosphatase SHP-1 immunoreactivity increases in a subset of astrocytes following deafferentation of the chicken auditory brainstem.

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.

Temporal, spatial, and morphologic features of hair cell regeneration in the avian basilar papilla.

Hair cell-selective antibodies were used in combination with the nucleotide bromode-oxyuridine (BrdU) to examine the temporal, spatial, and morphologic progression of auditory hair cell regeneration in chicks after a single gentamicin injection. New hair cells are first identifiable with an antibody to class III beta (beta) tubulin (TuJ1) by 14 hours after BrdU incorporation, but progenitor cells in S phase and M phase are TuJ1-negative. TuJ1 labeling reveals that new hair cells are first detected at 3 days after gentamicin, in the base, and the emergence and maturation of regenerating hair cells spreads apically over time. Differentiation of regenerating hair cells consists of a progressive series of morphologic changes. During early differentiation (14 hours to 1 day after BrdU), regenerating hair cells are round or fusiform and remain near the lumen, where they are generated. During intermediate differentiation (2-4 days after BrdU), regenerating hair cells resemble support cells; their somata are elongated, their nuclei are in the support cell layer, and they appear to contact both the lumenal surface and the basal lamina. The 275-kDa hair cell antigen is first expressed in regenerating hair cells during this period. During late differentiation (7 days after BrdU and later), TuJ1-positive cells acquire the globose shape of mature hair cells. Labeling with antibodies to hair cell antigen, calmodulin, and ribosomal RNA confirms this morphologic progression. Examination of sister cells born at 3 days post-gentamicin reveals that there is equal likelihood that they will assume the hair cell or support cell fate (i.e., both asymmetric and symmetric differentiation occur).

Characterization of damage and regeneration in cultured avian utricles.

Hair cell regeneration occurs spontaneously throughout life and following hair cell injury in the vestibular epithelia of mature birds and other nonmammalian vertebrates. We examined hair cell regeneration in post-hatch chick utricles that were cultured in media with or without the ototoxin, streptomycin, for various periods. The goal of our study was to characterize the dose- and time-dependent effects of streptomycin on hair cell loss and regeneration in vitro. Utricles that were cultured with streptomycin for 1 day displayed a dose-dependent loss of hair cells in spatial patterns and levels that were consistent with those observed in comparable experimental paradigms in vivo. Incorporation of the nucleotide analog bromodeoxyuridine (BrdU) demonstrated that supporting cell proliferation is decreased during the first day of culture in the presence of streptomycin, but it increases over time when cultures are subsequently placed in streptomycin-free media. Utricles cultured for 1 day with streptomycin followed by 2-4 more days without streptomycin had numerous bundles of immature stereocilia, suggesting that new hair cells were generated in vitro. We tested this hypothesis by culturing utricles with BrdU for 3 or 5 days and double-labeling them to detect BrdU and the hair cell-specific antigen, TuJ1. Numerous BrdU-positive/TuJ1-positive cells with phenotypic characteristics of immature hair cells were present in the cultures, and the number of such cells increased between 3 and 5 days in vitro, in a dose-dependent manner.

GABAergic inhibition in nucleus magnocellularis: implications for phase locking in the avian auditory brainstem.

In the avian auditory brainstem, nucleus magnocellularis (NM) functions to relay phase-locked signals to nucleus laminaris for binaural coincidence detection. Although many studies have revealed that NM neurons exhibit intrinsic physiological and anatomical specializations for this purpose, the role of inhibition has not been fully explored. The present study characterizes the organization of GABAergic feedback to NM. Anterograde and retrograde labeling methods showed that NM receives a prominent projection from the ipsilateral superior olivary nucleus (SON). The functional features of this projection were explored in a brain slice preparation. Stimulating fibers from the SON evoked long-lasting, depolarizing responses in NM neurons that were blockable by bicuculline, a GABA(A) receptor antagonist. The slow time course of these responses allowed them to undergo temporal summation during repetitive stimulation. The summed GABAergic response was capable of blocking spikes generated in NM neurons by suprathreshold current injection. This inhibitory effect was attributable to a large reduction in input resistance caused by a combination of the opening of a GABAergic Cl(-) conductance and the recruitment of a low-voltage activated K(+) conductance. This large reduction of input resistance increased the amount of current necessary to drive NM neurons to threshold. The results lead us to propose that GABAergic inhibition enhances phase-locking fidelity of NM neurons, which is essential to binaural coincidence detection in nucleus laminaris.

Ontogenetic expression of trk neurotrophin receptors in the chick auditory system.

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.

Life and death in otolaryngology: mechanisms of apoptosis and its role in the pathology and treatment of disease.

OBJECTIVES: To review recent advances in our understanding of programmed cell death, or apoptosis, and discuss implications of these basic science advances in our understanding of causes and potential treatments of a variety of diseases of the head and neck. DATA SOURCES: Basic science literature relevant to the study of apoptosis and its clinical implications. CONCLUSIONS: Apoptosis is now understood to be important in the normal development and survival of all multicellular organisms. Deregulation of this normally tightly controlled process underlies a variety of disease states, including neoplasia, autoimmune disease, and disorders of the central nervous system. A better understanding of this process and its regulation may help otolaryngologists better understand diseases relevant to this specialty and will lead to improved therapeutic interventions.

Glutamate regulates IP3-type and CICR stores in the avian cochlear nucleus.

Neurons of the avian cochlear nucleus, nucleus magnocellularis (NM), are activated by glutamate released from auditory nerve terminals. If this stimulation is removed, the intracellular calcium ion concentration ([Ca2+]i) of NM neurons rises and rapid atrophic changes ensue. We have been investigating mechanisms that regulate [Ca2+]i in these neurons based on the hypothesis that loss of Ca2+ homeostasis causes the cascade of cellular changes that results in neuronal atrophy and death. In the present study, video-enhanced fluorometry was used to monitor changes in [Ca2+]i stimulated by agents that mobilize Ca2+ from intracellular stores and to study the modulation of these responses by glutamate. Homobromoibotenic acid (HBI) was used to stimulate inositol trisphosphate (IP3)-sensitive stores, and caffeine was used to mobilize Ca2+ from Ca2+-induced Ca2+ release (CICR) stores. We provide data indicating that Ca2+ responses attributable to IP3- and CICR-sensitive stores are inhibited by glutamate, acting via a metabotropic glutamate receptor (mGluR). We also show that activation of C-kinase by a phorbol ester will reduce HBI-stimulated calcium responses. Although the protein kinase A accumulator, Sp-cAMPs, did not have an effect on HBI-induced responses. CICR-stimulated responses were not consistently attenuated by either the phorbol ester or the Sp-cAMPs. We have previously shown that glutamate attenuates voltage-dependent changes in [Ca2+]i. Coupled with the present findings, this suggests that in these neurons mGluRs serve to limit fluctuations in intracellular Ca2+ rather than increase [Ca2+]i. This system may play a role in protecting highly active neurons from calcium toxicity resulting in apoptosis.

Recovery of the vestibulocolic reflex after aminoglycoside ototoxicity in domestic chickens.

Avian auditory and vestibular hair cells regenerate after damage by ototoxic drugs, but until recently there was little evidence that regenerated vestibular hair cells function normally. In an earlier study we showed that the vestibuloocular reflex (VOR) is eliminated with aminoglycoside antibiotic treatment and recovers as hair cells regenerate. The VOR, which stabilizes the eye in the head, is an open-loop system that is thought to depend largely on regularly firing afferents. Recovery of the VOR is highly correlated with the regeneration of type I hair cells. In contrast, the vestibulocolic reflex (VCR), which stabilizes the head in space, is a closed-loop, negative-feedback system that seems to depend more on irregularly firing afferent input and is thought to be subserved by different circuitry than the VOR. We examined whether this different reflex also of vestibular origin would show similar recovery after hair cell regeneration. Lesions of the vestibular hair cells of 10-day-old chicks were created by a 5-day course of streptomycin sulfate. One day after completion of streptomycin treatment there was no measurable VCR gain, and total hair cell density was approximately 35% of that in untreated, age-matched controls. At 2 wk postlesion there was significant recovery of the VCR; at this time two subjects showed VCR gains within the range of control chicks. At 3 wk postlesion all subjects showed VCR gains and phase shifts within the normal range. These data show that the VCR recovers before the VOR. Unlike VOR gain, recovering VCR gain correlates equally well with the density of regenerating type I and type II vestibular hair cells, except at high frequencies. Several factors other than hair cell regeneration, such as length of stereocilia, reafferentation of hair cells, and compensation involving central neural pathways, may be involved in behavioral recovery. Our data suggest that one or more of these factors differentially affect the recovery of these two vestibular reflexes.

The superior olivary nucleus and its influence on nucleus laminaris: a source of inhibitory feedback for coincidence detection in the avian auditory brainstem.

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.