Experience-Dependent Intrinsic Plasticity During Auditory Learning.

Song learning in zebra finches (Taeniopygia guttata) requires exposure to the song of a tutor, resulting in an auditory memory. This memory is the foundation for later sensorimotor learning, resulting in the production of a copy of the tutor's song. The cortical premotor nucleus HVC (proper name) is necessary for auditory and sensorimotor learning as well as the eventual production of adult song. We recently discovered that the intrinsic physiology of HVC neurons changes across stages of song learning, but are those changes the result of learning or are they experience-independent developmental changes? To test the role of auditory experience in driving intrinsic changes, patch-clamp experiments were performed comparing HVC neurons in juvenile birds with varying amounts of tutor exposure. The intrinsic physiology of HVC neurons changed as a function of tutor exposure. Counterintuitively, tutor deprivation resulted in juvenile HVC neurons showing an adult-like phenotype not present in tutor-exposed juveniles. Biophysical models were developed to predict which ion channels were modulated by experience. The models indicate that tutor exposure transiently suppressed the Ih and T-type Ca2+ currents in HVC neurons that target the basal ganglia, whereas tutor exposure increased the resting membrane potential and decreased the spike amplitude in HVC neurons that drive singing. Our findings suggest that intrinsic plasticity may be part of the mechanism for auditory learning in the HVC. More broadly, models of learning and memory should consider intrinsic plasticity as a possible mechanism by which the nervous system encodes the lasting effects of experience.SIGNIFICANCE STATEMENT It is well established that learning involves plasticity of the synapses between neurons. However, the activity of a neural circuit can also be dramatically altered by changes in the intrinsic properties (ion channels) of the component neurons. The present experiments show experience-dependent changes in the intrinsic physiology of neurons in the cortical premotor nucleus HVC (proper name) in juvenile zebra finches (Taeniopygia guttata) during auditory learning of a tutor's song. Tutor deprivation does not "arrest" development of intrinsic properties, but rather results in neurons with a premature adult-like physiological phenotype. It is possible that auditory learning involves a form of nonsynaptic plasticity and that experience-dependent suppression of specific ion channels may work in concert with synaptic plasticity to promote vocal learning.

Intrinsic physiological properties underlie auditory response diversity in the avian cochlear nucleus.

Sensory systems exploit parallel processing of stimulus features to enable rapid, simultaneous extraction of information. Mechanisms that facilitate this differential extraction of stimulus features can be intrinsic or synaptic in origin. A subdivision of the avian cochlear nucleus, nucleus angularis (NA), extracts sound intensity information from the auditory nerve and contains neurons that exhibit diverse responses to sound and current injection. NA neurons project to multiple regions ascending the auditory brain stem including the superior olivary nucleus, lateral lemniscus, and avian inferior colliculus, with functional implications for inhibitory gain control and sound localization. Here we investigated whether the diversity of auditory response patterns in NA can be accounted for by variation in intrinsic physiological features. Modeled sound-evoked auditory nerve input was applied to NA neurons with dynamic clamp during in vitro whole cell recording at room temperature. Temporal responses to auditory nerve input depended on variation in intrinsic properties, and the low-threshold K+ current was implicated as a major contributor to temporal response diversity and neuronal input-output functions. An auditory nerve model of acoustic amplitude modulation produced synchrony coding of modulation frequency that depended on the intrinsic physiology of the individual neuron. In Primary-Like neurons, varying low-threshold K+ conductance with dynamic clamp altered temporal modulation tuning bidirectionally. Taken together, these data suggest that intrinsic physiological properties play a key role in shaping auditory response diversity to both simple and more naturalistic auditory stimuli in the avian cochlear nucleus. NEW & NOTEWORTHY This article addresses the question of how the nervous system extracts different information in sounds. Neurons in the cochlear nucleus show diverse responses to acoustic stimuli that may allow for parallel processing of acoustic features. The present studies suggest that diversity in intrinsic physiological features of individual neurons, including levels of a low voltage-activated K+ current, play a major role in regulating the diversity of auditory responses.

Intrinsic physiology of inhibitory neurons changes over auditory development.

During auditory development, changes in membrane properties promote the ability of excitatory neurons in the brain stem to code aspects of sound, including the level and timing of a stimulus. Some of these changes coincide with hearing onset, suggesting that sound-driven neural activity produces developmental plasticity of ion channel expression. While it is known that the coding properties of excitatory neurons are modulated by inhibition in the mature system, it is unknown whether there are also developmental changes in the membrane properties of brain stem inhibitory neurons. We investigated the primary source of inhibition in the avian auditory brain stem, the superior olivary nucleus (SON). The present studies test the hypothesis that, as in excitatory neurons, the membrane properties of these inhibitory neurons change after hearing onset. We examined SON neurons at different stages of auditory development: embryonic days 14-16 (E14-E16), a time at which cochlear ganglion neurons are just beginning to respond to sound; later embryonic stages (E18-E19); and after hatching (P0-P2). We used in vitro whole cell patch electrophysiology to explore physiological changes in SON. Age-related changes were observed at the level of a single spike and in multispiking behavior. In particular, tonic behavior, measured as a neuron's ability to sustain tonic firing over a range of current steps, became more common later in development. Voltage-clamp recordings and biophysical models were employed to examine how age-related increases in ion currents enhance excitability in SON. Our findings suggest that concurrent increases in sodium and potassium currents underlie the emergence of tonic behavior. NEW & NOTEWORTHY This article is the first to examine heterogeneity of neuronal physiology in the inhibitory nucleus of the avian auditory system and demonstrate that tonic firing here emerges over development. By pairing computer simulations with physiological data, we show that increases in both sodium and potassium channels over development are necessary for the emergence of tonic firing.

Interhemispheric dominance switching in a neural network model for birdsong.

Male zebra finches produce a sequence-invariant set of syllables, separated by short inspiratory gaps. These songs are learned from an adult tutor and maintained throughout life, making them a tractable model system for learned, sequentially ordered behaviors, particularly speech production. Moreover, much is known about the cortical, thalamic, and brain stem areas involved in producing this behavior, with the premotor cortical nucleus HVC (proper name) being of primary importance. In a previous study, our group developed a behavioral neural network model for birdsong constrained by the structural connectivity of the song system, the signaling properties of individual neurons and circuits, and circuit-breaking behavioral studies. Here we describe a more computationally tractable model and use it to explain the behavioral effects of unilateral cooling and electrical stimulations of HVC on song production. The model demonstrates that interhemispheric switching of song control is sufficient to explain these results, consistent with the hypotheses proposed when the experiments were initially conducted. Finally, we use the model to make testable predictions that can be used to validate the model framework and explain the effects of other perturbations of the song system, such as unilateral ablations of the primary input and output nuclei of HVC. NEW & NOTEWORTHY In this report, we propose a two-hemisphere neural network model for the bilaterally symmetrical song system underlying birdsong in the male zebra finch. This model captures the behavioral effects of unilateral cooling and electrical stimulations of the premotor cortical nucleus HVC during song production, supporting the hypothesis of interhemispheric switching of song control. We use the model to make testable predictions regarding the behavioral effects of other unilateral perturbations to the song system.

A distributed neural network model for the distinct roles of medial and lateral HVC in zebra finch song production.

Male zebra finches produce a song consisting of a canonical sequence of syllables, learned from a tutor and repeated throughout its adult life. Much of the neural circuitry responsible for this behavior is located in the cortical premotor region HVC (acronym is name). In a recent study from our laboratory, we found that partial bilateral ablation of the medial portion of HVC has effects on the song that are qualitatively different from those of bilateral ablation of the lateral portion. In this report we describe a neural network organization that can explain these data, and in so doing suggests key roles for other brain nuclei in the production of song. We also suggest that syllables and the gaps between them are each coded separately by neural chains within HVC, and that the timing mechanisms for syllables and gaps are distinct. The design principles underlying this model assign distinct roles for medial and lateral HVC circuitry that explain the data on medial and lateral ablations. In addition, despite the fact that the neural coding of song sequence is distributed among several brain nuclei in our model, it accounts for data showing that cooling of HVC stretches syllables uniformly and to a greater extent than gaps. Finally, the model made unanticipated predictions about details of the effects of medial and lateral HVC ablations that were then confirmed by reanalysis of these previously acquired behavioral data.NEW & NOTEWORTHY Zebra finch song consists of a string of syllables repeated in a nearly invariant sequence. We propose a neural network organization that can explain recent data indicating that the medial and lateral portions of the premotor cortical nucleus HVC have different roles in zebra finch song production. Our model explains these data, as well as data on the effects on song of cooling HVC, and makes predictions that we test in the singing bird.

Independent premotor encoding of the sequence and structure of birdsong in avian cortex.

How the brain coordinates rapid sequences of learned behavior, such as human speech, remains a fundamental problem in neuroscience. Birdsong is a model of such behavior, which is learned and controlled by a neural circuit that spans avian cortex, basal ganglia, and thalamus. The songs of adult male zebra finches (Taeniopygia guttata), produced as rapid sequences of vocal gestures (syllables), are encoded by the cortical premotor region HVC (proper name). While the motor encoding of song within HVC has traditionally been viewed as unitary and distributed, we used an ablation technique to ask whether the sequence and structure of song are processed independently within HVC. Results revealed a functional topography across the medial-lateral axis of HVC. Bilateral ablation of medial HVC induced a positive disruption of song (increase in atypical syllable sequences), whereas bilateral ablation of lateral HVC induced a negative disruption (omission of individual syllables). Bilateral ablation of central HVC either had no effect on song or induced syllable omission, similar to lateral HVC ablation. We then investigated HVC connectivity and found parallel afferent and efferent pathways that transit medial and lateral HVC and converge at vocal motor cortex. In light of recent evidence that syntactic and lexical components of human speech are processed independently by neighboring regions of cortex (Menenti et al., 2012), our demonstration of anatomically distinct pathways that differentially process the sequence and structure of birdsong in parallel suggests that the vertebrate brain relies on a common approach to encode rapid sequences of vocal gestures.

Axial organization of a brain region that sequences a learned pattern of behavior.

Neural activity within HVC (proper name), a premotor nucleus of the songbird telencephalon analogous to premotor cortical regions in mammals, controls the temporal structure of learned song in male zebra finches (Taeniopygia guttata). HVC is composed of a superficially isomorphic neuronal mosaic, implying that song is encoded in a distributed network within HVC. Here, we combined HVC microlesions (10% focal ablation) with singing-driven immediate-early gene (IEG) labeling to explore the network architecture of HVC during singing. Microlesions produce a transient disruption of HVC activity that results in a temporary (≈ 1 week) loss of vocal patterning. Results showed an asymmetrical reduction in the density of IEG-labeled cells 3-5 d after microlesions: swaths of unlabeled cells extended rostrally and/or caudally depending on the position of the HVC microlesion. Labeling returned once birds recovered their songs. Axial swaths of unlabeled cells occurred whether microlesions were located at rostral or caudal poles of HVC, indicating that the localized reduction in IEG labeling could not be attributable solely to transection of afferents that enter HVC rostrally. The asymmetrical pattern of reduced IEG labeling could be explained if synaptic connectivity within HVC is organized preferentially within the rostrocaudal axis. In vivo retrograde tracer injections and in vitro stimulation and recording experiments in horizontal slices of HVC confirmed a rostrocaudal organization of HVC neural connectivity. Our findings suggest that HVC contains an axially organized network architecture that may encode the temporal structure of song.

Electrophysiological characterization and computational models of HVC neurons in the zebra finch.

The nucleus HVC (proper name) within the avian analog of mammal premotor cortex produces stereotyped instructions through the motor pathway leading to precise, learned vocalization by songbirds. Electrophysiological characterization of component HVC neurons is an important requirement in building a model to understand HVC function. The HVC contains three neural populations: neurons that project to the RA (robust nucleus of arcopallium), neurons that project to Area X (of the avian basal ganglia), and interneurons. These three populations are interconnected with specific patterns of excitatory and inhibitory connectivity, and they fire with characteristic patterns both in vivo and in vitro. We performed whole cell current-clamp recordings on HVC neurons within brain slices to examine their intrinsic firing properties and determine which ionic currents are responsible for their characteristic firing patterns. We also developed conductance-based models for the different neurons and calibrated the models using data from our brain slice work. These models were then used to generate predictions about the makeup of the ionic currents that are responsible for the different responses to stimuli. These predictions were then tested and verified in the slice using pharmacological manipulations. The model and the slice work highlight roles of a hyperpolarization-activated inward current (Ih), a low-threshold T-type Ca(2+) current (ICa-T), an A-type K(+) current (IA), a Ca(2+)-activated K(+) current (ISK), and a Na(+)-dependent K(+) current (IKNa) in driving the characteristic neural patterns observed in the three HVC neuronal populations. The result is an improved characterization of the HVC neurons responsible for song production in the songbird.

Cannabinoid receptor 1 signaling in embryo neurodevelopment.

In utero exposure to tetrahydrocannabinol, the psychoactive component of marijuana, is associated with an increased risk for neurodevelopmental defects in the offspring by interfering with the functioning of the endocannabinoid (eCB) system. At the present time, it is not clearly known whether the eCB system is present before neurogenesis. Using an array of biochemical techniques, we analyzed the levels of CB1 receptors, eCBs (AEA and 2-AG), and the enzymes (NAPE-PLD, DAGLα, DAGLß, MAGL, and FAAH) involved in the metabolism of the eCBs in chick and mouse models during development. The findings demonstrate the presence of eCB system in early embryo before neurogenesis. The eCB system might play a critical role in early embryogenesis and there might be adverse developmental consequences of in utero exposure to marijuana and other drugs of abuse during this period.

Network dynamics underlie learning and performance of birdsong.

Understanding the sensorimotor control of the endless variety of human speech patterns stands as one of the apex problems in neuroscience. The capacity to learn - through imitation - to rapidly sequence vocal sounds in meaningful patterns is clearly one of the most derived of human behavioral traits. Selection pressure produced an analogous capacity in numerous species of vocal-learning birds, and due to an increasing appreciation for the cognitive and computational flexibility of avian cortex and basal ganglia, a general understanding of the forebrain network that supports the learning and production of birdsong is beginning to emerge. Here, we review recent advances in experimental studies of the zebra finch (Taeniopygia guttata), which offer new insights into the network dynamics that support this surprising analogue of human speech learning and production.

Female zebra finches do not sing yet share neural pathways necessary for singing in males.

Adult female zebra finches (Taeniopygia guttata), which do not produce learned songs, have long been thought to possess only vestiges of the forebrain network that supports learned song in males. This view ostensibly explains why females do not sing-many of the neural populations and pathways that make up the male song control network appear rudimentary or even missing in females. For example, classic studies of vocal-premotor cortex (HVC, acronym is name) in male zebra finches identified prominent efferent pathways from HVC to vocal-motor cortex (RA, robust nucleus of the arcopallium) and from HVC to the avian basal ganglia (Area X). In females, by comparison, the efferent targets of HVC were thought to be only partially innervated by HVC axons (RA) or absent (Area X). Here, using a novel visually guided surgical approach to target tracer injections with precision, we mapped the extrinsic connectivity of the adult female HVC. We find that female HVC shows a mostly male-typical pattern of afferent and efferent connectivity, including robust HVC innervation of RA and Area X. As noted by earlier investigators, we find large sex differences in the volume of many regions that control male singing (male > female). However, sex differences in volume were diminished in regions that convey ascending afferent input to HVC. Our findings do not support a vestigial interpretation of the song control network in females. Instead, our findings support the emerging view that the song control network may have an altogether different function in nonsinging females.

Effects of lithium and deafferentation on expression of glycogen synthase kinase-3beta, NFkappaB, beta-catenin and pCreb in the chick cochlear nucleus.

The avian brainstem serves as a useful model to answer the question of how afferent activity influences the viability of target neurons. Approximately 20-30% of neurons in the avian cochlear nucleus, nucleus magnocellularis (NM) die following deafferentation (i.e., deafness produced by cochlea removal). Interestingly, Bcl-2 mRNA (but not protein) is upregulated in 20-30% of NM neurons following deafferentation. We have recently shown that chronic treatments of lithium upregulates the neuroprotective protein Bcl-2 and increases neuronal survival following deafferentation. The pathways leading to the upregulation of Bcl-2 expression following these two manipulations are unknown. The present experiments examine changes in glycogen synthase kinase-3 beta (Gsk-3beta), and transcription factors nuclear factor kappaB (NFkappaB), beta-catenin, and pCreb following lithium administration and following deafferentation. These molecules are known to be influenced by lithium and to regulate Bcl-2 expression in other model systems. Lithium decreased immunolabeling for Gsk-3beta and increased expression for all three transcription factors. Deafferentation, however, did not alter Gsk-3beta or NFkappaB, resulted in lower beta-catenin expression, but did increase pCreb immunoreactivity. While it is possible that pCreb is a common link in the regulation of Bcl-2 following these two manipulations, the timing and distribution of pCreb labeling suggests that it is not the sole determinant of Bcl-2 upregulation following deafferentation. It is likely that the regulation of Bcl-2 gene expression by lithium and by deafferentation involves different molecular pathways.

Orthogonal topography in the parallel input architecture of songbird HVC.

Neural activity within the cortical premotor nucleus HVC (acronym is name) encodes the learned songs of adult male zebra finches (Taeniopygia guttata). HVC activity is driven and/or modulated by a group of five afferent nuclei (the Medial Magnocellular nucleus of the Anterior Nidopallium, MMAN; Nucleus Interface, NIf; nucleus Avalanche, Av; the Robust nucleus of the Arcopallium, RA; the Uvaeform nucleus, Uva). While earlier evidence suggested that HVC receives a uniformly distributed and nontopographic pattern of afferent input, recent evidence suggests this view is incorrect (Basista et al., ). Here, we used a double-labeling strategy (varying both the distance between and the axial orientation of dual tracer injections into HVC) to reveal a massively parallel and in some cases topographic pattern of afferent input. Afferent neurons target only one rostral or caudal location within medial or lateral HVC, and each HVC location receives convergent input from each afferent nucleus in parallel. Quantifying the distributions of single-labeled cells revealed an orthogonal topography in the organization of afferent input from MMAN and NIf, two cortical nuclei necessary for song learning. MMAN input is organized across the lateral-medial axis whereas NIf input is organized across the rostral-caudal axis. To the extent that HVC activity is influenced by afferent input during the learning, perception, or production of song, functional models of HVC activity may need revision to account for the parallel input architecture of HVC, along with the orthogonal input topography of MMAN and NIf.

Neuronal Intrinsic Physiology Changes During Development of a Learned Behavior.

Juvenile male zebra finches learn their songs over distinct auditory and sensorimotor stages, the former requiring exposure to an adult tutor song pattern. The cortical premotor nucleus HVC (acronym is name) plays a necessary role in both learning stages, as well as the production of adult song. Consistent with neural network models where synaptic plasticity mediates developmental forms of learning, exposure to tutor song drives changes in the turnover, density, and morphology of HVC synapses during vocal development. A network's output, however, is also influenced by the intrinsic properties (e.g., ion channels) of the component neurons, which could change over development. Here, we use patch clamp recordings to show cell-type-specific changes in the intrinsic physiology of HVC projection neurons as a function of vocal development. Developmental changes in HVC neurons that project to the basal ganglia include an increased voltage sag response to hyperpolarizing currents and an increased rebound depolarization following hyperpolarization. Developmental changes in HVC neurons that project to vocal-motor cortex include a decreased resting membrane potential and an increased spike amplitude. HVC interneurons, however, show a relatively stable range of intrinsic features across vocal development. We used mathematical models to deduce possible changes in ionic currents that underlie the physiological changes and to show that the magnitude of the observed changes could alter HVC circuit function. The results demonstrate developmental plasticity in the intrinsic physiology of HVC projection neurons and suggest that intrinsic plasticity may have a role in the process of song learning.

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

To better understand the effects of deafness on the brain, these experiments examine how disrupted balance between excitatory and inhibitory neurotransmission following the loss of excitatory input from the auditory nerve alters the central auditory system. In the avian cochlear nucleus, nucleus magnocellularis (NM), deprivation of excitatory input induced by deafness triggers neuronal death. While this neuronal death was previously accredited to the loss of excitatory drive, the present experiments examine an alternative hypothesis: that inhibitory input to NM, which may also be affected by deafness, contributes to neuronal death in NM. Using an in vitro slice preparation in which excitatory input from the auditory nerve is absent, we pharmacologically altered GABA receptor activation in NM, and assayed an early marker of neuronal health, antigenicity for the ribosomal antibody Y10B (Y10B-ir). We found that GABA decreases Y10B-ir, and that GABAA activation is necessary for the GABA-induced effect. We further found that endogenous GABAA activation similarly decreases Y10B-ir and this decrease requires extracellular Ca(2+). Our results suggest that, in the absence of excitatory input, endogenous activation of ionotropic GABAA receptors is detrimental to NM neurons.

The analysis of interaural time differences in the chick brain stem.

The brain stem auditory system of the chick has proven to be a useful model system for analyzing how the brain encodes temporal information. This paper reviews some of the work on a circuit in the brain stem that compares the timing of information coming from the two ears to determine the location of a sound source. The contralateral projection from the cochlear nucleus, nucleus magnocellularis (NM), to nucleus laminaris (NL) forms a delay line as it proceeds from medial to lateral across NL. NL neurons function like coincidence detectors in that they respond maximally when input from the two ears arrive simultaneously. This arrangement may allow NL to code sound space by the relative level of activity across the nucleus. The head anatomy of the chick allows for enhancement of the functional interaural time differences. Comparing the functional interaural time differences to the length of the neural delay line suggests that each NL can encode approximately one hemifield of sound space. Finally it is suggested that inhibitory input into the NM-NL circuit may provide a means to dynamically adjust the gain of the circuit to allow accurate coding of sound location despite changes in overall sound intensity.

Rapid deafferentation-induced upregulation of bcl-2 mRNA in the chick cochlear nucleus.

Neuronal survival in developing animals is often dependent on afferent activity. In the posthatch chick, approximately 30% of the neurons in the avian cochlear nucleus, nucleus magnocellularis (NM) die following elimination of VIIIth nerve activity. The factors that influence death or survival of an individual NM neuron are largely unknown. Previous studies indicate that both cell death and cell survival mechanisms compete to determine cell fate. One factor that has been shown to suppress cell death cascades in a variety of systems is bcl-2. If this gene product plays a role in regulating cell survival following deafferentation, then one might expect the expression of this gene to be influenced by removal of afferent input. In the present study, in situ hybridization revealed a rapid and transient increase in bcl-2 mRNA in NM neurons following deafferentation. Enhanced bcl-2 mRNA expression was observed at 6 and 12 h following deafferentation, but not at 3 or 24 h. Surprisingly, the upregulation of bcl-2 mRNA was limited to a subpopulation (20-30%) of deafferented neurons corresponding to the number of NM neurons that eventually die following cochlea removal. The robust and rapid upregulation of this gene suggests that cell death cascades regulated by bcl-2 may be initiated following deafferentation.

Group I and II metabotropic glutamate receptors are necessary for the activity-dependent regulation of ribosomes in chick auditory neurons.

Elimination of eighth-nerve activity results in the death of 30% of the neurons in the chick cochlear nucleus, nucleus magnocellularis (NM). One early event in this cell death cascade is the disruption of ribosomes in NM neurons which can be observed within 1 h following deafferentation. These rapid changes in ribosomes can be visualized using Y10B, a monoclonal antibody that recognizes ribosomal RNA. Previous studies using a brain slice preparation of the avian brain stem auditory system have shown that activation of metabotropic glutamate receptors (mGluRs) is necessary for the activity-dependent maintenance of Y10B antigenicity. The purpose of the present study was to determine if group I and/or II mGluRs are necessary for this activity-dependent regulation. This was accomplished by selectively blocking group I or II receptors while unilaterally stimulating the auditory nerve in vitro. In normal media, unilateral stimulation of the auditory nerve resulted in darker Y10B immunolabeling of NM neurons on the stimulated side of the slice. The group I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) and the group II antagonists LY341495 and (S)-alpha-ethylglutamic acid (EGLU) all prevented the activity-dependent difference in Y10B immunolabeling. These data suggest that both group I and II mGluRs play vital roles in the activity-dependent regulation of ribosomes in NM.

Activation of metabotropic glutamate receptors regulates ribosomes of cochlear nucleus neurons.

The brain stem auditory system of the chick is an advantageous model for examining changes that occur as a result of deafness. Elimination of acoustic input through cochlear ablation results in the eventual death of approximately 30% of neurons in the chick cochlear nucleus, nucleus magnocellularis (NM). One early change following deafness is an alteration in NM ribosomes, evidenced both by a decrease in protein synthesis and reduction in antigenicity for Y10B, a monoclonal antibody that recognizes a ribosomal epitope. Previous studies have shown that mGluR activation is necessary to maintain Y10B antigenicity and NM viability. What is still unclear, however, is whether or not mGluR activation is sufficient to prevent deafness-induced changes in these neurons, or if other activity-dependent factors are also necessary. The current study investigated the ability of mGluR activation to regulate cochlear nucleus ribosomes in the absence of auditory nerve input. In vitro methods were employed to periodically pressure eject glutamate or mGluR agonists over neurons on one side of a slice preparation leaving the opposite side of the same slice untreated. Immunohistochemistry was then performed using Y10B in order to assess ribosomal changes. Application of glutamate and both group I and II selective mGluR agonists effectively rescued ribosomal antigenicity on the treated side of the slice in comparison to ribosomes on the untreated side. These findings suggest that administration of mGluR agonists is sufficient to reduce the early interruption of normal ribosomal integrity that is typically seen following loss of auditory nerve activity.

In vivo analysis of the role of metabotropic glutamate receptors in the afferent regulation of chick cochlear nucleus neurons.

Cochlea removal results in the death of approximately 20-30% of neurons in the chick nucleus magnocellularis (NM). One early event in NM neuronal degradation is the disruption of their ribosomes. This can be visualized in the first few hours following cochlea removal using Y10B, an antibody that recognizes ribosomal RNA. Previous studies using a brain slice preparation suggest that maintenance of ribosomal integrity in NM neurons requires metabotropic glutamate receptor (mGluR) activation. Isolating the brain slice in vitro, however, may eliminate other potential sources of trophic support and only allows for evaluation of the early changes that occur in NM neurons following deafferentation. Consequently, it is not known if mGluR activation is truly required for the maintenance of NM neurons in the intact system. The current experiments evaluated the importance of mGluRs in vivo. The effects of short-term receptor blockade were assessed through Y10B labeling and the effects of long-term blockade were assessed through stereological counting of NM neurons in Nissl-stained tissue. mGluR antagonists or vehicle were administered intracerebroventricularly following unilateral cochlea removal. Vehicle-treated subjects replicated the previously reported effects of cochlea removal, showing lighter Y10B labeling and fewer Nissl-stained NM neurons on the deafened side of the brain. Blockade of mGluRs prevented the rapid activity-dependent difference in Y10B labeling, and in some cases, had the reverse effect, yielding lighter labeling of NM neurons on the intact side of the brain. Similarly, mGluR blockade over longer survival periods resulted in a reduction in number of cells on both intact and deafferented sides of the brain, and in some cases, yielded a reverse effect of fewer neurons on the intact side versus deafened side. These data are consistent with in vitro findings and suggest that mGluR activation plays a vital role in the afferent maintenance of NM neurons.