Medial Nucleus Of The Trapezoid Body Cells Research Articles

Transcriptional profiling reveals roles of intercellular Fgf9 signaling in astrocyte maturation and synaptic refinement during brainstem development

Neural tissue maturation is a coordinated process under tight transcriptional control. We previously analyzed the kinetics of gene expression in the medial nucleus of the trapezoid body (MNTB) in the brainstem during the critical postnatal phase of its development. While this work revealed timed execution of transcriptional programs, it was blind to the specific cells where gene expression changes occurred. Here, we utilized single-cell RNA-Seq to determine transcriptional profiles of each major MNTB cell type. We discerned directional signaling patterns between neuronal, glial, and vascular-associated cells for VEGF, TGFβ, and Delta-Notch pathways during a robust period of vascular remodeling in the MNTB. Furthermore, we describe functional outcomes of the disruption of neuron-astrocyte fibroblast growth factor 9 (Fgf9) signaling. We used a conditional KO (cKO) approach to genetically delete Fgf9 from principal neurons in the MNTB, which led to an early onset of glial fibrillary acidic protein (Gfap) expression in astrocytes. In turn, Fgf9 cKO mice show increased levels of astrocyte-enriched brevican (Bcan), a component of the perineuronal net matrix that ensheaths principal neurons in the MNTB and the large calyx of Held terminal, while levels of the neuron-enriched hyaluronan and proteoglycan link protein 1 (Hapln1) were unchanged. Finally, volumetric analysis of vesicular glutamate transporters 1 and 2 (Vglut1/2), which serves as a proxy for terminal size, revealed an increase in calyx of Held volume in the Fgf9 cKO. Overall, we demonstrate a coordinated neuron-astrocyte Fgf9 signaling network that functions to regulate astrocyte maturation, perineuronal net structure, and synaptic refinement.

Maintenance of neuronal size gradient in MNTB requires sound-evoked activity

The medial nucleus of the trapezoid body (MNTB) is an important source of inhibition during the computation of sound location. It transmits fast and precisely timed action potentials at high frequencies; this requires an efficient calcium clearance mechanism, in which plasma membrane calcium ATPase 2 (PMCA2) is a key component. Deafwaddler (dfw2J ) mutant mice have a null mutation in PMCA2 causing deafness in homozygotes (dfw2J /dfw2J ) and high-frequency hearing loss in heterozygotes (+/dfw2J ). Despite the deafness phenotype, no significant differences in MNTB volume or cell number were observed in dfw2J homozygous mutants, suggesting that PMCA2 is not required for MNTB neuron survival. The MNTB tonotopic axis encodes high to low sound frequencies across the medial to lateral dimension. We discovered a cell size gradient along this axis: lateral neuronal somata are significantly larger than medially located somata. This size gradient is decreased in +/dfw2J and absent in dfw2J /dfw2J The lack of acoustically driven input suggests that sound-evoked activity is required for maintenance of the cell size gradient. This hypothesis was corroborated by selective elimination of auditory hair cell activity with either hair cell elimination in Pou4f3 DTR mice or inner ear tetrodotoxin (TTX) treatment. The change in soma size was reversible and recovered within 7 days of TTX treatment, suggesting that regulation of the gradient is dependent on synaptic activity and that these changes are plastic rather than permanent.NEW & NOTEWORTHY Neurons of the medial nucleus of the trapezoid body (MNTB) act as fast-spiking inhibitory interneurons within the auditory brain stem. The MNTB is topographically organized, with low sound frequencies encoded laterally and high frequencies medially. We discovered a cell size gradient along this axis: lateral neurons are larger than medial neurons. The absence of this gradient in deaf mice lacking plasma membrane calcium ATPase 2 suggests an activity-dependent, calcium-mediated mechanism that controls neuronal soma size.

Natural and lesion‐induced decrease in cell proliferation in the medial nucleus of the trapezoid body during hearing development

ABSTRACTThe functional interactions between neurons and glial cells that are important for nervous system function are presumably established during development from the activity of progenitor cells. In this study we examined proliferation of progenitor cells in the medial nucleus of the trapezoid body (MNTB) located in the rat auditory brainstem. We performed DNA synthesis labeling experiments to demonstrate changes in cell proliferation activity during postnatal stages of development. An increase in cell proliferation correlated with MNTB growth and the presence of S100β‐positive astrocytes among MNTB neurons. In additional experiments we analyzed the fate of newly born cells. At perinatal ages, newly born cells colabeled with the astrocyte marker S100β in higher numbers than when cells were generated at postnatal day 6. Furthermore, we identified newly born cells that were colabeled with caspase‐3 immunohistochemistry and performed comparative experiments to demonstrate that there is a natural decrease in cell proliferation activity during postnatal development in rats, mice, gerbils, and ferrets. Lastly, we found that there is a stronger decrease in MNTB cell proliferation after performing bilateral lesions of the auditory periphery in rats. Altogether, these results identify important stages in the development of astrocytes in the MNTB and provide evidence that the proliferative activity of the progenitor cells is developmentally regulated. We propose that the developmental reduction in cell proliferation may reflect coordinated signaling between the auditory brainstem and the auditory periphery. J. Comp. Neurol. 522:971–985, 2014. © 2013 Wiley Periodicals, Inc.

Natural and lesion‐induced decrease in cell proliferation in the medial nucleus of the trapezoid body during hearing development

The functional interactions between neurons and glial cells that are important for nervous system function are presumably established during development from the activity of progenitor cells. In this study we examined proliferation of progenitor cells in the medial nucleus of the trapezoid body (MNTB) located in the rat auditory brainstem. We performed DNA synthesis labeling experiments to demonstrate changes in cell proliferation activity during postnatal stages of development. An increase in cell proliferation correlated with MNTB growth and the presence of S100β-positive astrocytes among MNTB neurons. In additional experiments we analyzed the fate of newly born cells. At perinatal ages, newly born cells colabeled with the astrocyte marker S100β in higher numbers than when cells were generated at postnatal day 6. Furthermore, we identified newly born cells that were colabeled with caspase-3 immunohistochemistry and performed comparative experiments to demonstrate that there is a natural decrease in cell proliferation activity during postnatal development in rats, mice, gerbils, and ferrets. Lastly, we found that there is a stronger decrease in MNTB cell proliferation after performing bilateral lesions of the auditory periphery in rats. Altogether, these results identify important stages in the development of astrocytes in the MNTB and provide evidence that the proliferative activity of the progenitor cells is developmentally regulated. We propose that the developmental reduction in cell proliferation may reflect coordinated signaling between the auditory brainstem and the auditory periphery. J. Comp. Neurol. 522:971–985, 2014.

Unique features of extracellular matrix in the mouse medial nucleus of trapezoid body – Implications for physiological functions

The medial nucleus of the trapezoid body (MNTB) is a vital structure of sound localization circuits in the auditory brainstem. Each principal cell of MNTB is contacted by a very large presynaptic glutamatergic terminal, the calyx of Held. The MNTB principal cells themselves are surrounded by extracellular matrix components forming prominent perineuronal nets (PNs). Throughout the CNS, PNs, which form lattice-like structures around the somata and proximal dendrites, are associated with distinct types of neurons. PNs are highly enriched in hyaluronan and chondroitin sulfate proteoglycans therefore providing a charged surface structure surrounding the cell body and proximal neurites of these neurons. The localization and composition of PNs have lead investigators to a number of hypotheses about their functions including: creating a specific extracellular ionic milieu around these neurons, stabilizing synapses, and influencing the outgrowth of axons. However, presently the precise functions of PNs are still quite unclear primarily due to the lack of an ideal experimental model system that is highly enriched in PNs and in which the synaptic transmission properties can be precisely measured. The MNTB principal cells could offer such a model, since they have been extensively characterized electrophysiologically. However, extracellular matrix (ECM) in these neurons has not yet been precisely detailed. The present study gives a detailed examination of the ECM organization and structural differences in PNs of the mouse MNTB. The different PN components and their distribution pattern are scrutinized throughout the MNTB. The data are complemented by electron microscopic investigations of the unique ultrastructural localization of PN-components and their interrelation with distinct pre- and postsynaptic MNTB cell structures. Therefore, we believe this work identifies the MNTB as an ideal system for studying PN function.

Astroglial glutamate transporters trigger glutaminergic gliotransmission

Rapid exchange of molecular signals between synapses and the surrounding astroglia has recently emerged as an essential player in regulating neural circuit activities in the brain. This conceptual leap was prompted in large part by early findings demonstrating that electrically non-excitable glia could, in fact, generate and propagate relatively rapid, physiologically informative signals via internal Ca2+ waves (Parpura et al. 1994). Astrocytes have been found to respond with internal Ca2+ transients to the common fast neurotransmitters glutamate and GABA through the target receptors expressed in their plasma membranes. In turn, astroglia could generate and dispatch their own Ca2+-dependent messages to neurons, by releasing a variety of signalling molecules including ATP, glutamate, d-serine or tumour necrosis factor α, although the cellular mechanisms of such actions remain under intense investigation (Hamilton & Attwell, 2010). Outshined by this newly acquired prominence of astrocytes, their long-established, classical functions have been somewhat moved from centre stage. In fact, the ubiquitous processes of glutamate uptake and potassium buffering by astroglia have long been known to occur in the brain on a scale that must ensure their substantial contribution to neural function. In their recent elegant work in this issue of The Journal of Physiology, Uwechue and colleagues (2012) have focused on the immediate consequences of glutamate uptake in astrocytes that surround the giant glutamatergic synapse of the calyx of Held in the rat medial nucleus of the trapezoid body (MNTB). This classical subject of synaptic studies enables direct access to readily identifiable pre- and postsynaptic elements of the synapse and is therefore ideally suited for controlled probing of mechanisms involved in astroglia–synapse exchange. It has long been proposed that replenishment of glutamate in excitatory synaptic terminals depends mainly on local uptake of glutamine supplied by glia. Intra-glial conversion of transported glutamate into glutamine completes this metabolic cycle. The study by Uwechue and colleagues unveils a previously unrecognised and potentially important aspect of this machinery. The authors found that, by engaging neuronal system A transporters, glutamine released by astroglia can rapidly depolarise the local neuronal membrane. This observation appears to suggest that mere metabolic glutamine supply by astroglia actually triggers what could be considered as an excitatory feedback signal promoted by local synaptic discharges. The authors have ruled out the involvement of glutamate in this feedback signal by dissecting pharmacologically the known glutamate receptors and transporters. Conversely, to probe astroglial transporter currents in isolation they used application of d-aspartate, a non-glutamate activator of glutamate transporters, thus excluding the involvement of glutamate receptors in transporter-dependent glial glutamine release. By combining these approaches with astrocytic sodium imaging and dual-patch recordings from both an MNTB cell and an adjacent astrocyte they were able to demonstrate convincingly that glutamate transporter activation in an astrocyte induces a depolarising current in the nearby neuron. The inward current induced by neuronal glutamine uptake following activation of astroglial glutamate transporters was relatively small, in the ∼10 pA range (Uwechue et al. 2012). However, the local current density in such cases is likely to depend strongly on the juxtaposition of glial and synaptic membranes. It is not, therefore, inconceivable that local glutamine uptake may evoke relatively strong depolarisation in small cellular compartments, such as dendritic spines or presynaptic axonal boutons covered by astroglia (Fig. 1). By the same token, enhanced glutamate uptake within local hotspots of glial glutamate transporters (Zheng et al. 2008) might, in principle, boost localised efflux of glutamine. Whether the detected depolarisation has any significant impact on the function of MNTB synapses and whether such influences indeed occur at other central synapses, pre- or postsynaptically, remains an open and intriguing question. In particular, it would be important to understand whether some of the reported changes in synaptic efficacy induced by astroglial activity (Perea & Araque, 2010) involve the action of glial glutamate and neuronal glutamine transporters in the synaptic microenvironment. In any case, the findings of Uwechue and colleagues shed new light on the classical function of astroglia, with some unexpected implications for our understanding of mechanisms that may contribute to regulatory signals sent by astrocytes to their neuronal counterparts. Figure 1 A possible astrocyte–neuron feedback signalling mechanism involving glial glutamate transporters

Embryonic assembly of auditory circuits: spiral ganglion and brainstem

During early development, peripheral sensory systems generate physiological activity prior to exposure to normal environmental stimuli. This activity is thought to facilitate maturation of these neurons and their connections, perhaps even promoting efficacy or modifying downstream circuitry. In the mammalian auditory system, initial connections form at embryonic ages, but the functional characteristics of these early neural connections have not been assayed. We investigated processes of embryonic auditory development using a whole-head slice preparation that preserved connectivity between peripheral and brainstem stations of the auditory pathway. Transgenic mice expressing fluorescent protein provided observation of spiral ganglion and cochlear nucleus neurons to facilitate targeted electrophysiological recording. Here we demonstrate an apparent peripheral-to-central order for circuit maturation. Spiral ganglion cells acquire action potential-generating capacity at embryonic day 14 (E14), the earliest age tested, and action potential waveforms begin to mature in advance of comparable states for neurons of the ventral cochlear nucleus (VCN) and medial nucleus of the trapezoid body (MNTB). In accordance, auditory nerve synapses in the VCN are functional at E15, prior to VCN connectivity with the MNTB, which occurs at least 1 day later. Spiral ganglion neurons exhibit spontaneous activity at least by E14 and are able to drive third-order auditory brainstem neurons by E17. This activity precedes cochlear-generated wave activity by 4 days and ear canal opening by at least 2 weeks. Together, these findings reveal a previously unknown initial developmental phase for auditory maturation, and further implicate the spiral ganglion as a potential controlling centre in this process.

Activation of glutamate transport evokes rapid glutamine release from perisynaptic astrocytes

Stimulation of astrocytes by neuronal activity and the subsequent release of neuromodulators is thought to be an important regulator of synaptic communication. In this study we show that astrocytes juxtaposed to the glutamatergic calyx of Held synapse in the rat medial nucleus of the trapezoid body (MNTB) are stimulated by the activation of glutamate transporters and consequently release glutamine on a very rapid timescale. MNTB principal neurones express electrogenic system A glutamine transporters, and were exploited as glutamine sensors in this study. By simultaneous whole-cell voltage clamping astrocytes and neighbouring MNTB neurones in brainstem slices, we show that application of the excitatory amino acid transporter (EAAT) substrate d-aspartate stimulates astrocytes to rapidly release glutamine, which is detected by nearby MNTB neurones. This release is significantly reduced by the toxins L-methionine sulfoximine and fluoroacetate, which reduce glutamine concentrations specifically in glial cells. Similarly, glutamine release was also inhibited by localised inactivation of EAATs in individual astrocytes, using internal DL-threo-β-benzyloxyaspartic acid (TBOA) or dissipating the driving force by modifying the patch-pipette solution. These results demonstrate that astrocytes adjacent to glutamatergic synapses can release glutamine in a temporally precise, controlled manner in response to glial glutamate transporter activation. Since glutamine can be used by neurones as a precursor for glutamate and GABA synthesis, this represents a potential feedback mechanism by which astrocytes can respond to synaptic activation and react in a way that sustains or enhances further communication. This would therefore represent an additional manifestation of the tripartite relationship between synapses and astrocytes.

Maturation of synaptic partners: functional phenotype and synaptic organization tuned in synchrony

Maturation of principal neurons of the medial nucleus of the trapezoid body (MNTB) was assessed in the context of the developmental organization and activity of their presynaptic afferents, which grow rapidly to form calyces of Held and to establish mono-innervation between postnatal days (P)2 and 4. MNTB neurons and their inputs were studied from embryonic day (E)17, when the nucleus was first discernable, until P14 after the onset of hearing. Using a novel slice preparation containing portions of the cochlea, cochlear nucleus and MNTB, we determined that synaptic inputs form onto MNTB neurons at E17 and stimulation of the cochlear nucleus can evoke action potentials (APs) and Ca(2+) signals. We analysed converging inputs onto individual MNTB neurons and found that competition among inputs was resolved quickly, as a single large input, typically larger than 4 nA, emerged from P3-P4. During calyx growth but before hearing onset, MNTB cells acquired their mature, phasic firing property and quantitative real-time PCR confirmed a coincident increase in low threshold K(+) channel mRNA. These events occurred in concert with an increase in somatic surface area and a 7-fold increase in the current threshold (30 to >200 pA) required to evoke action potentials, as input resistance (R(in)) settled from embryonic values greater than 1 GΩ to approximately 200 MΩ. We postulate that the postsynaptic transition from hyperexcitability to decreased excitability during calyx growth could provide a mechanism to establish the mature 1:1 innervation by selecting the winning calyceal input based on synaptic strength. By comparing biophysical maturation of the postsynaptic cell to alterations in presynaptic organization, we propose that maturation of synaptic partners is coordinated by synaptic activity in a process that is likely to generalize to other neural systems.

Sound stimulation modulates high-threshold K+ currents in mouse auditory brainstem neurons

The auditory system provides a valuable experimental model to investigate the role of sensory activity in regulating neuronal membrane properties. In this study, we have investigated the role of activity directly by measuring changes in medial nucleus of the trapezoid body (MNTB) neurons in normal hearing mice subjected to 1-h sound stimulation. Broadband (4-12 kHz) chirps were used to activate MNTB neurons tonotopically restricted to the lateral MNTB, as confirmed by c-Fos-immunoreactivity. Following 1-h sound stimulation a substantial increase in Kv3.1b-immunoreactivity was measured in the lateral region of the MNTB, which lasted for 2 h before returning to control levels. Electrophysiological patch-clamp recordings in brainstem slices revealed an increase in high-threshold potassium currents in the lateral MNTB of sound-stimulated mice. Current-clamp and dynamic-clamp experiments showed that MNTB cells from the sound-stimulated mice were able to maintain briefer action potentials during high-frequency firing than cells from control mice. These results provide evidence that acoustically driven auditory activity can selectively regulate high-threshold potassium currents in the MNTB of normal hearing mice, likely due to an increased membrane expression of Kv3.1b channels.

A novel role for MNTB neuron dendrites in regulating action potential amplitude and cell excitability during repetitive firing

Principal cells of the medial nucleus of the trapezoid body (MNTB) are simple round neurons that receive a large excitatory synapse (the calyx of Held) and many small inhibitory synapses on the soma. Strangely, these neurons also possess one or two short tufted dendrites, whose function is unknown. Here we assess the role of these MNTB cell dendrites using patch-clamp recordings, imaging and immunohistochemistry techniques. Using outside-out patches and immunohistochemistry, we demonstrate the presence of dendritic Na+ channels. Current-clamp recordings show that tetrodotoxin applied onto dendrites impairs action potential (AP) firing. Using Na+ imaging, we show that the dendrite may serve to maintain AP amplitudes during high-frequency firing, as Na+ clearance indendritic compartments is faster than axonal compartments. Prolonged high-frequency firing can diminish Na+ gradients in the axon while the dendritic gradient remains closer to resting conditions; therefore, the dendrite can provide additional inward current during prolonged firing. Using electron microscopy, we demonstrate that there are small excitatory synaptic boutons on dendrites. Multi-compartment MNTB cell simulations show that, with an active dendrite, dendritic excitatory postsynaptic currents (EPSCs) elicit delayed APs compared with calyceal EPSCs. Together with high- and low-threshold voltage-gated K+ currents, we suggest that the function of the MNTB dendrite is to improve high-fidelity firing, and our modelling results indicate that an active dendrite could contribute to a 'dual' firing mode for MNTB cells (an instantaneous response to calyceal inputs and a delayed response to non-calyceal dendritic excitatory postsynaptic potentials).

Dynamic coupling of excitatory and inhibitory responses in the medial nucleus of the trapezoid body

The neuronal representation of acoustic amplitude modulations is an important prerequisite for understanding the processing of natural sounds. We investigated this representation in the medial nucleus of the trapezoid body (MNTB) of the Mongolian gerbil using sinusoidal amplitude modulations (SAM). Depending on the SAM's carrier frequency (f(C)) MNTB cells either increase or decrease their discharge rates, indicating underlying excitatory and inhibitory/suppressive mechanisms. As natural sounds typically are composed of multiple spectral components we investigated how stimuli containing two spectral components are represented in the MNTB, especially when they have opposing effects on the discharge rate. Three conditions were compared: SAM stimuli (1) with rate-increasing f(C), (2) with rate-increasing f(C) and an additional unmodulated rate-decreasing pure tone, and (3) with rate-decreasing f(C) and an unmodulated, rate-increasing pure tone. We found that responses under all three conditions showed comparable strength of phase-locking. Adding a rate-decreasing tone to a rate-increasing SAM increased phase-locking for modulation frequencies (f(AM)) of < or = 600 Hz. A comparison of two possible coding strategies--phase-locking vs. envelope reproduction--indicates that both strategies are realized to different degrees depending on the f(AM). We measured latencies for following modulations in rate-increasing and rate-decreasing SAMs using a modified reverse correlation approach. Although latencies varied between 2.5 and 5 ms between cells, a decrease in rate consistently followed an increase in rate with a delay of about 0.2 ms in each cell. These results suggest a temporally precise representation of rate-increasing and rate-decreasing stimuli at the level of the MNTB during dynamic stimulation.

Response patterns to sound associated with labeled globular/bushy cells in cat

The mammalian cochlear nucleus (CN) consists of a diverse set of neurons both physiologically and morphologically that are involved in processing different aspects of the sound signal. One class of CN neurons that is located near the entrance of the auditory nerve (AN) to the CN has an oval soma with an eccentric nucleus and a short-bushy dendritic tree and is called a globular/bushy cell (GBC). They contact the principal cells of the medial nucleus of the trapezoid body (MNTB) with the very large calyx of Held that is one of the most secure synapses in the brain. Because MNTB cells provide an inhibitory input to the lateral superior olive (LSO), a structure purported to play a role in lateralizing high frequency sounds, GBC physiology is of great interest. Results were obtained with intracellular recording and subsequent labeling with neurobiotin of 32 GBCs along with a number of cells characterized extracellularly as likely GBCs in the cochlear nucleus (CN) of cat. Their poststimulus discharge response pattern to repeated tones varies from a primarylike pattern, i.e. similar to the AN, to a primarylike pattern with a 0.5–2 ms notch after the initial spike, to an onset pattern with a low-sustained rate. They can represent low frequency tones and amplitude modulated signals exceptionally well with a temporal code.

Glycinergic Transmission Shaped by the Corelease of GABA in a Mammalian Auditory Synapse

The firing pattern of neurons is shaped by the convergence of excitation and inhibition, each with finely tuned magnitude and duration. In an auditory brainstem nucleus, glycinergic inhibition features fast decay kinetics, the mechanism of which is unknown. By applying glycine to native or recombinant glycine receptors, we show that response decay times are accelerated by addition of GABA, a weak partial agonist of glycine receptors. Systematic variation in agonist exposure time revealed that fast synaptic time course may be achieved with submillisecond exposures to mixtures of glycine and GABA at physiological concentrations. Accordingly, presynaptic terminals generally contained both transmitters, and depleting terminals of GABA slowed glycinergic synaptic currents. Thus, coreleased GABA accelerates glycinergic transmission by acting directly on glycine receptors, narrowing the time window for effective inhibition. Packaging both weak and strong agonists in vesicles may be a general means by which presynaptic neurons regulate the duration of postsynaptic responses.

Hyperpolarization‐activated currents are differentially expressed in mice brainstem auditory nuclei

The hyperpolarization-activated cation current (I(h)) may influence precise auditory processing by modulating resting membrane potential and cell excitability. We used electrophysiology and immunohistochemistry to investigate the properties of I(h) in three auditory brainstem nuclei in mice: the anteroventral cochlear nucleus (AVCN), the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO). I(h) amplitude varied considerably between these cell types, with the order of magnitude LSO > AVCN > MNTB. Kinetically, I(h) is faster in LSO neurons, and more active at rest, compared with AVCN and MNTB cells. The half-activation voltage is -10 mV more hyperpolarized for AVCN and MNTB cells compared with LSO neurons. HCN1 immunoreactivity strongly labelled AVCN and LSO neurons, while HCN2 staining was more diffuse in all nuclei. The HCN4 subunit displayed robust membrane staining in AVCN and MNTB cells but weak labelling of the LSO. We used a dynamic clamp, after blocking I(h), to reinsert I(h) to the different cell types. Our results indicate that the native I(h) for each cell type influences the resting membrane potential and can delay the generation of action potentials in response to injected current. Native I(h) increases rebound depolarizations following hyperpolarizations in all cell types, and increases the likelihood of rebound action potentials (particularly in multiple-firing LSO neurons). This systematic comparison shows that I(h) characteristics vary considerably between different brainstem nuclei, and that these differences significantly affect the response properties of cells within these nuclei.

Altered sodium currents in auditory neurons of congenitally deaf mice

Sodium currents are essential for action potential generation and propagation in most excitable cells. Appropriate tuning of these currents can be modulated both developmentally and in response to activity. Here we use a mouse model of congenital deafness (dn/dn- asymptomatic deafness associated with hair cell degeneration) to investigate the effect of lack of activity in the expression of Na(+) currents in neurons from the medial nucleus of the trapezoid body (MNTB). Patch-clamp recordings show that at postnatal day (P) 14, both normal and deaf mice display a significant amount of persistent and resurgent Na(+) currents. However, the persistent current is greater in deaf mice than in normal mice, and resurgent current kinetics are slower in deaf mice. At P7, resurgent currents are not present in either group. MNTB immunohistochemistry demonstrates that Nav1.1 subunits are expressed postsynaptically in both P14 normal and deaf mice, while postsynaptic Nav1.6 staining was only observed in deaf mice. Labelling of Nav1.6 subunits in different age groups revealed that at younger ages (P7), both normal and deaf mice express this protein. Nav1.6 staining was not observed in MNTB neurons of P28 normal mice, whereas it is maintained in deaf mice cells until much later (P28). At P7, none of the groups displayed resurgent currents (despite the detection of Nav1.6 subunits at this age group); this suggests that factors other than alpha subunits are important for modulating these currents in MNTB cells. Our results emphasize the importance of activity during development in regulating Na(+) channels.

Decreased Temporal Precision of Auditory Signaling inKcna1-Null Mice: An Electrophysiological StudyIn Vivo

The voltage-gated potassium (Kv) channel subunit Kv1.1, encoded by the Kcna1 gene, is expressed strongly in the ventral cochlear nucleus (VCN) and the medial nucleus of the trapezoid body (MNTB) of the auditory pathway. To examine the contribution of the Kv1.1 subunit to the processing of auditory information, in vivo single-unit recordings were made from VCN neurons (bushy cells), axonal endings of bushy cells at MNTB cells (calyces of Held), and MNTB neurons of Kcna1-null (-/-) mice and littermate control (+/+) mice. Thresholds and spontaneous firing rates of VCN and MNTB neurons were not different between genotypes. At higher sound intensities, however, evoked firing rates of VCN and MNTB neurons were significantly lower in -/- mice than +/+ mice. The SD of the first-spike latency (jitter) was increased in VCN neurons, calyces, and MNTB neurons of -/- mice compared with +/+ controls. Comparison along the ascending pathway suggests that the increased jitter found in -/- MNTB responses arises mostly in the axons of VCN bushy cells and/or their calyceal terminals rather than in the MNTB neurons themselves. At high rates of sinusoidal amplitude modulations, -/- MNTB neurons maintained high vector strength values but discharged on significantly fewer cycles of the amplitude-modulated stimulus than +/+ MNTB neurons. These results indicate that in Kcna1-null mice the absence of the Kv1.1 subunit results in a loss of temporal fidelity (increased jitter) and the failure to follow high-frequency amplitude-modulated sound stimulation in vivo.

Noradrenaline increases high-frequency firing at the calyx of Held synapse during development by inhibiting glutamate release.

The mammalian auditory brain stem receives profuse adrenergic innervation, whose function is poorly understood. Here we investigate, during postnatal development, the effect of noradrenaline (NA) at the calyx of Held synapse in the rat medial nucleus of the trapezoid body (MNTB). We observed that NA inhibits the large glutamatergic EPSC, evoked by afferent fiber stimulation, in a dose-dependent manner. The inhibition was maximal (approximately 48%) at the concentration of 2 microM. It was antagonized by yohimbine and mimicked by the alpha2-adrenergic specific agonist UK14304. Both AMPA and NMDA receptor-mediated EPSCs were inhibited in parallel by NA, suggesting a presynaptic effect. Presynaptic recordings showed that NA inhibits the action potential (AP) generated Ca current by about 20%; however, NA did not significantly affect the presynaptic AP waveform. We thus conclude that the calyx of Held presynaptic terminal expresses alpha2-adrenergic receptors that inhibit its Ca current and thus glutamate release. Noradrenaline was effective in all cells tested from postnatal days 6 to 7 (P6-P7), and thereafter the number of responsive cells diminished, although half of the P14 cells tested still had EPSCs that were inhibited by NA. By contrast, activation by L-2-amino-5-phosphonovaleric acid-sensitive metabotropic glutamate receptors strongly inhibited the EPSCs of all cells tested from P6 to P14. The effect of NA on postsynaptic action potential firing was dependent on the stimulus frequency. At 10 Hz, NA had no effect on firing probability; however, NA helped MNTB cells fire more action potentials during a 100-Hz train of stimuli, even though it did not increase the steady-state depressed EPSC, because it produced a smaller N-methyl-D-aspartate (NMDA) receptor-activated depolarizing plateau. We therefore suggest that the reduction by NA of the first few EPSCs in a train leads to a smaller NMDA depolarizing plateau and thus to increased firing probability at 100 Hz in young synapses. Surprisingly, the inhibition of glutamate release by NA can thus actually increase the excitability of MNTB neurons during early postnatal development.

Organization of the disynaptic pathway from the anteroventral cochlear nucleus to the lateral superior olivary nucleus in the ferret.

The medial nucleus of the trapezoid body (MNTB) is one of three major nuclei of the superior olivary complex and provides an important inhibitory input from the contralateral ear to the lateral superior olivary nucleus (LSO) in the initial binaural pathway for coding interaural intensity differences. The major input to the MNTB from the contralateral anteroventral cochlear nucleus (AVCN) involves giant, calyx-like endings that have a one-to-one relationship with cells in the MNTB as confirmed in the ferret in this study. The main objective of the present study was to define the subsequent organization of projections from cells receiving these calyx-like endings. Several anatomical tracers (Phaseolus vulgaris leucoagglutinin, dextran-biotin, and biocytin) were used that are transported both anterogradely and retrogradely within neuronal projections in order to define the organization of MNTB connections with the LSO in the adult ferret. Analysis focused on determining the topography in both the transverse and longitudinal planes of the projections. Focal tracer injections in the LSO resulted in retrograde labeling of a long, narrow column of cells in the MNTB. The orientation and location of labeled cells was dependent on the medial-lateral position of the injection site. In the rostral-caudal dimension of MNTB, there was no such topographic relation between the injection site and the position of labeled cells. Labeled cells in the MNTB were distributed more or less evenly in a longitudinal column regardless of whether the injection site was restricted to the rostral, middle or caudal part of the LSO. In keeping with this pattern, tracer injections in the MNTB resulted in bands of labeled axons that distributed endings throughout the rostral-caudal axis of the LSO. These bands or sheets varied in medial-lateral position relative to the location of the injection site, but lacked any such rostral-caudal gradient. Thus, overall the MNTB-LSO projections have a convergent-divergent pattern of organization. While MNTB cells receive singular calyx-like endings from the AVCN, LSO cells receive projections from a long column of cells in the MNTB. Implications for processing interaural intensity differences are discussed.

Anatomy and physiology of principal cells of the medial nucleus of the trapezoid body (MNTB) of the cat.

We have recorded from principal cells of the medial nucleus of the trapezoid body (MNTB) in the cat's superior olivary complex using either glass micropipettes filled with Neurobiotin or horseradish peroxidase for intracellular recording and subsequent labeling or extracellular metal microelectrodes relying on prepotentials and electrode location. Labeled principal cells had cell bodies that usually gave rise to one or two primary dendrites, which branched profusely in the vicinity of the cell. At the electron microscopic (EM) level, there was a dense synaptic terminal distribution on the cell body and proximal dendrites. Up to half the measured cell surface could be covered with excitatory terminals, whereas inhibitory terminals consistently covered about one-fifth. The distal dendrites were very sparsely innervated. The thick myelinated axon originated from the cell body and innervated nuclei exclusively in the ipsilateral auditory brain stem. These include the lateral superior olive (LSO), ventral nucleus of the lateral lemniscus, medial superior olive, dorsomedial and ventromedial periolivary nuclei, and the MNTB itself. At the EM level the myelinated collaterals gave rise to terminals that contained nonround vesicles and, in the LSO, were seen terminating on cell bodies and primary dendrites. Responses of MNTB cells were similar to their primary excitatory input, the globular bushy cell (GBC), in a number of ways. The spontaneous spike rate of MNTB cells with low characteristic frequencies (CFs) was low, whereas it tended to be higher for higher CF units. In response to short tones, a low frequency MNTB cell showed enhanced phase-locking abilities, relative to auditory nerve fibers. For cells with CFs >1 kHz, the short tone response often resembled the primary-like with notch response seen in many globular bushy cells, with a well-timed onset component. Exceptions to and variations of this standard response were also noted. When compared with GBCs with comparable CFs, the latency of the MNTB cell response was delayed slightly, as would be expected given the synapse interposed between the two cell types. Our data thus confirm that, in the cat, the MNTB receives and converts synaptic inputs from globular bushy cells into a reasonably accurate reproduction of the bushy cell spike response. This MNTB cell output then becomes an important inhibitory input to a number of ipsilateral auditory brain stem nuclei.