A Developmental Switch in Cholinergic Mechanisms of Modulation in the Medial Nucleus of the Trapezoid Body.

The medial nucleus of the trapezoid body (MNTB) has been intensively investigated as a primary source of inhibition in brainstem auditory circuitry. MNTB-derived inhibition plays a critical role in the computation of sound location, as temporal features of sounds are precisely conveyed through the calyx of Held/MNTB synapse. In adult gerbils, cholinergic signaling influences sound-evoked responses of MNTB neurons via nicotinic acetylcholine receptors (nAChRs; Zhang et al., 2021) establishing a modulatory role for cholinergic input to this nucleus. However, the cellular mechanisms through which acetylcholine (ACh) mediates this modulation in the MNTB remain obscure. To investigate these mechanisms, we used whole-cell current and voltage-clamp recordings to examine cholinergic physiology in MNTB neurons from Mongolian gerbils (Meriones unguiculatus) of both sexes. Membrane excitability was assessed in brain slices, in pre-hearing (postnatal days 9-13) and post-hearing onset (P18-20) MNTB neurons during bath application of agonists and antagonists of nicotinic (nAChRs) and muscarinic receptors (mAChRs). Muscarinic activation induced a potent increase in excitability most prominently prior to hearing onset with nAChR modulation emerging at later time points. Pharmacological manipulations further demonstrated that the voltage-gated K+ channel KCNQ (Kv7) is the downstream effector of mAChR activation that impacts excitability early in development. Cholinergic modulation of Kv7 reduces outward K+ conductance and depolarizes resting membrane potential. Immunolabeling revealed expression of Kv7 channels as well as mAChRs containing M1 and M3 subunits. Together, our results suggest that mAChR modulation is prominent but transient in the developing MNTB and that cholinergic modulation functions to shape auditory circuit development.

Fast Inhibition Slows and Desynchronizes Mouse Auditory Efferent Neuron Activity.

The encoding of acoustic stimuli requires precise neuron timing. Auditory neurons in the cochlear nucleus (CN) and brainstem are well suited for accurate analysis of fast acoustic signals, given their physiological specializations of fast membrane time constants, fast axonal conduction, and reliable synaptic transmission. The medial olivocochlear (MOC) neurons that provide efferent inhibition of the cochlea reside in the ventral brainstem and participate in these fast neural circuits. However, their modulation of cochlear function occurs over time scales of a slower nature. This suggests the presence of mechanisms that reduce MOC inhibition of cochlear function. To determine how monaural excitatory and inhibitory synaptic inputs integrate to affect the timing of MOC neuron activity, we developed a novel in vitro slice preparation ("wedge-slice"). The wedge-slice maintains the ascending auditory nerve root, the entire CN and projecting axons, while preserving the ability to perform visually guided patch-clamp electrophysiology recordings from genetically identified MOC neurons. The "in vivo-like" timing of the wedge-slice demonstrates that the inhibitory pathway accelerates relative to the excitatory pathway when the ascending circuit is intact, and the CN portion of the inhibitory circuit is precise enough to compensate for reduced precision in later synapses. When combined with machine learning PSC analysis and computational modeling, we demonstrate a larger suppression of MOC neuron activity when the inhibition occurs with in vivo-like timing. This delay of MOC activity may ensure that the MOC system is only engaged by sustained background sounds, preventing a maladaptive hypersuppression of cochlear activity.

Metabolic response of auditory brainstem neurons to their broad physiological activity range.

Neurons exhibit a high energetic need, and the question arises as how they metabolically adapt to changing activity states. This is relevant for interpreting functional neuroimaging in different brain areas. Particularly, neurons with a broad firing range might exhibit metabolic adaptations. Therefore, we studied MNTB (medial nucleus of the trapezoid body) principal neurons, which generate action potentials (APs) at frequencies up to several hundred hertz. We performed the experiments in acute brainstem slices of the Mongolian gerbil (Meriones unguiculatus) at 22.5-24.5°C. Upon electrical stimulation of afferent MNTB fibres with 400 stimuli at varying frequencies, we monitored autofluorescence levels of NAD(P)H and FAD and determined the extremum amplitudes of their biphasic response. Additionally, we compared these data with alterations in O2 concentrations measured with an electrochemical sensor. These O2 changes are prominent since MNTB neurons rely on oxidative phosphorylation as shown by our pharmacological experiments. We calculated the O2 consumption rate as change in O2 concentration divided by stimulus durations, because these periods varied inversely with stimulus frequency as a result of the constant number of 400 stimuli applied. The O2 consumption rate increased with stimulation frequency up to a constant value at 600 Hz; that is, energy demand depends on temporal characteristics of activity despite the same number of stimuli. The rates showed no correlation with peak amplitudes of NAD(P)H or FAD, whilst the values of the two molecules were linearly correlated. This points at the complexity of analysing autofluorescence imaging for quantitative metabolic studies, because these values report only relative net changes of many superimposed oxidative and reductive processes. Monitoring O2 concentration rates is, thus, an important tool to improve the interpretation of NAD(P)H/FAD autofluorescence data, as they do not under all conditions and in all systems appropriately reflect the metabolic activity or energy demand.

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, TGFß, 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.

Synaptic plasticity of inhibitory synapses onto medial olivocochlear efferent neurons.

The descending auditory system modulates the ascending system at every level. The final descending, or efferent, stage comprises lateral olivocochlear and medial olivocochlear (MOC) neurons. MOC somata in the ventral brainstem project axons to the cochlea to synapse onto outer hair cells (OHC), inhibiting OHC-mediated cochlear amplification. MOC suppression of OHC function is implicated in cochlear gain control with changing sound intensity, detection of salient stimuli, attention and protection against acoustic trauma. Thus, sound excites MOC neurons to provide negative feedback of the cochlea. Sound also inhibits MOC neurons via medial nucleus of the trapezoid body (MNTB) neurons. However, MNTB-MOC synapses exhibit short-term depression, suggesting reduced MNTB-MOC inhibition during sustained stimuli. Further, due to high rates of both baseline and sound-evoked activity in MNTB neurons in vivo, MNTB-MOC synapses may be tonically depressed. To probe this, we characterized short-term plasticity of MNTB-MOC synapses in mouse brain slices. We mimicked in vivo-like temperature and extracellular calcium conditions, and in vivo-like activity patterns of fast synaptic activation rates, sustained activation and prior tonic activity. Synaptic depression was sensitive to extracellular calcium concentration and temperature. During rapid MNTB axon stimulation, postsynaptic currents in MOC neurons summated but with concurrent depression, resulting in smaller, sustained currents, suggesting tonic inhibition of MOC neurons during rapid circuit activity. Low levels of baseline MNTB activity did not significantly reduce responses to subsequent rapid activity that mimics sound stimulation, indicating that, in vivo, MNTB inhibition of MOC neurons persists despite tonic synaptic depression. KEY POINTS: Inhibitory synapses from the medial nucleus of the trapezoid body (MNTB) onto medial olivocochlear (MOC) neurons exhibit short-term plasticity that is sensitive to calcium and temperature, with enhanced synaptic depression occurring at higher calcium concentrations and at room temperature. High rates of background synaptic activity that mimic the upper limits of spontaneous MNTB activity cause tonic synaptic depression of MNTB-MOC synapses that limits further synaptic inhibition. High rates of activity at MNTB-MOC synapses cause synaptic summation with concurrent depression to yield a response with an initial large amplitude that decays to a tonic inhibition.

Synaptic Inhibition of Medial Olivocochlear Efferent Neurons by Neurons of the Medial Nucleus of the Trapezoid Body.

Medial olivocochlear (MOC) efferent neurons in the brainstem comprise the final stage of descending control of the mammalian peripheral auditory system through axon projections to the cochlea. MOC activity adjusts cochlear gain and frequency tuning, and protects the ear from acoustic trauma. The neuronal pathways that activate and modulate the MOC somata in the brainstem to drive these cochlear effects are poorly understood. Evidence suggests that MOC neurons are primarily excited by sound stimuli in a three-neuron activation loop from the auditory nerve via an intermediate neuron in the cochlear nucleus. Anatomical studies suggest that MOC neurons receive diverse synaptic inputs, but the functional effect of additional synaptic influences on MOC neuron responses is unknown. Here we use patch-clamp electrophysiological recordings from identified MOC neurons in brainstem slices from mice of either sex to demonstrate that in addition to excitatory glutamatergic synapses, MOC neurons receive inhibitory GABAergic and glycinergic synaptic inputs. These synapses are activated by electrical stimulation of axons near the medial nucleus of the trapezoid body (MNTB). Focal glutamate uncaging confirms MNTB neurons as a source of inhibitory synapses onto MOC neurons. MNTB neurons inhibit MOC action potentials, but this effect depresses with repeat activation. This work identifies a new pathway of connectivity between brainstem auditory neurons and indicates that MOC neurons are both excited and inhibited by sound stimuli received at the same ear. The pathway depression suggests that the effect of MNTB inhibition of MOC neurons diminishes over the course of a sustained sound.SIGNIFICANCE STATEMENT Medial olivocochlear (MOC) neurons are the final stage of descending control of the mammalian auditory system and exert influence on cochlear mechanics to modulate perception of acoustic stimuli. The brainstem pathways that drive MOC function are poorly understood. Here we show for the first time that MOC neurons are inhibited by neurons of the MNTB, which may suppress the effects of MOC activity on the cochlea.

Mechanisms Underlying Enhancement of Spontaneous Glutamate Release by Group I mGluRs at a Central Auditory Synapse.

One emerging concept in neuroscience states that synaptic vesicles and the molecular machinery underlying spontaneous transmitter release are different from those underlying action potential-driven synchronized transmitter release. Differential neuromodulation of these two distinct release modes by metabotropic glutamate receptors (mGluRs) constitutes critical supporting evidence. However, the mechanisms underlying such a differential modulation are not understood. Here, we investigated the mechanisms of the modulation by group I mGluRs (mGluR Is) on spontaneous glutamate release in the medial nucleus of the trapezoid body (MNTB), an auditory brainstem nucleus critically involved in sound localization. Whole-cell patch recordings from brainstem slices of mice of both sexes were performed. Activation of mGluR I by 3,5-dihydroxyphenylglycine (3,5-DHPG; 200 µm) produced an inward current at -60 mV and increased spontaneous glutamate release in MNTB neurons. Pharmacological evidence indicated involvement of both mGluR1 and mGluR5, which was further supported for mGluR5 by immunolabeling results. The modulation was eliminated by blocking NaV channels (tetrodotoxin, 1 µm), persistent Na+ current (INaP; riluzole, 10 µm), or CaV channels (CdCl2, 100 µm). Presynaptic calyx recordings revealed that 3,5-DHPG shifted the activation of INaP to more hyperpolarized voltages and increased INaP at resting membrane potential. Our data indicate that mGluR I enhances spontaneous glutamate release via regulation of INaP and subsequent Ca2+-dependent processes under resting condition.SIGNIFICANCE STATEMENT For brain cells to communicate with each other, neurons release chemical messengers, termed neurotransmitters, in response to action potential invasion (evoked release). Neurons also release neurotransmitters spontaneously. Recent work has revealed different release machineries underlying these two release modes, and their different roles in synaptic development and plasticity. Our recent work discovered differential neuromodulation of these two release modes, but the mechanisms are not well understood. The present study showed that activation of group I metabotropic glutamate receptors enhanced spontaneous glutamate release in an auditory brainstem nucleus, while suppressing evoked release. The modulation is dependent on a persistent Na+ current and involves subsequent Ca2+ signaling, providing insight into the mechanisms underlying the different release modes in auditory processing.

Presynaptic Diversity Revealed by Ca2+-Permeable AMPA Receptors at the Calyx of Held Synapse.

GluA2-lacking Ca2+-permeable AMPARs (CP-AMPARs) play integral roles in synaptic plasticity and can mediate excitotoxic cellular signaling at glutamatergic synapses. However, the developmental profile of functional CP-AMPARs at the auditory brainstem remains poorly understood. Through a combination of electrophysiological and live-cell Ca2+ imaging from mice of either sex, we show that the synaptic release of glutamate from the calyx of Held nerve terminal activates CP-AMPARs in the principal cells of the medial nucleus of the trapezoid body in the brainstem. This leads to significant Ca2+ influx through these receptors before the onset of hearing at postnatal day 12 (P12). Using a selective open channel blocker of CP-AMPARs, IEM-1460, we estimate that ∼80% of the AMPAR population are permeable to Ca2+ at immature P4-P5 synapses. However, after the onset of hearing, Ca2+ influx through these receptors was greatly reduced. We estimate that CP-AMPARs comprise approximately 40% and 33% of the AMPAR population at P18-P22 and P30-P34, respectively. By quantifying the rate of EPSC block by IEM-1460, we found an increased heterogeneity in glutamate release probability for adult-like calyces (P30-P34). Using tetraethylammonium (TEA), a presynaptic potassium channel blocker, we show that the apparent reduction of CP-AMPARs in more mature synapses is not a consequence of presynaptic action potential (AP) speeding. Finally, through postsynaptic AP recordings, we show that inhibition of CP-AMPARs reduces spike fidelity in juvenile synapses, but not in more mature synapses. We conclude that the expression of functional CP-AMPARs declines over early postnatal development in the calyx of Held synapse.SIGNIFICANCE STATEMENT The calyx of Held synapse is pivotal to the circuitry that computes sound localization. Postsynaptic Ca2+ influx via AMPARs may be critical for signaling the maturation of this brainstem synapse. The GluA4 subunit may dominate the AMPAR complex at mature synapses because of its fast gating kinetics and large unitary conductance. The expectation is that AMPARs dominated by GluA4 subunits should be highly Ca2+ permeable. However, we find that Ca2+-permeable AMPAR expression declines during postnatal development. Using the rate of EPSC block by IEM-1460, an open channel blocker of Ca2+-permeable AMPARs, we propose a novel method to determine glutamate release probability and uncover an increased heterogeneity in release probability for more mature calyces of Held nerve terminals.

Strengthening of the Efferent Olivocochlear System Leads to Synaptic Dysfunction and Tonotopy Disruption of a Central Auditory Nucleus.

The auditory system in many mammals is immature at birth but precisely organized in adults. Spontaneous activity in the inner ear plays a critical role in guiding this maturation process. This is shaped by an efferent pathway that descends from the brainstem and makes transient direct synaptic contacts with inner hair cells. In this work, we used an α9 cholinergic nicotinic receptor knock-in mouse model (of either sex) with enhanced medial efferent activity (Chrna9L9'T, L9'T) to further understand the role of the olivocochlear system in the correct establishment of auditory circuits. Wave III of auditory brainstem responses (which represents synchronized activity of synapses within the superior olivary complex) was smaller in L9'T mice, suggesting a central dysfunction. The mechanism underlying this functional alteration was analyzed in brain slices containing the medial nucleus of the trapezoid body (MNTB), where neurons are topographically organized along a mediolateral (ML) axis. The topographic organization of MNTB physiological properties observed in wildtype (WT) was abolished in L9'T mice. Additionally, electrophysiological recordings in slices indicated MNTB synaptic alterations. In vivo multielectrode recordings showed that the overall level of MNTB activity was reduced in the L9'T The present results indicate that the transient cochlear efferent innervation to inner hair cells during the critical period before the onset of hearing is involved in the refinement of topographic maps as well as in setting the properties of synaptic transmission at a central auditory nucleus.SIGNIFICANCE STATEMENT Cochlear inner hair cells of altricial mammals display spontaneous electrical activity before hearing onset. The pattern and firing rate of these cells are crucial for the correct maturation of the central auditory pathway. A descending efferent innervation from the CNS contacts the hair cells during this developmental window. The present work shows that genetic enhancement of efferent function disrupts the orderly topographic distribution of biophysical and synaptic properties in the auditory brainstem and causes severe synaptic dysfunction. This work adds to the notion that the transient efferent innervation to the cochlea is necessary for the correct establishment of the central auditory circuitry.

Mechanisms and functional impact of Group I metabotropic glutamate receptor modulation of excitability in mouse MNTB neurons.

We examined effects of Group I metabotropic glutamate receptors on the excitability of mouse medial nucleus of the trapezoid body (MNTB) neurons. The selective agonist, S-3,5-dihydroxyphenylglycine (DHPG), evoked a dose-dependent depolarization of the resting potential, increased membrane resistance, increased sag depolarization, and promoted rebound action potential firing. Under voltage-clamp, DHPG evoked an inward current, referred to as IDHPG , which was developmentally stable through postnatal day P56. IDHPG had low temperature dependence in the range 25-34°C, consistent with a channel mechanism. However, the I-V relationship took the form of an inverted U that did not reverse at the calculated Nernst potential for K+ or Cl- . Thus, it is likely that more than one ion type contributes to IDHPG and the mix may be voltage dependent. IDHPG was resistant to the Na+ channel blockers tetrodotoxin and amiloride, and to inhibitors of iGluR (CNQX and MK801). IDHPG was inhibited 21% by Ba2+ (500 µM), 60% by ZD7288 (100 µM) and 73% when the two antagonists were applied together, suggesting that KIR channels and HCN channels contribute to the current. Voltage clamp measurements of IH indicated a small (6%) increase in Gmax by DHPG with no change in the voltage dependence. DHPG reduced action potential rheobase and reduced the number of post-synaptic AP failures during high frequency stimulation of the calyx of Held. Thus, activation of post-synaptic Group I mGlu receptors modifies the excitability of MNTB neurons and contributes to the reliability of high frequency firing in this auditory relay nucleus.

Resistance to action potential depression of a rat axon terminal in vivo.

The shape of the presynaptic action potential (AP) has a strong impact on neurotransmitter release. Because of the small size of most terminals in the central nervous system, little is known about the regulation of their AP shape during natural firing patterns in vivo. The calyx of Held is a giant axosomatic terminal in the auditory brainstem, whose biophysical properties have been well studied in slices. Here, we made whole-cell recordings from calyceal terminals in newborn rat pups. The calyx showed a characteristic burst firing pattern, which has previously been shown to originate from the cochlea. Surprisingly, even for frequencies over 200 Hz, the AP showed little or no depression. Current injections showed that the rate of rise of the AP depended strongly on its onset potential, and that the membrane potential after the AP (Vafter) was close to the value at which no depression would occur during high-frequency activity. Immunolabeling revealed that Nav1.6 is already present at the calyx shortly after its formation, which was in line with the fast recovery from AP depression that we observed in slice recordings. Our findings thus indicate that fast recovery from depression and an inter-AP membrane potential that minimizes changes on the next AP in vivo, together enable high timing precision of the calyx of Held already shortly after its formation.

Neurotransmitter- and Release-Mode-Specific Modulation of Inhibitory Transmission by Group I Metabotropic Glutamate Receptors in Central Auditory Neurons of the Mouse.

Neuromodulation mediated by metabotropic glutamate receptors (mGluRs) regulates many brain functions. However, the functions of mGluRs in the auditory system under normal and diseased states are not well understood. The medial nucleus of the trapezoid body (MNTB) is a critical nucleus in the auditory brainstem nuclei involved in sound localization. In addition to the classical calyx excitatory inputs, MNTB neurons also receive synaptic inhibition and it remains entirely unknown how this inhibition is regulated. Here, using whole-cell voltage clamp in brain slices, we investigated group I mGluR (mGluR I)-mediated modulation of the glycinergic and GABAergic inputs to MNTB neurons in both WT mice and a fragile X syndrome (FXS) mouse model (both sexes) in which the fragile X mental retardation gene 1 is knocked out (Fmr1 KO), causing exaggerated activity of mGluR I and behavioral phenotypes. Activation of mGluR I by (RS)-3,5-dihydroxyphenylglycine (3,5-DHPG) increased the frequency and amplitude of glycinergic spontaneous IPSCs (sIPSCs) in both WT and Fmr1 KO neurons in a voltage-gated sodium channel-dependent fashion, but did not modulate glycinergic evoked IPSCs (eIPSCs). In contrast, 3,5-DHPG did not affect GABAergic sIPSCs, but did suppress eIPSCs in WT neurons via endocannabinoid signaling. In the KO, the effect of 3,5-DHPG on GABAergic eIPSCs was highly variable, which supports the notion of impaired GABAergic signaling in the FXS model. The differential modulation of sIPSC and eIPSC and differential modulation of glycinergic and GABAergic transmission suggest distinct mechanisms responsible for spontaneous and evoked release of inhibitory transmitters and their modulation through the mGluR I signaling pathway.SIGNIFICANCE STATEMENT Neurons communicate with each other through the release of neurotransmitters, which assumes two basic modes, spontaneous and evoked release. These two release modes are believed to function using the same vesicle pool and machinery. Recent works have challenged this dogma, pointing to distinct vesicle release mechanisms underlying the two release modes. Here, we provide the first evidence in the central auditory system supporting this novel concept. We discovered neural-transmitter- and release-mode-specific neuromodulation of inhibitory transmission by metabotropic glutamate receptors and revealed part of the signaling pathways underlying this differential modulation. The results establish the foundation for a multitude of directions to study physiological significance of different release modes in auditory processing.

Sound-Evoked Activity Influences Myelination of Brainstem Axons in the Trapezoid Body.

Plasticity of myelination represents a mechanism to tune the flow of information by balancing functional requirements with metabolic and spatial constraints. The auditory system is heavily myelinated and operates at the upper limits of action potential generation frequency and speed observed in the mammalian CNS. This study aimed to characterize the development of myelin within the trapezoid body, a central auditory fiber tract, and determine the influence sensory experience has on this process in mice of both sexes. We find that in vitro conduction speed doubles following hearing onset and the ability to support high-frequency firing increases concurrently. Also in this time, the diameter of trapezoid body axons and the thickness of myelin double, reaching mature-like thickness between 25 and 35 d of age. Earplugs were used to induce ∼50 dB elevation in auditory thresholds. If introduced at hearing onset, trapezoid body fibers developed thinner axons and myelin than age-matched controls. If plugged during adulthood, the thickest trapezoid body fibers also showed a decrease in myelin. These data demonstrate the need for sensory activity in both development and maintenance of myelin and have important implications in the study of myelin plasticity and how this could relate to sensorineural hearing loss following peripheral impairment.SIGNIFICANCE STATEMENT The auditory system has many mechanisms to maximize the dynamic range of its afferent fibers, which operate at the physiological limit of action potential generation, precision, and speed. In this study we demonstrate for the first time that changes in peripheral activity modifies the thickness of myelin in sensory neurons, not only in development but also in mature animals. The current study suggests that changes in CNS myelination occur as a downstream mechanism following peripheral deficit. Given the required submillisecond temporal precision for binaural auditory processing, reduced myelination might augment sensorineural hearing impairment.

SK Channels Regulate Resting Properties and Signaling Reliability of a Developing Fast-Spiking Neuron.

Reliable and precise signal transmission is essential in circuits of the auditory brainstem to encode timing with submillisecond accuracy. Globular bushy cells reliably and faithfully transfer spike signals to the principal neurons of the medial nucleus of the trapezoid body (MNTB) through the giant glutamatergic synapse, the calyx of Held. Thus, the MNTB works as a relay nucleus that preserves the temporal pattern of firing at high frequency. Using whole-cell patch-clamp recordings, we observed a K+ conductance mediated by small-conductance calcium-activated potassium (SK) channels in the MNTB neurons from rats of either sex. SK channels were activated by intracellular Ca2+ sparks and mediated spontaneous transient outward currents in developing MNTB neurons. SK channels were also activated by Ca2+ influx through voltage-gated Ca2+ channels and synaptically activated NMDA receptors. Blocking SK channels with apamin depolarized the resting membrane potential, reduced resting conductance, and affected the responsiveness of MNTB neurons to signal inputs. Moreover, SK channels were activated by action potentials and affected the spike afterhyperpolarization. Blocking SK channels disrupted the one-to-one signal transmission from presynaptic calyces to postsynaptic MNTB neurons and induced extra postsynaptic action potentials in response to presynaptic firing. These data reveal that SK channels play crucial roles in regulating the resting properties and maintaining reliable signal transmission of MNTB neurons.SIGNIFICANCE STATEMENT Reliable and precise signal transmission is required in auditory brainstem circuits to localize the sound source. The calyx of Held synapse in the mammalian medial nucleus of the trapezoid body (MNTB) plays an important role in sound localization. We investigated the potassium channels that shape the reliability of signal transfer across the calyceal synapse and observed a potassium conductance mediated by small-conductance calcium-activated potassium (SK) channels in rat MNTB principal neurons. We found that SK channels are tonically activated and contribute to the resting membrane properties of MNTB neurons. Interestingly, SK channels are transiently activated by calcium sparks and calcium influx during action potentials and control the one-to-one signal transmission from presynaptic calyces to postsynaptic MNTB neurons.

Excitation by Axon Terminal GABA Spillover in a Sound Localization Circuit.

Synapses from neurons of the medial nucleus of the trapezoid body (MNTB) onto neurons of the lateral superior olive (LSO) in the auditory brainstem are glycinergic in maturity, but also GABAergic and glutamatergic in development. The role for this neurotransmitter cotransmission is poorly understood. Here we use electrophysiological recordings in brainstem slices from P3-P21 mice to demonstrate that GABA release evoked from MNTB axons can spill over to neighboring MNTB axons and cause excitation by activating GABAAR. This spillover excitation generates patterns of staggered neurotransmitter release from different MNTB axons resulting in characteristic "doublet" postsynaptic currents in LSO neurons. Postembedding immunogold labeling and electron microscopy provide evidence that GABAARs are localized at MNTB axon terminals. Photolytic uncaging of p-hydroxyphenacyl (pHP) GABA demonstrates backpropagation of GABAAR-mediated depolarizations from MNTB axon terminals to the soma, some hundreds of microns away. These somatic depolarizations enhanced somatic excitability by increasing the probability of action potential generation. GABA spillover excitation between MNTB axon terminals may entrain neighboring MNTB neurons, which may play a role in the developmental refinement of the MNTB-LSO pathway. Axonal spillover excitation persisted beyond the second postnatal week, suggesting that this mechanism may play a role in sound localization, by providing new avenues of communication between MNTB neurons via their distal axonal projections. Significance statement: In this study, a new mechanism of neuronal communication between auditory synapses in the mammalian sound localization pathway is described. Evidence is provided that the inhibitory neurotransmitter GABA can spill over between axon terminals to cause excitation of nearby synapses to further stimulate neurotransmitter release. Excitatory GABA spillover between inhibitory axon terminals may have important implications for the development and refinement of this auditory circuit and may play a role in the ability to precisely localize sound sources.

Enhancement of phase-locking in rodents. I. An axonal recording study in gerbil.

The trapezoid body (TB) contains axons of neurons in the anteroventral cochlear nucleus projecting to monaural and binaural nuclei in the superior olivary complex (SOC). Characterization of these monaural inputs is important for the interpretation of response properties of SOC neurons. In particular, understanding of the sensitivity to interaural time differences (ITDs) in neurons of the medial and lateral superior olive requires knowledge of the temporal firing properties of the monaural excitatory and inhibitory inputs to these neurons. In recent years, studies of ITD sensitivity of SOC neurons have made increasing use of small animal models with good low-frequency hearing, particularly the gerbil. We presented stimuli as used in binaural studies to monaural neurons in the TB and studied their temporal coding. We found that general trends as have been described in the cat are present in gerbil, but with some important differences. Phase-locking to pure tones tends to be higher in TB axons and in neurons of the medial nucleus of the TB (MNTB) than in the auditory nerve for neurons with characteristic frequencies (CFs) below 1 kHz, but this enhancement is quantitatively more modest than in cat. Stronger enhancement is common when TB neurons are stimulated at low frequencies below CF. It is rare for TB neurons in gerbil to entrain to low-frequency stimuli, i.e., to discharge a well-timed spike on every stimulus cycle. Also, complex phase-locking behavior, with multiple modes of increased firing probability per stimulus cycle, is common in response to low frequencies below CF.NEW & NOTEWORTHY Phase-locking is an important property of neurons in the early auditory pathway: it is critical for the sensitivity to time differences between the two ears enabling spatial hearing. Studies in cat have shown an improvement in phase-locking from the peripheral to the central auditory nervous system. We recorded from axons in an output tract of the cochlear nucleus and show that a similar but more limited form of temporal enhancement is present in gerbil.

Complexin stabilizes newly primed synaptic vesicles and prevents their premature fusion at the mouse calyx of held synapse.

Complexins (Cplxs) are small synaptic proteins that cooperate with SNARE-complexes in the control of synaptic vesicle (SV) fusion. Studies involving genetic mutation, knock-down, or knock-out indicated two key functions of Cplx that are not mutually exclusive but cannot easily be reconciled, one in facilitating SV fusion, and one in "clamping" SVs to prevent premature fusion. Most studies on the role of Cplxs in mammalian synapse function have relied on cultured neurons, heterologous expression systems, or membrane fusion assays in vitro, whereas little is known about the function of Cplxs in native synapses. We therefore studied consequences of genetic ablation of Cplx1 in the mouse calyx of Held synapse, and discovered a developmentally exacerbating phenotype of reduced spontaneous and evoked transmission but excessive asynchronous release after stimulation, compatible with combined facilitating and clamping functions of Cplx1. Because action potential waveforms, Ca(2+) influx, readily releasable SV pool size, and quantal size were unaltered, the reduced synaptic strength in the absence of Cplx1 is most likely a consequence of a decreased release probability, which is caused, in part, by less tight coupling between Ca(2+) channels and docked SV. We found further that the excessive asynchronous release in Cplx1-deficient calyces triggered aberrant action potentials in their target neurons, and slowed-down the recovery of EPSCs after depleting stimuli. The augmented asynchronous release had a delayed onset and lasted hundreds of milliseconds, indicating that it predominantly represents fusion of newly recruited SVs, which remain unstable and prone to premature fusion in the absence of Cplx1.

Developmental profiles of the intrinsic properties and synaptic function of auditory neurons in preterm and term baboon neonates.

The human fetus starts to hear and undergoes major developmental changes in the auditory system during the third trimester of pregnancy. Although there are significant data regarding development of the auditory system in rodents, changes in intrinsic properties and synaptic function of auditory neurons in developing primate brain at hearing onset are poorly understood. We performed whole-cell patch-clamp recordings of principal neurons in the medial nucleus of trapezoid body (MNTB) in preterm and term baboon brainstem slices to study the structural and functional maturation of auditory synapses. Each MNTB principal neuron received an excitatory input from a single calyx of Held terminal, and this one-to-one pattern of innervation was already formed in preterm baboons delivered at 67% of normal gestation. There was no difference in frequency or amplitude of spontaneous excitatory postsynaptic synaptic currents between preterm and term MNTB neurons. In contrast, the frequency of spontaneous GABA(A)/glycine receptor-mediated inhibitory postsynaptic synaptic currents, which were prevalent in preterm MNTB neurons, was significantly reduced in term MNTB neurons. Preterm MNTB neurons had a higher input resistance than term neurons and fired in bursts, whereas term MNTB neurons fired a single action potential in response to suprathreshold current injection. The maturation of intrinsic properties and dominance of excitatory inputs in the primate MNTB allow it to take on its mature role as a fast and reliable relay synapse.

Neurexins control the strength and precise timing of glycinergic inhibition in the auditory brainstem.

Neurexins play diverse functions as presynaptic organizers in various glutamatergic and GABAergic synapses. However, it remains unknown whether and how neurexins are involved in shaping functional properties of the glycinergic synapses, which mediate prominent inhibition in the brainstem and spinal cord. To address these issues, we examined the role of neurexins in a model glycinergic synapse between the principal neuron in the medial nucleus of the trapezoid body (MNTB) and the principal neuron in the lateral superior olive (LSO) in the auditory brainstem. Combining RNAscope with stereotactic injection of AAV-Cre in the MNTB of neurexin1/2/3 conditional triple knockout mice, we showed that MNTB neurons highly express all isoforms of neurexins although their expression levels vary remarkably. Selective ablation of all neurexins in MNTB neurons not only reduced the amplitude but also altered the kinetics of the glycinergic synaptic transmission at LSO neurons. The synaptic dysfunctions primarily resulted from an impaired Ca2+ sensitivity of release and a loosened coupling between voltage-gated Ca2+ channels and synaptic vesicles. Together, our current findings demonstrate that neurexins are essential in controlling the strength and temporal precision of the glycinergic synapse, which therefore corroborates the role of neurexins as key presynaptic organizers in all major types of fast chemical synapses.

Sex-specific loss of mitochondrial membrane integrity and mass in the auditory brainstem of a mouse model of Fragile X syndrome.

Sound sensitivity is one of the most common sensory complaints for people with autism spectrum disorders (ASDs). How and why sounds are perceived as overwhelming by affected people is unknown. To process sound information properly, the brain requires high activity and fast processing, as seen in areas like the medial nucleus of the trapezoid body (MNTB) of the auditory brainstem. Recent work has shown dysfunction in mitochondria, which are the primary source of energy in cells, in a genetic model of ASD, Fragile X syndrome (FXS). Whether mitochondrial functions are also altered in sound-processing neurons, has not been characterized yet. To address this question, we imaged the MNTB in a mouse model of FXS. We stained MNTB brain slices from wild-type and FXS mice with two mitochondrial markers, TOMM20 and PMPCB, located on the Outer Mitochondrial Membrane and in the matrix, respectively. These markers allow exploration of mitochondrial subcompartments. Our integrated imaging pipeline reveals significant sex-specific differences in the degree of mitochondrial length in FXS. Significant differences are also observable in the overall number of mitochondria in male FXS mice, however, colocalization analyses between TOMM20 and PMPCB reveal that the integrity of these compartments is most disrupted in female FXS mice. We highlight a quantitative fluorescence microscopy pipeline to monitor mitochondrial functions in the MNTB from control or FXS mice and provide four complementary readouts. Our approach paves the way to understanding how cellular mechanisms important to sound encoding are altered in ASDs.