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.

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.

Group I metabotropic glutamate receptor-triggered temporally patterned action potential-dependent spontaneous synaptic transmission in mouse MNTB neurons.

Rhythmic action potentials (AP) are generated via intrinsic ionic mechanisms in pacemaking neurons, producing synaptic responses of regular inter-event intervals (IEIs) in their targets. In auditory processing, evoked temporally patterned activities are induced when neural responses timely lock to a certain phase of the sound stimuli. Spontaneous spike activity, however, is a stochastic process, rendering the prediction of the exact timing of the next event completely based on probability. Furthermore, neuromodulation mediated by metabotropic glutamate receptors (mGluRs) is not commonly associated with patterned neural activities. Here, we report an intriguing phenomenon. In a subpopulation of medial nucleus of the trapezoid body (MNTB) neurons recorded under whole-cell voltage-clamp mode in acute mouse brain slices, temporally patterned AP-dependent glycinergic sIPSCs and glutamatergic sEPSCs were elicited by activation of group I mGluRs with 3,5-DHPG (200 µM). Auto-correlation analyses revealed rhythmogenesis in these synaptic responses. Knockout of mGluR5 largely eliminated the effects of 3,5-DHPG. Cell-attached recordings showed temporally patterned spikes evoked by 3,5-DHPG in potential presynaptic VNTB cells for synaptic inhibition onto MNTB. The amplitudes of sEPSCs enhanced by 3,5-DHPG were larger than quantal size but smaller than spike-driven calyceal inputs, suggesting that non-calyceal inputs to MNTB might be responsible for the temporally patterned sEPSCs. Finally, immunocytochemical studies identified expression and localization of mGluR5 and mGluR1 in the VNTB-MNTB inhibitory pathway. Our results imply a potential central mechanism underlying the generation of patterned spontaneous spike activity in the brainstem sound localization circuit.

Species-Specific Adaptation for Ongoing High-Frequency Action Potential Generation in MNTB Neurons.

Comparative analysis of evolutionarily conserved neuronal circuits between phylogenetically distant mammals highlights the relevant mechanisms and specific adaptations to information processing. The medial nucleus of the trapezoid body (MNTB) is a conserved mammalian auditory brainstem nucleus relevant for temporal processing. While MNTB neurons have been extensively investigated, a comparative analysis of phylogenetically distant mammals and the spike generation is missing. To understand the suprathreshold precision and firing rate, we examined the membrane, voltage-gated ion channel and synaptic properties in Phyllostomus discolor (bat) and in Meriones unguiculatus (rodent) of either sex. Between the two species, the membrane properties of MNTB neurons were similar at rest with only minor differences, while larger dendrotoxin (DTX)-sensitive potassium currents were found in gerbils. Calyx of Held-mediated EPSCs were smaller and frequency dependence of short-term plasticity (STP) less pronounced in bats. Simulating synaptic train stimulations in dynamic clamp revealed that MNTB neurons fired with decreasing success rate near conductance threshold and at increasing stimulation frequency. Driven by STP-dependent conductance decrease, the latency of evoked action potentials increased during train stimulations. The spike generator showed a temporal adaptation at the beginning of train stimulations that can be explained by sodium current inactivation. Compared with gerbils, the spike generator of bats sustained higher frequency input-output functions and upheld the same temporal precision. Our data mechanistically support that MNTB input-output functions in bats are suited to sustain precise high-frequency rates, while for gerbils, temporal precision appears more relevant and an adaptation to high output-rates can be spared.SIGNIFICANCE STATEMENT Neurons in the mammalian medial nucleus of the trapezoid body (MNTB) convey precise, faithful inhibition vital for binaural hearing and gap detection. The MNTB's structure and function appear evolutionarily well conserved. We compared the cellular physiology of MNTB neurons in bat and gerbil. Because of their adaptations to echolocation or low frequency hearing both species are model systems for hearing research, yet with largely overlapping hearing ranges. We find that bat neurons sustain information transfer with higher ongoing rates and precision based on synaptic and biophysical differences in comparison to gerbils. Thus, even in evolutionarily conserved circuits species-specific adaptations prevail, highlighting the importance for comparative research to differentiate general circuit functions and their specific adaptations.

Electrical signaling in cochlear efferents is driven by an intrinsic neuronal oscillator.

Efferent neurons are believed to play essential roles in maintaining auditory function. The lateral olivocochlear (LOC) neurons-which project from the brainstem to the inner ear, where they release multiple transmitters including peptides, catecholamines, and acetylcholine-are the most numerous yet least understood elements of efferent control of the cochlea. Using in vitro calcium imaging and patch-clamp recordings, we found that LOC neurons in juvenile and young adult mice exhibited extremely slow waves of activity (∼0.1 Hz). These seconds-long bursts of Na+ spikes were driven by an intrinsic oscillator dependent on L-type Ca2+ channels and were not observed in prehearing mice, suggesting an age-dependent mechanism underlying the intrinsic oscillator. Using optogenetic approaches, we identified both ascending (T-stellate cells of the cochlear nucleus) and descending (auditory cortex) sources of synaptic excitation, as well as the synaptic receptors used for such excitation. Additionally, we identified potent inhibition originating in the glycinergic medial nucleus of trapezoid body (MNTB). Conductance-clamp experiments revealed an unusual mechanism of electrical signaling in LOC neurons, in which synaptic excitation and inhibition served to switch on and off the intrinsically generated spike burst mechanism, allowing for prolonged periods of activity or silence controlled by brief synaptic events. Protracted bursts of action potentials may be essential for effective exocytosis of the diverse transmitters released by LOC fibers in the cochlea.

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.

Brain-Derived Neurotrophic Factor Is Involved in Activity-Dependent Tonotopic Refinement of MNTB Neurons.

In the mammalian brain, auditory brainstem nuclei are arranged topographically according to acoustic frequency responsiveness. During postnatal development, the axon initial segment (AIS) of principal neurons undergoes structural refinement depending on location along the tonotopic axis within the medial nucleus of the trapezoid body (MNTB). However, the molecular mechanisms underlying the structural refinement of the AIS along the tonotopic axis in the auditory brainstem have not been explored. We tested the hypothesis that brain-derived neurotrophic factor (BDNF) is a molecular mediator of the structural development of the MNTB in an activity-dependent manner. Using BDNF heterozygous mutant (BDNF+/- ) mice, we examined the impact of global BDNF reduction on structural and functional development of MNTB neurons by assessing AIS structure and associated intrinsic neuronal properties. BDNF reduction inhibits the structural and functional differentiation of principal neurons along the tonotopic axis in the MNTB. Augmented sound input during the critical period of development has been shown to enhance the structural refinement of the AIS of MNTB neurons. However, in BDNF +/- mice, MNTB neurons did not show this activity-dependent structural modification of the AIS following repeated sound stimulation. In addition, BDNF+/- mice lacked a defined isofrequency band of neuronal activity following exposure to 16 kHz sound, suggesting degradation of tonotopy. Taken together, structural development and functional refinement of auditory brainstem neurons require physiological levels of BDNF to establish proper tonotopic gradients.

Using ephaptic coupling to estimate the synaptic cleft resistivity of the calyx of Held synapse.

At synapses, the pre- and postsynaptic cells get so close that currents entering the cleft do not flow exclusively along its conductance, gcl. A prominent example is found in the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB), where the presynaptic action potential can be recorded in the postsynaptic cell in the form of a prespike. Here, we developed a theoretical framework for ephaptic coupling via the synaptic cleft, and we tested its predictions using the MNTB prespike recorded in voltage-clamp. The shape of the prespike is predicted to resemble either the first or the second derivative of the inverted presynaptic action potential if cleft currents dissipate either mostly capacitively or resistively, respectively. We found that the resistive dissipation scenario provided a better description of the prespike shape. Its size is predicted to scale with the fourth power of the radius of the synapse, explaining why intracellularly recorded prespikes are uncommon in the central nervous system. We show that presynaptic calcium currents also contribute to the prespike shape. This calcium prespike resembled the first derivative of the inverted calcium current, again as predicted by the resistive dissipation scenario. Using this calcium prespike, we obtained an estimate for gcl of ~1 µS. We demonstrate that, for a circular synapse geometry, such as in conventional boutons or the immature calyx of Held, gcl is scale-invariant and only defined by extracellular resistivity, which was ~75 Ωcm, and by cleft height. During development the calyx of Held develops fenestrations. We show that these fenestrations effectively minimize the cleft potentials generated by the adult action potential, which might otherwise interfere with calcium channel opening. We thus provide a quantitative account of the dissipation of currents by the synaptic cleft, which can be readily extrapolated to conventional, bouton-like synapses.

Projections from the ventral nucleus of the lateral lemniscus to the cochlea in the mouse.

Auditory efferents originate in the central auditory system and project to the cochlea. Although the specific anatomy of the olivocochlear (OC) efferents can vary between species, two types of auditory efferents have been identified based upon the general location of their cell bodies and their distinctly different axon terminations in the organ of Corti. In the mouse, the relatively small somata of the lateral (LOC) efferents reside in the lateral superior olive (LSO), have unmyelinated axons, and terminate around ipsilateral inner hair cells (IHCs), primarily against the afferent processes of type I auditory nerve fibers. In contrast, the larger somata of the medial (MOC) efferents are distributed in the ventral nucleus of the trapezoid body (VNTB), have myelinated axons, and terminate bilaterally against the base of multiple outer hair cells (OHCs). Using in vivo retrograde cell body marking, anterograde axon tracing, immunohistochemistry, and electron microscopy, we have identified a group of efferent neurons in mouse, whose cell bodies reside in the ventral nucleus of the lateral lemniscus (VNLL). By virtue of their location, we call them dorsal efferent (DE) neurons. Labeled DE cells were immuno-negative for tyrosine hydroxylase, glycine, and GABA, but immuno-positive for choline acetyltransferase. Morphologically, DEs resembled LOC efferents by their small somata, unmyelinated axons, and ipsilateral projection to IHCs. These three classes of efferent neurons all project axons directly to the cochlea and exhibit cholinergic staining characteristics. The challenge is to discover the contributions of this new population of neurons to auditory efferent function.

Endogenous Cholinergic Signaling Modulates Sound-Evoked Responses of the Medial Nucleus of the Trapezoid Body.

The medial nucleus of trapezoid body (MNTB) is a major source of inhibition in auditory brainstem circuitry. The MNTB projects well-timed inhibitory output to principal sound-localization nuclei in the superior olive (SOC) as well as other computationally important centers. Acoustic information is conveyed to MNTB neurons through a single calyx of Held excitatory synapse arising from the cochlear nucleus. The encoding efficacy of this large synapse depends on its activity rate, which is primarily determined by sound intensity and stimulus frequency. However, MNTB activity rate is additionally influenced by inhibition and possibly neuromodulatory inputs, albeit their functional role is unclear. Happe and Morley (2004) discovered prominent expression of α7 nAChRs in rat SOC, suggesting possible engagement of ACh-mediated modulation of neural activity in the MNTB. However, the existence and nature of this putative modulation have never been physiologically demonstrated. We probed nicotinic cholinergic influences on acoustic responses of MNTB neurons from adult gerbils (Meriones unguiculatus) of either sex. We recorded tone-evoked MNTB single-neuron activity in vivo using extracellular single-unit recording. Piggyback multibarrel electrodes enabled pharmacological manipulation of nAChRs by reversibly applying antagonists to two receptor types, α7 and α4ß2. We observed that tone-evoked responses are dependent on ACh modulation by both nAChR subtypes. Spontaneous activity was not affected by antagonist application. Functionally, we demonstrate that ACh contributes to sustaining high discharge rates and enhances signal encoding efficacy. Additionally, we report anatomic evidence revealing novel cholinergic projections to MNTB arising from pontine and superior olivary nuclei.SIGNIFICANCE STATEMENT This study is the first to physiologically probe how acetylcholine, a pervasive neuromodulator in the brain, influences the encoding of acoustic information by the medial nucleus of trapezoid body, the most prominent source of inhibition in brainstem sound-localization circuitry. We demonstrate that this cholinergic input enhances neural discrimination of tones from noise stimuli, which may contribute to processing important acoustic signals, such as speech. Additionally, we describe novel anatomic projections providing cholinergic input to the MNTB. Together, these findings shed new light on the contribution of neuromodulation to fundamental computational processes in auditory brainstem circuitry and to a more holistic understanding of modulatory influences in sensory processing.

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.

Computational principles of neural adaptation for binaural signal integration.

Adaptation to statistics of sensory inputs is an essential ability of neural systems and extends their effective operational range. Having a broad operational range facilitates to react to sensory inputs of different granularities, thus is a crucial factor for survival. The computation of auditory cues for spatial localization of sound sources, particularly the interaural level difference (ILD), has long been considered as a static process. Novel findings suggest that this process of ipsi- and contra-lateral signal integration is highly adaptive and depends strongly on recent stimulus statistics. Here, adaptation aids the encoding of auditory perceptual space of various granularities. To investigate the mechanism of auditory adaptation in binaural signal integration in detail, we developed a neural model architecture for simulating functions of lateral superior olive (LSO) and medial nucleus of the trapezoid body (MNTB) composed of single compartment conductance-based neurons. Neurons in the MNTB serve as an intermediate relay population. Their signal is integrated by the LSO population on a circuit level to represent excitatory and inhibitory interactions of input signals. The circuit incorporates an adaptation mechanism operating at the synaptic level based on local inhibitory feedback signals. The model's predictive power is demonstrated in various simulations replicating physiological data. Incorporating the innovative adaptation mechanism facilitates a shift in neural responses towards the most effective stimulus range based on recent stimulus history. The model demonstrates that a single LSO neuron quickly adapts to these stimulus statistics and, thus, can encode an extended range of ILDs in the ipsilateral hemisphere. Most significantly, we provide a unique measurement of the adaptation efficacy of LSO neurons. Prerequisite of normal function is an accurate interaction of inhibitory and excitatory signals, a precise encoding of time and a well-tuned local feedback circuit. We suggest that the mechanisms of temporal competitive-cooperative interaction and the local feedback mechanism jointly sensitize the circuit to enable a response shift towards contra-lateral and ipsi-lateral stimuli, respectively.

Physiological and anatomical development of glycinergic inhibition in the mouse superior paraolivary nucleus following hearing onset.

Neural circuits require balanced synaptic excitation and inhibition to ensure accurate neural computation. Our knowledge about the development and maturation of inhibitory synaptic inputs is less well developed than that concerning excitation. Here we describe the maturation of an inhibitory circuit within the mammalian auditory brainstem where counterintuitively, inhibition drives action potential firing of principal neurons. With the use of combined anatomical tracing and electrophysiological recordings from mice, neurons of the superior paraolivary nucleus (SPN) are shown to receive converging glycinergic input from at least four neurons of the medial nucleus of the trapezoid body (MNTB). These four axons formed 30.71 ± 2.72 (means ± SE) synaptic boutons onto each SPN neuronal soma, generating a total inhibitory conductance of 80 nS. Such strong inhibition drives the underlying postinhibitory rebound firing mechanism, which is a hallmark of SPN physiology. In contrast to inhibitory projections to the medial and lateral superior olives, the inhibitory projection to the SPN does not exhibit experience-dependent synaptic refinement following the onset of hearing. These findings emphasize that the development and function of neural circuits cannot be inferred from one synaptic target to another, even if both originate from the same neuron.NEW & NOTEWORTHY Neuronal activity regulates development and maturation of neural circuits. This activity can include spontaneous burst firing or firing elicited by sensory input during early development. For example, auditory brainstem circuits involved in sound localization require acoustically evoked activity to form properly. Here we show, that an inhibitory circuit, involved in processing sound offsets, gaps, and rhythmically modulated vocal communication signals, matures before the onset of acoustically evoked activity.

Delayed expression of activity-dependent gating switch in synaptic AMPARs at a central synapse.

Developing central synapses exhibit robust plasticity and undergo experience-dependent remodeling. Evidently, synapses in sensory systems such as auditory brainstem circuits mature rapidly to achieve high-fidelity neurotransmission for sound localization. This depends on a developmental switch in AMPAR composition from slow-gating GluA1-dominant to fast-gating GluA4-dominant, but the mechanisms underlying this switch remain unknown. We hypothesize that patterned stimuli mimicking spontaneous/sound evoked activity in the early postnatal stage drives this gating switch. We examined activity-dependent changes in evoked and miniature excitatory postsynaptic currents (eEPSCs and mEPSCs) at the calyx of Held synapse by breaking through the postsynaptic membrane at different time points following 2 min of theta burst stimulation (TBS) to afferents in mouse brainstem slices. We found the decay time course of eEPSCs accelerated, but this change was not apparent until > 30 min after TBS. Histogram analyses of the decay time constants of mEPSCs for naive and tetanized synapses revealed two populations centered around τfast ≈ 0.4 and 0.8 ms, but the relative weight of the τ0.4 population over the τ0.8 population increased significantly only in tetanized synapses. Such changes are blocked by NMDAR or mGluR1/5 antagonists or inhibitors of CaMKII, PKC and protein synthesis, and more importantly precluded in GluA4-/- synapses, suggesting GluA4 is the substrate underlying the acceleration. Our results demonstrate a novel form of plasticity working through NMDAR and mGluR activation to trigger a gating switch of AMPARs with a temporally delayed onset of expression, ultimately enhancing the development of high-fidelity synaptic transmission.

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.

Microtubule and Actin Differentially Regulate Synaptic Vesicle Cycling to Maintain High-Frequency Neurotransmission.

Cytoskeletal filaments such as microtubules (MTs) and filamentous actin (F-actin) dynamically support cell structure and functions. In central presynaptic terminals, F-actin is expressed along the release edge and reportedly plays diverse functional roles, but whether axonal MTs extend deep into terminals and play any physiological role remains controversial. At the calyx of Held in rats of either sex, confocal and high-resolution microscopy revealed that MTs enter deep into presynaptic terminal swellings and partially colocalize with a subset of synaptic vesicles (SVs). Electrophysiological analysis demonstrated that depolymerization of MTs specifically prolonged the slow-recovery time component of EPSCs from short-term depression induced by a train of high-frequency stimulation, whereas depolymerization of F-actin specifically prolonged the fast-recovery component. In simultaneous presynaptic and postsynaptic action potential recordings, depolymerization of MTs or F-actin significantly impaired the fidelity of high-frequency neurotransmission. We conclude that MTs and F-actin differentially contribute to slow and fast SV replenishment, thereby maintaining high-frequency neurotransmission.SIGNIFICANCE STATEMENT The presence and functional role of MTs in the presynaptic terminal are controversial. Here, we demonstrate that MTs are present near SVs in calyceal presynaptic terminals and that MT depolymerization specifically prolongs the slow-recovery component of EPSCs from short-term depression. In contrast, F-actin depolymerization specifically prolongs fast-recovery component. Depolymerization of MT or F-actin has no direct effect on SV exocytosis/endocytosis or basal transmission, but significantly impairs the fidelity of high-frequency transmission, suggesting that presynaptic cytoskeletal filaments play essential roles in SV replenishment for the maintenance of high-frequency neurotransmission.

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.

Topographic map refinement and synaptic strengthening of a sound localization circuit require spontaneous peripheral activity.

KEY POINTS: Loss of the calcium sensor otoferlin disrupts neurotransmission from inner hair cells. Central auditory nuclei are functionally denervated in otoferlin knockout mice (Otof KOs) via gene ablation confined to the periphery. We employed juvenile and young adult Otof KO mice (postnatal days (P)10-12 and P27-49) as a model for lacking spontaneous activity and deafness, respectively. We studied the impact of peripheral activity on synaptic refinement in the sound localization circuit from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO). MNTB in vivo recordings demonstrated drastically reduced spontaneous spiking and deafness in Otof KOs. Juvenile KOs showed impaired synapse elimination and strengthening, manifested by broader MNTB-LSO inputs, imprecise MNTB-LSO topography and weaker MNTB-LSO fibres. The impairments persisted into young adulthood. Further functional refinement after hearing onset was undetected in young adult wild-types. Collectively, activity deprivation confined to peripheral protein loss impairs functional MNTB-LSO refinement during a critical prehearing period. ABSTRACT: Circuit refinement is critical for the developing sound localization pathways in the auditory brainstem. In prehearing mice (hearing onset around postnatal day (P)12), spontaneous activity propagates from the periphery to central auditory nuclei. At the glycinergic projection from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO) of neonatal mice, super-numerous MNTB fibres innervate a given LSO neuron. Between P4 and P9, MNTB fibres are functionally eliminated, whereas the remaining fibres are strengthened. Little is known about MNTB-LSO circuit refinement after P20. Moreover, MNTB-LSO refinement upon activity deprivation confined to the periphery is largely unexplored. This leaves a considerable knowledge gap, as deprivation often occurs in patients with congenital deafness, e.g. upon mutations in the otoferlin gene (OTOF). Here, we analysed juvenile (P10-12) and young adult (P27-49) otoferlin knockout (Otof KO) mice with respect to MNTB-LSO refinement. MNTB in vivo recordings revealed drastically reduced spontaneous activity and deafness in knockouts (KOs), confirming deprivation. As RNA sequencing revealed Otof absence in the MNTB and LSO of wild-types, Otof loss in KOs is specific to the periphery. Functional denervation impaired MNTB-LSO synapse elimination and strengthening, which was assessed by glutamate uncaging and electrical stimulation. Impaired elimination led to imprecise MNTB-LSO topography. Impaired strengthening was associated with lower quantal content per MNTB fibre. In young adult KOs, the MNTB-LSO circuit remained unrefined. Further functional refinement after P12 appeared absent in wild-types. Collectively, we provide novel insights into functional MNTB-LSO circuit maturation governed by a cochlea-specific protein. The central malfunctions in Otof KOs may have implications for patients with sensorineuronal hearing loss.

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.

GABA is a modulator, rather than a classical transmitter, in the medial nucleus of the trapezoid body-lateral superior olive sound localization circuit.

KEY POINTS: The lateral superior olive (LSO), a brainstem hub involved in sound localization, integrates excitatory and inhibitory inputs from the ipsilateral and the contralateral ear, respectively. In gerbils and rats, inhibition to the LSO reportedly shifts from GABAergic to glycinergic within the first three postnatal weeks. Surprisingly, we found no evidence for synaptic GABA signalling during this time window in mouse LSO principal neurons. However, we found that presynaptic GABAB Rs modulate Ca2+ influx into medial nucleus of the trapezoid body axon terminals, resulting in reduced synaptic strength. Moreover, GABA elicited strong responses in LSO neurons that were mediated by extrasynaptic GABAA Rs. RNA sequencing revealed highly abundant δ subunits, which are characteristic of extrasynaptic receptors. Whereas GABA increased the excitability of neonatal LSO neurons, it reduced the excitability around hearing onset. Collectively, GABA appears to control the excitability of mouse LSO neurons via extrasynaptic and presynaptic signalling. Thus, GABA acts as a modulator, rather than as a classical transmitter. ABSTRACT: GABA and glycine mediate fast inhibitory neurotransmission and are coreleased at several synapse types. Here we assessed the contribution of GABA and glycine in synaptic transmission between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO), two nuclei involved in sound localization. Whole-cell patch-clamp experiments in acute mouse brainstem slices at postnatal days (P) 4 and 11 during pharmacological blockade of GABAA receptors (GABAA Rs) and/or glycine receptors demonstrated no GABAergic synaptic component on LSO principal neurons. A GABAergic component was absent in evoked inhibitory postsynaptic currents and miniature events. Coimmunofluorescence experiments revealed no codistribution of the presynaptic GABAergic marker GAD65/67 with gephyrin, a postsynaptic marker for GABAA Rs, corroborating the conclusion that GABA does not act synaptically in the mouse LSO. Imaging experiments revealed reduced Ca2+ influx into MNTB axon terminals following activation of presynaptic GABAB Rs. GABAB R activation reduced the synaptic strength at P4 and P11. GABA appears to act on extrasynaptic GABAA Rs as demonstrated by application of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, a δ-subunit-specific GABAA R agonist. RNA sequencing showed high mRNA levels for the δ-subunit in the LSO. Moreover, GABA transporters GAT-1 and GAT-3 appear to control extracellular GABA. Finally, we show an age-dependent effect of GABA on the excitability of LSO neurons. Whereas tonic GABA increased the excitability at P4, leading to spike facilitation, it decreased the excitability at P11 via shunting inhibition through extrasynaptic GABAA Rs. Taken together, we demonstrate a modulatory role of GABA in the murine LSO, rather than a function as a classical synaptic transmitter.