Effects of Age on Responses of Principal Cells of the Mouse Anteroventral Cochlear Nucleus in Quiet and Noise.

Older listeners often report difficulties understanding speech in noisy environments. It is important to identify where in the auditory pathway hearing-in-noise deficits arise to develop appropriate therapies. We tested how encoding of sounds is affected by masking noise at early stages of the auditory pathway by recording responses of principal cells in the anteroventral cochlear nucleus (AVCN) of aging CBA/CaJ and C57BL/6J mice in vivo. Previous work indicated that masking noise shifts the dynamic range of single auditory nerve fibers (ANFs), leading to elevated tone thresholds. We hypothesized that such threshold shifts could contribute to increased hearing-in-noise deficits with age if susceptibility to masking increased in AVCN units. We tested this by recording the responses of AVCN principal neurons to tones in the presence and absence of masking noise. Surprisingly, we found that masker-induced threshold shifts decreased with age in primary-like units and did not change in choppers. In addition, spontaneous activity decreased in primary-like and chopper units of old mice, with no change in dynamic range or tuning precision. In C57 mice, which undergo early-onset hearing loss, units showed similar changes in threshold and spontaneous rate at younger ages, suggesting they were related to hearing loss and not simply aging. These findings suggest that sound information carried by AVCN principal cells remains largely unchanged with age. Therefore, hearing-in-noise deficits may result from other changes during aging, such as distorted across-channel input from the cochlea and changes in sound coding at later stages of the auditory pathway.

Low Intensity Noise Exposure Enhanced Auditory Loudness and Temporal Processing by Increasing Excitability of DCN.

Sound stimulation is generally used for tinnitus and hyperacusis treatment. Recent studies found that long-term noise exposure can change synaptic and firing properties in the central auditory system, which will be detected by the acoustic startle reflex. However, the perceptual consequences of long-term low-intensity sound exposure are indistinct. This study will detect the effects of moderate-level noise exposure (83 dB SPL) on auditory loudness, and temporal processing was evaluated using CBA/CaJ mice. C-Fos staining was used to detect neural activity changes in the central auditory pathway. With two weeks of 83 dB SPL noise exposure (8 hours per day), no persistent threshold shift of the auditory brainstem response (ABR) was identified. On the other hand, noise exposure enhanced the acoustic startle response (ASR) and gap-induced prepulse inhibition significantly (gap-PPI). Low-level noise exposure, according to the findings, can alter temporal acuity. Noise exposure increased the number of c-Fos labeled neurons in the dorsal cochlear nucleus (DCN) and caudal pontine reticular nucleus (PnC) but not at a higher level in the central auditory nuclei. Our results suggested that noise stimulation can change acoustical temporal processing presumably by increasing the excitability of auditory brainstem neurons.

Induction of Activity-Dependent Plasticity at Auditory Nerve Synapses.

Exposure to nontraumatic noise in vivo drives long-lasting changes in auditory nerve synapses, which may influence hearing, but the induction mechanisms are not known. We mimicked activity in acute slices of the cochlear nucleus from mice of both sexes by treating them with high potassium, after which voltage-clamp recordings from bushy cells indicated that auditory nerve synapses had reduced EPSC amplitude, quantal size, and vesicle release probability (P r). The effects of high potassium were prevented by blockers of nitric oxide (NO) synthase and protein kinase A. Treatment with the NO donor, PAPA-NONOate, also decreased P r, suggesting NO plays a central role in inducing synaptic changes. To identify the source of NO, we activated auditory nerve fibers specifically using optogenetics. Strobing for 2 h led to decreased EPSC amplitude and P r, which was prevented by antagonists against ionotropic glutamate receptors and NO synthase. This suggests that the activation of AMPA and NMDA receptors in postsynaptic targets of auditory nerve fibers drives release of NO, which acts retrogradely to cause long-term changes in synaptic function in auditory nerve synapses. This may provide insight into preventing or treating disorders caused by noise exposure.SIGNIFICANCE STATEMENT Auditory nerve fibers undergo long-lasting changes in synaptic properties in response to noise exposure in vivo, which may contribute to changes in hearing. Here, we investigated the cellular mechanisms underlying induction of synaptic changes using high potassium and optogenetic stimulation in vitro and identified important signaling pathways using pharmacology. Our results suggest that auditory nerve activity drives postsynaptic depolarization through AMPA and NMDA receptors, leading to the release of nitric oxide, which acts retrogradely to regulate presynaptic neurotransmitter release. These experiments revealed that auditory nerve synapses are unexpectedly sensitive to activity and can show dramatic, long-lasting changes in a few hours that could affect hearing.

Induction of synapse formation by de novo neurotransmitter synthesis.

A vital question in neuroscience is how neurons align their postsynaptic structures with presynaptic release sites. Although synaptic adhesion proteins are known to contribute in this process, the role of neurotransmitters remains unclear. Here we inquire whether de novo biosynthesis and vesicular release of a noncanonical transmitter can facilitate the assembly of its corresponding postsynapses. We demonstrate that, in both stem cell-derived human neurons as well as in vivo mouse neurons of purely glutamatergic identity, ectopic expression of GABA-synthesis enzymes and vesicular transporters is sufficient to both produce GABA from ambient glutamate and transmit it from presynaptic terminals. This enables efficient accumulation and consistent activation of postsynaptic GABAA receptors, and generates fully functional GABAergic synapses that operate in parallel but independently of their glutamatergic counterparts. These findings suggest that presynaptic release of a neurotransmitter itself can signal the organization of relevant postsynaptic apparatus, which could be directly modified to reprogram the synapse identity of neurons.

Time Course of Activity-Dependent Changes in Auditory Nerve Synapses Reveals Multiple Underlying Cellular Mechanisms.

Abnormal levels of acoustic activity can result in hearing problems such as tinnitus and language processing disorders, but the underlying cellular and synaptic changes triggered by abnormal activity are not well understood. To address this issue, we studied the time course of activity-dependent changes that occur at auditory nerve synapses in mice of both sexes after noise exposure and conductive hearing loss. We found that EPSC amplitude and synaptic depression decreased within 2 d of noise exposure through a decrease in the probability of vesicle release (Pr). This was followed by a gradual increase in EPSC amplitude through a larger pool of releasable vesicles (N). Occlusion of the ear canal led to a rapid decrease in EPSC amplitude through a decrease in N, which was followed by an increase in EPSC amplitude and synaptic depression through an increase in Pr After returning to normal sound levels, synaptic depression recovered to control levels within 1-2 d. However, repeated exposure to noise for as little as 8 h/d caused synaptic changes after 7 d, suggesting recovery did not fully offset changes. Thus, there appear to be three activity-dependent mechanisms in auditory nerve synapses-bidirectional changes in Pr in 1-2 d, slower bidirectional changes in N through synaptic growth or retraction, and rapid downregulation of N with low activity. The dynamic changes indicate that multiple mechanisms are present to fine-tune synaptic fidelity across different acoustic conditions in a simple relay.SIGNIFICANCE STATEMENT Hearing impairments can arise from exposure to noise or conductive hearing loss. This appears to result from changes in the brain, but the mechanisms are not well understood. We study this issue by studying the synapses made by auditory nerve fibers called endbulbs of Held. These synapses undergo bidirectional changes in size and release probability of neurotransmitter in response to increased or decreased activity. Here, we made a close examination of how quickly these synaptic characteristics change, which indicates there are at least three cellular mechanisms underlying changes. Furthermore, repeated exposure to brief periods of noise can produce cumulative effects. These changes could significantly affect hearing, especially because they occur at the start of the central auditory pathway.

Mechanisms and Functional Consequences of Presynaptic Homeostatic Plasticity at Auditory Nerve Synapses.

Multiple forms of homeostasis influence synaptic function under diverse activity conditions. Both presynaptic and postsynaptic forms of homeostasis are important, but their relative impact on fidelity is unknown. To address this issue, we studied auditory nerve synapses onto bushy cells in the cochlear nucleus of mice of both sexes. These synapses undergo bidirectional presynaptic and postsynaptic homeostatic changes with increased and decreased acoustic stimulation. We found that both young and mature synapses exhibit similar activity-dependent changes in short-term depression. Experiments using chelators and imaging both indicated that presynaptic Ca2+ influx decreased after noise exposure, and increased after ligating the ear canal. By contrast, Ca2+ cooperativity was unaffected. Experiments using specific antagonists suggest that occlusion leads to changes in the Ca2+ channel subtypes driving neurotransmitter release. Furthermore, dynamic-clamp experiments revealed that spike fidelity primarily depended on changes in presynaptic depression, with some contribution from changes in postsynaptic intrinsic properties. These experiments indicate that presynaptic Ca2+ influx is homeostatically regulated in vivo to enhance synaptic fidelity.SIGNIFICANCE STATEMENT Homeostatic mechanisms in synapses maintain stable function in the face of different levels of activity. Both juvenile and mature auditory nerve synapses onto bushy cells modify short-term depression in different acoustic environments, which raises the question of what the underlying presynaptic mechanisms are and the relative importance of presynaptic and postsynaptic contributions to the faithful transfer of information. Changes in short-term depression under different acoustic conditions were a result of changes in presynaptic Ca2+ influx. Spike fidelity was affected by both presynaptic and postsynaptic changes after ear occlusion and was only affected by presynaptic changes after noise-rearing. These findings are important for understanding regulation of auditory synapses under normal conditions and also in disorders following noise exposure or conductive hearing loss.

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.

Effects of Non-traumatic Noise and Conductive Hearing Loss on Auditory System Function.

The effects of traumatic noise-exposure and deafening on auditory system function have received a great deal of attention. However, lower levels of noise as well as temporary conductive hearing loss also have consequences on auditory physiology and hearing. Here we review how abnormal acoustic experience at early ages affects the ascending and descending auditory pathways, as well as hearing behavior.

Changes in Properties of Auditory Nerve Synapses following Conductive Hearing Loss.

Auditory activity plays an important role in the development of the auditory system. Decreased activity can result from conductive hearing loss (CHL) associated with otitis media, which may lead to long-term perceptual deficits. The effects of CHL have been mainly studied at later stages of the auditory pathway, but early stages remain less examined. However, changes in early stages could be important because they would affect how information about sounds is conveyed to higher-order areas for further processing and localization. We examined the effects of CHL at auditory nerve synapses onto bushy cells in the mouse anteroventral cochlear nucleus following occlusion of the ear canal. These synapses, called endbulbs of Held, normally show strong depression in voltage-clamp recordings in brain slices. After 1 week of CHL, endbulbs showed even greater depression, reflecting higher release probability. We observed no differences in quantal size between control and occluded mice. We confirmed these observations using mean-variance analysis and the integration method, which also revealed that the number of release sites decreased after occlusion. Consistent with this, synaptic puncta immunopositive for VGLUT1 decreased in area after occlusion. The level of depression and number of release sites both showed recovery after returning to normal conditions. Finally, bushy cells fired fewer action potentials in response to evoked synaptic activity after occlusion, likely because of increased depression and decreased input resistance. These effects appear to reflect a homeostatic, adaptive response of auditory nerve synapses to reduced activity. These effects may have important implications for perceptual changes following CHL. SIGNIFICANCE STATEMENT: Normal hearing is important to everyday life, but abnormal auditory experience during development can lead to processing disorders. For example, otitis media reduces sound to the ear, which can cause long-lasting deficits in language skills and verbal production, but the location of the problem is unknown. Here, we show that occluding the ear causes synapses at the very first stage of the auditory pathway to modify their properties, by decreasing in size and increasing the likelihood of releasing neurotransmitter. This causes synapses to deplete faster, which reduces fidelity at central targets of the auditory nerve, which could affect perception. Temporary hearing loss could cause similar changes at later stages of the auditory pathway, which could contribute to disorders in behavior.

Preparing Brain Slices to Study Basic Synaptic Properties.

This protocol describes how to prepare brain slices for electrophysiology, with an emphasis on synaptic physiology in voltage clamp. This approach remains the gold standard for understanding the properties of individual neurons, as well as the connections between neurons.

Measuring the Basic Physiological Properties of Synapses.

Studying synaptic physiology remains an important endeavor for understanding the mechanisms that underlie neurotransmitter release as well as synaptic development and refinement. It is also critical to understanding brain function. The basic conceptual framework for synaptic physiology was worked out by Katz and colleagues and has coalesced around three factors: the number of releasable vesicles (N), the probability of release (P), and quantal size (Q). Here, I discuss a few key experiments related to these quantities and how they are assessed.

Low Somatic Sodium Conductance Enhances Action Potential Precision in Time-Coding Auditory Neurons.

Auditory nerve fibers encode sounds in the precise timing of action potentials (APs), which is used for such computations as sound localization. Timing information is relayed through several cell types in the auditory brainstem that share an unusual property: their APs are not overshooting, suggesting that the cells have very low somatic sodium conductance (gNa). However, it is not clear how gNa influences temporal precision. We addressed this by comparing bushy cells (BCs) in the mouse cochlear nucleus with T-stellate cells (SCs), which do have normal overshooting APs. BCs play a central role in both relaying and refining precise timing information from the auditory nerve, whereas SCs discard precise timing information and encode the envelope of sound amplitude. Nucleated-patch recording at near-physiological temperature indicated that the Na current density was 62% lower in BCs, and the voltage dependence of gNa inactivation was 13 mV hyperpolarized compared with SCs. We endowed BCs with SC-like gNa using two-electrode dynamic clamp and found that synaptic activity at physiologically relevant rates elicited APs with significantly lower probability, through increased activation of delayed rectifier channels. In addition, for two near-simultaneous synaptic inputs, the window of coincidence detection widened significantly with increasing gNa, indicating that refinement of temporal information by BCs is degraded by gNa Thus, reduced somatic gNa appears to be an adaption for enhancing fidelity and precision in time-coding neurons. SIGNIFICANCE STATEMENT: Proper hearing depends on analyzing temporal aspects of sounds with high precision. Auditory neurons that specialize in precise temporal information have a suite of unusual intrinsic properties, including nonovershooting action potentials and few sodium channels in the soma. However, it was not clear how low sodium channel availability in the soma influenced the temporal precision of action potentials initiated in the axon initial segment. We studied this using dynamic clamp to mimic sodium channels in the soma, which yielded normal, overshooting action potentials. Increasing somatic sodium conductance had major negative consequences: synaptic activity evoked action potentials with lower fidelity, and the precision of coincidence detection was degraded. Thus, low somatic sodium channel availability appears to enhance fidelity and temporal precision.

Audiograms, gap detection thresholds, and frequency difference limens in cannabinoid receptor 1 knockout mice.

The cannabinoid receptor 1 (CB1R) is found at several stages in the auditory pathway, but its role in hearing is unknown. Hearing abilities were measured in CB1R knockout mice and compared to those of wild-type mice. Operant conditioning and the psychophysical Method of Constant Stimuli were used to measure audiograms, gap detection thresholds, and frequency difference limens in trained mice using the same methods and stimuli as in previous experiments. CB1R knockout mice showed deficits at frequencies above 8 kHz in their audiograms relative to wild-type mice. CB1R knockouts showed enhancements for detecting gaps in low-pass noisebursts relative to wild-type mice, but were similar for other noise conditions. Finally, the two groups of mice did not differ in their frequency discrimination abilities as measured by the frequency difference limens task. These experiments suggest that the CB1R is involved in auditory processing and lay the groundwork for future physiological experiments.

Skipped-stimulus approach reveals that short-term plasticity dominates synaptic strength during ongoing activity.

All synapses show activity-dependent changes in strength, which affect the fidelity of postsynaptic spiking. This is particularly important at auditory nerve synapses, where the presence and timing of spikes carry information about a sound's structure, which must be passed along for proper processing. However, it is not clear how synaptic plasticity influences spiking during ongoing activity. Under these conditions, conventional analyses erroneously suggest that synaptic plasticity has no influence on EPSC amplitude or spiking. Therefore, we developed new approaches to study how ongoing activity influences synaptic strength, using voltage- and current-clamp recordings from bushy cells in brain slices from mouse anteroventral cochlear nucleus. We applied identical trains of stimuli, except for one skipped stimulus, and found that EPSC amplitude was affected for 60 ms following a skipped stimulus. We further showed that the initial probability of release, calcium-dependent mechanisms of recovery, and desensitization all play a role even during ongoing activity. Current-clamp experiments indicated that these processes had a significant effect on postsynaptic spiking, as did the refractory period to a smaller extent. Thus short-term plasticity has real, important functional consequences.

Activity-dependent, homeostatic regulation of neurotransmitter release from auditory nerve fibers.

Information processing in the brain requires reliable synaptic transmission. High reliability at specialized auditory nerve synapses in the cochlear nucleus results from many release sites (N), high probability of neurotransmitter release (Pr), and large quantal size (Q). However, high Pr also causes auditory nerve synapses to depress strongly when activated at normal rates for a prolonged period, which reduces fidelity. We studied how synapses are influenced by prolonged activity by exposing mice to constant, nondamaging noise and found that auditory nerve synapses changed to facilitating, reflecting low Pr. For mice returned to quiet, synapses recovered to normal depression, suggesting that these changes are a homeostatic response to activity. Two additional properties, Q and average excitatory postsynaptic current (EPSC) amplitude, were unaffected by noise rearing, suggesting that the number of release sites (N) must increase to compensate for decreased Pr. These changes in N and Pr were confirmed physiologically using the integration method. Furthermore, consistent with increased N, endbulbs in noise-reared animals had larger VGlut1-positive puncta, larger profiles in electron micrographs, and more release sites per profile. In current-clamp recordings, noise-reared BCs had greater spike fidelity even during high rates of synaptic activity. Thus, auditory nerve synapses regulate excitability through an activity-dependent, homeostatic mechanism, which could have major effects on all downstream processing. Our results also suggest that noise-exposed bushy cells would remain hyperexcitable for a period after returning to normal quiet conditions, which could have perceptual consequences.

Different pools of glutamate receptors mediate sensitivity to ambient glutamate in the cochlear nucleus.

Ambient glutamate plays an important role in pathological conditions, such as stroke, but its role during normal activity is not clear. In addition, it is not clear how ambient glutamate acts on glutamate receptors with varying affinities or subcellular localizations. To address this, we studied "endbulb of Held" synapses, which are formed by auditory nerve fibers onto bushy cells (BCs) in the anteroventral cochlear nucleus. When ambient glutamate was increased by applying the glutamate reuptake inhibitor TFB-TBOA, BCs depolarized as a result of activation of N-methyl-D-aspartate receptors (NMDARs) and group I metabotropic glutamate receptors (mGluRs). Application of antagonists against NMDARs (in 0 Mg(2+)) or mGluRs caused hyperpolarization, indicating that these receptors were bound by a tonic source of glutamate. AMPA receptors did not show these effects, consistent with their lower glutamate affinity. We also evaluated the subcellular localization of the receptors activated by ambient glutamate. The mGluRs were not activated by synaptic stimulation and thus appear to be exclusively extrasynaptic. By contrast, NMDARs in both synaptic and extrasynaptic compartments were activated by ambient glutamate, as shown using the use-dependent antagonist MK-801. Levels of ambient glutamate appeared to be regulated in a spike-independent manner, and glia likely play a major role. These low levels of ambient glutamate likely have functional consequences, as even low concentrations of TBOA caused significant increases in BC spiking following synaptic stimulation. These results indicate that normal resting potential appears to be poised in the region of maximal sensitivity to small changes in ambient glutamate.

High-speed dynamic-clamp interface.

The dynamic-clamp technique is highly useful for mimicking synaptic or voltage-gated conductances. However, its use remains rare in part because there are few systems, and they can be expensive and difficult for less-experienced programmers to implement. Furthermore, some conductances (such as sodium channels) can be quite rapid or may have complex voltage sensitivity, so high speeds are necessary. To address these issues, we have developed a new interface that uses a common personal computer platform with National Instruments data acquisition and WaveMetrics IGOR to provide a simple user interface. This dynamic clamp implements leak and linear synaptic conductances as well as a voltage-dependent synaptic conductance and kinetic channel conductances based on Hodgkin-Huxley or Markov models. The speed of the system can be assayed using a testing mode, and currently speeds of >100 kHz (10 µs per cycle) are achievable with short latency and little jitter.

Stochastic properties of neurotransmitter release expand the dynamic range of synapses.

Release of neurotransmitter is an inherently random process, which could degrade the reliability of postsynaptic spiking, even at relatively large synapses. This is particularly important at auditory synapses, where the rate and precise timing of spikes carry information about sounds. However, the functional consequences of the stochastic properties of release are unknown. We addressed this issue at the mouse endbulb of Held synapse, which is formed by auditory nerve fibers onto bushy cells (BCs) in the anteroventral cochlear nucleus. We used voltage clamp to characterize synaptic variability. Dynamic clamp was used to compare BC spiking with stochastic or deterministic synaptic input. The stochastic component increased the responsiveness of the BC to conductances that were on average subthreshold, thereby increasing the dynamic range of the synapse. This had the benefit that BCs relayed auditory nerve activity even when synapses showed significant depression during rapid activity. However, the precision of spike timing decreased with stochastic conductances, suggesting a trade-off between encoding information in spike timing versus probability. These effects were confirmed in fiber stimulation experiments, indicating that they are physiologically relevant, and that synaptic randomness, dynamic range, and jitter are causally related.

How do short-term changes at synapses fine-tune information processing?

Synaptic transmission is highly dependent on recent activity and can lead to depression or facilitation of synaptic strength. This phenomenon is called "short-term synaptic plasticity" and is shown at all synapses. While much work has been done to understand the mechanisms of short-term changes in the state of synapses, short-term plasticity is often thought of as a mechanistic consequence of the design of a synapse. This review will attempt to go beyond this view and discuss how, on one hand, complex neuronal activity affects the short-term state of synapses, but also how these dynamic changes in synaptic strength affect information processing in return.