Matthew A. Xu-Friedman

Complexin-I Is Required for High-Fidelity Transmission at the Endbulb of Held Auditory Synapse

Complexins (CPXs I–IV) presumably act as regulators of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, but their function in the intact mammalian nervous system is not well established. Here, we explored the role of CPXs in the mouse auditory system. Hearing was impaired in CPX I knock-out mice but normal in knock-out mice for CPXs II, III, IV, and III/IV as measured by auditory brainstem responses. Complexins were not detectable in cochlear hair cells but CPX I was expressed in spiral ganglion neurons (SGNs) that give rise to the auditory nerve. Ca2+-dependent exocytosis of inner hair cells and sound encoding by SGNs were unaffected in CPX I knock-out mice. In the absence of CPX I, the resting release probability in the endbulb of Held synapses of the auditory nerve fibers with bushy cells in the cochlear nucleus was reduced. As predicted by computational modeling, bushy cells had decreased spike rates at sound onset as well as longer and more variable first spike latencies explaining the abnormal auditory brainstem responses. In addition, we found synaptic transmission to outlast the stimulus at many endbulb of Held synapses in vitro and in vivo, suggesting impaired synchronization of release to stimulus offset. Although sound encoding in the cochlea proceeds in the absence of complexins, CPX I is required for faithful processing of sound onset and offset in the cochlear nucleus.

A Low-Affinity Antagonist Reveals Saturation and Desensitization in Mature Synapses in the Auditory Brain Stem

Postsynaptic receptor desensitization has been observed to contribute to depression in immature synapses. However, it is not clear whether desensitization persists and causes depression in mature synapses. We investigate this issue at the endbulb of Held, the synapse made by auditory nerve (AN) fibers onto bushy cells (BCs) of the anteroventral cochlear nucleus, where depression could influence the processing of sound information. Experiments using cyclothiazide (CTZ) have implicated desensitization in endbulbs from postnatal day 16 (P16) to P21 mice, but application of γ-D-glutamylglycine (DGG) did not reveal desensitization in endbulbs >P22. To reconcile these findings, we have studied the effects of both CTZ and DGG on endbulbs from P5 to P40 CBA/CaJ mice. In paired-pulse protocols, both CTZ and DGG reduced depression in all ages at intervals <10 ms, consistent with their effects preventing desensitization. However, DGG increased depression at intervals >20 ms, consistent with DGGs use to prevent saturation. DGG application revealed receptor saturation even under conditions of very low release probability. Preventing desensitization by CTZ occluded the effects of DGG on desensitization and revealed the effects of saturation at short intervals. We developed an approach to separate DGGs effect on saturation from its effect on desensitization, which showed that desensitization has an impact during bursts of auditory nerve activity. Dynamic-clamp experiments indicated that desensitization can reduce BC spike probability and increase latency and jitter. Thus desensitization may affect sound processing in the mature auditory system.

Neuromodulation by GABA Converts a Relay Into a Coincidence Detector

Modulation of synaptic strength by γ-aminobutyric acid receptors (GABARs) is a common feature in sensory pathways that contain relay cell types. However, the functional impact of these receptors on information processing is not clear. We considered this issue at bushy cells (BCs) in the cochlear nucleus, which relay auditory nerve (AN) activity to higher centers. BCs express GABA(A)Rs, and synaptic inputs to BCs express GABA(B)Rs. We tested the effects of GABAR activation on the relaying of AN activity using patch-clamp recordings in mature mouse brain slices at 34°C. GABA affected BC firing in response to trains of AN activity at concentrations as low as 10 μM. GABA(A)Rs reduced firing primarily late in high-frequency trains, whereas GABA(B)Rs reduced firing early and in low-frequency trains. BC firing was significantly restored when two converging AN inputs were activated simultaneously, with maximal effect over a window of <0.5 ms. Thus GABA could adjust the function of BCs, to suppress the relaying of individual inputs and require coincident activity of multiple inputs.

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.

Context-Dependent Effects of NMDA Receptors on Precise Timing Information at the Endbulb of Held in the Cochlear Nucleus

Many synapses contain both AMPA receptors (AMPAR) and N-methyl-d-aspartate receptors (NMDAR), but their different roles in synaptic computation are not clear. We address this issue at the auditory nerve fiber synapse (called the endbulb of Held), which is formed on bushy cells of the cochlear nucleus. The endbulb refines and relays precise temporal information to nuclei responsible for sound localization. The endbulb has a number of specializations that aid precise timing, including AMPAR-mediated excitatory postsynaptic currents (EPSCs) with fast kinetics. Voltage-clamp experiments in mouse brain slices revealed that slow NMDAR EPSCs are maintained at mature endbulbs, contributing a peak conductance of around 10% of the AMPAR-mediated EPSC. During repetitive synaptic activity, AMPAR EPSCs depressed and NMDAR EPSCs summated, thereby increasing the relative importance of NMDARs. This could impact temporal precision of bushy cells because of the slow kinetics of NMDARs. We tested this by blocking NMDARs and quantifying bushy cell spike timing in current clamp when single endbulbs were activated. These experiments showed that NMDARs contribute to an increased probability of firing, shorter latency, and reduced jitter. Dynamic-clamp experiments confirmed this effect and showed it was dose-dependent. Bushy cells can receive inputs from multiple endbulbs. When we applied multiple synaptic inputs in dynamic clamp, NMDARs had less impact on spike timing. NMDAR conductances much higher than mature levels could disrupt spiking, which may explain its downregulation during development. Thus mature NMDAR expression can support the conveying of precise temporal information at the endbulb, depending on the stimulus conditions.

Effects of sodium salicylate on spontaneous and evoked spike rate in the dorsal cochlear nucleus

Spontaneous hyperactivity in the dorsal cochlear nucleus (DCN), particularly in fusiform cells, has been proposed as a neural generator of tinnitus. To determine if sodium salicylate, a reliable tinnitus inducer, could evoke hyperactivity in the DCN, we measured the spontaneous and depolarization-evoked spike rate in fusiform and cartwheel cells during salicylate superfusion. Five minute treatment with 1.4 mM salicylate suppressed spontaneous and evoked firing in fusiform cells; this decrease partially recovered after salicylate washout. Less suppression and greater recovery occurred with 3 min treatment using 1.4 mM salicylate. In contrast, salicylate had no effect on the spontaneous or evoked firing of cartwheel cells indicating that salicylates suppressive effects are specific to fusiform cells. To determine if salicylates suppressive effects were a consequence of increased synaptic inhibition, spontaneous inhibitory postsynaptic currents (IPSC) were measured during salicylate treatment. Salicylate unexpectedly reduced IPSC thereby ruling out increased inhibition as a mechanism to explain the depressed firing rates in fusiform cells. The salicylate-induced suppression of fusiform spike rate apparently arises from unidentified changes in the cells intrinsic excitability.

Developmental Mechanisms for Suppressing the Effects of Delayed Release at the Endbulb of Held

Delayed release of neurotransmitter, also called asynchronous release, is commonly observed at synapses, yet its influence on transmission of spike information is unknown. We examined this issue at endbulb of Held synapses, which are formed by auditory nerve fibers onto bushy cells in the cochlear nucleus. Endbulbs from CBA/CaJ mice aged P6–P49 showed prominent delayed release when driven at physiologically relevant rates. In bushy cells from mice before the onset of hearing (P6–P12), spikes were driven by delayed release up to 100 ms after presynaptic activity. However, no such spikes were observed in bushy cells from mice after the onset of hearing (>P14). Dynamic-clamp experiments indicated that delayed release can drive spikes in older bushy cells provided synchronous release is absent, suggesting that activity normally suppresses these spikes. Application of apamin or α-dendrotoxin revealed late spikes in older bushy cells, suggesting that postsynaptic activation of KV1.x and SK channels during spiking suppresses the subsequent effects of delayed release. The developmental upregulation of these potassium channels would be highly adaptive for temporally precise auditory processing. Furthermore, delayed release appeared to influence synchronous neurotransmitter release. Enhancement of delayed release using strontium was correlated with lower firing probability in current clamp and smaller synchronous EPSCs in voltage clamp. EGTA-AM had the opposite effects. These effects were consistent with delayed and synchronous release competing for a single vesicle pool. Thus delayed release apparently has negative presynaptic and postsynaptic consequences at the endbulb, which are partly mitigated by postsynaptic potassium channel expression.

Excitatory modulation in the cochlear nucleus through group I metabotropic glutamate receptor activation.

Activation of group I metabotropic glutamate receptors (mGluRs) has been suggested to modulate development of auditory neurons. However, the acute effects of mGluR activation on physiological response properties are unclear. To address this, we studied the effects of mGluRs in bushy cells (BCs) of the mammalian anteroventral cochlear nucleus (AVCN). Activation of mGluRs with dihydroxyphenylglycine (DHPG) caused depolarization of BCs in mice as old as P42, but did not affect neurotransmitter release by presynaptic auditory nerve (AN) fibers. Application of mGluR antagonists indicated that mGluRs are tonically active, and are highly sensitive to small elevations in ambient glutamate by the glutamate reuptake blocker threo-β-benzyloxyaspartic acid (TBOA). mGluR-mediated depolarization enhanced the firing probability in response to AN stimulation, and reduced the latency and jitter. Furthermore, excitation through postsynaptic mGluRs can significantly counterbalance the inhibitory effects of presynaptic GABAB receptors. Thus, interaction between these two modulatory pathways may provide additional flexibility for fine-tuning the BC relay.

Emergence of coordinated plasticity in the cochlear nucleus and cerebellum.

Synapses formed by one cell type onto another cell type tend to show characteristic short-term plasticity, which varies from facilitating to depressing depending on the particular system. Within a population of synapses, plasticity can also be variable, and it is unknown how this plasticity is determined on a cell-by-cell level. We have investigated this in the mouse cochlear nucleus, where auditory nerve (AN) fibers contact bushy cells (BCs) at synapses called “endbulbs of Held.” Synapses formed by different AN fibers onto one BC had plasticity that was more similar than would be expected at random. Experiments using MK-801 indicated that this resulted in part from similarity in the presynaptic probability of release. The similarity was not present in immature synapses but emerged after the onset of hearing. In addition, the phenomenon occurred at excitatory synapses in the cerebellum. This indicates that postsynaptic cells coordinate the plasticity of their inputs, which suggests that plasticity is of fundamental importance to synaptic function.

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

Significance Synapses with high probability of neurotransmitter release (Pr) depress during prolonged activity, which reduces the faithful transfer of information. Auditory nerve synapses onto bushy cells show particularly strong depression at physiologically relevant rates of activity, which raises the question of how bushy cells transmit information when sound levels are high for a prolonged period. After rearing mice in constant, nondamaging noise, auditory nerve synapses changed from high to low Pr, with a corresponding increase in the number of release sites, which increased spike fidelity during high activity. Neither quantal size nor average excitatory postsynaptic current changed. After returning to control conditions, Pr recovered to high. These changes seem to reflect a homeostatic response to enhance fidelity. 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.