Ambient sound stimulation tunes axonal conduction velocity by regulating radial growth of myelin on an individual, axon-by-axon basis.

Adaptive myelination is the emerging concept of tuning axonal conduction velocity to the activity within specific neural circuits over time. Sound processing circuits exhibit structural and functional specifications to process signals with microsecond precision: a time scale that is amenable to adjustment in length and thickness of myelin. Increasing activity of auditory axons by introducing sound-evoked responses during postnatal development enhances myelin thickness, while sensory deprivation prevents such radial growth during development. When deprivation occurs during adulthood, myelin thickness was reduced. However, it is unclear whether sensory stimulation adjusts myelination in a global fashion (whole fiber bundles) or whether such adaptation occurs at the level of individual fibers. Using temporary monaural deprivation in mice provided an internal control for a) differentially tracing structural changes in active and deprived fibers and b) for monitoring neural activity in response to acoustic stimulation of the control and the deprived ear within the same animal. The data show that sound-evoked activity increased the number of myelin layers around individual active axons, even when located in mixed bundles of active and deprived fibers. Thicker myelination correlated with faster axonal conduction velocity and caused shorter auditory brainstem response wave VI-I delays, providing a physiologically relevant readout. The lack of global compensation emphasizes the importance of balanced sensory experience in both ears throughout the lifespan of an individual.

Serine 937 phosphorylation enhances KCC2 activity and strengthens synaptic inhibition.

The potassium chloride cotransporter KCC2 is crucial for Cl- extrusion from mature neurons and thus key to hyperpolarizing inhibition. Auditory brainstem circuits contain well-understood inhibitory projections and provide a potent model to study the regulation of synaptic inhibition. Two peculiarities of the auditory brainstem are (i) posttranslational activation of KCC2 during development and (ii) extremely negative reversal potentials in specific circuits. To investigate the role of the potent phospho-site serine 937 therein, we generated a KCC2 Thr934Ala/Ser937Asp double mutation, in which Ser937 is replaced by aspartate mimicking the phosphorylated state, and the neighbouring Thr934 arrested in the dephosphorylated state. This double mutant showed a twofold increased transport activity in HEK293 cells, raising the hypothesis that auditory brainstem neurons show lower [Cl-]i. and increased glycinergic inhibition. This was tested in a mouse model carrying the same KCC2 Thr934Ala/Ser937Asp mutation by the use of the CRISPR/Cas9 technology. Homozygous KCC2 Thr934Ala/Ser937Asp mice showed an earlier developmental onset of hyperpolarisation in the auditory brainstem. Mature neurons displayed stronger glycinergic inhibition due to hyperpolarized ECl-. These data demonstrate that phospho-regulation of KCC2 Ser937 is a potent way to interfere with the excitation-inhibition balance in neural circuits.

Kv3.3 subunits control presynaptic action potential waveform and neurotransmitter release at a central excitatory synapse.

Kv3 potassium currents mediate rapid repolarisation of action potentials (APs), supporting fast spikes and high repetition rates. Of the four Kv3 gene family members, Kv3.1 and Kv3.3 are highly expressed in the auditory brainstem and we exploited this to test for subunit-specific roles at the calyx of Held presynaptic terminal in the mouse. Deletion of Kv3.3 (but not Kv3.1) reduced presynaptic Kv3 channel immunolabelling, increased presynaptic AP duration and facilitated excitatory transmitter release; which in turn enhanced short-term depression during high-frequency transmission. The response to sound was delayed in the Kv3.3KO, with higher spontaneous and lower evoked firing, thereby reducing signal-to-noise ratio. Computational modelling showed that the enhanced EPSC and short-term depression in the Kv3.3KO reflected increased vesicle release probability and accelerated activity-dependent vesicle replenishment. We conclude that Kv3.3 mediates fast repolarisation for short precise APs, conserving transmission during sustained high-frequency activity at this glutamatergic excitatory synapse.

Expression Patterns of the Neuropeptide Urocortin 3 and Its Receptor CRFR2 in the Mouse Central Auditory System.

Sensory systems have to be malleable to context-dependent modulations occurring over different time scales, in order to serve their evolutionary function of informing about the external world while also eliciting survival-promoting behaviors. Stress is a major context-dependent signal that can have fast and delayed effects on sensory systems, especially on the auditory system. Urocortin 3 (UCN3) is a member of the corticotropin-releasing factor family. As a neuropeptide, UCN3 regulates synaptic activity much faster than the classic steroid hormones of the hypothalamic-pituitary-adrenal axis. Moreover, due to the lack of synaptic re-uptake mechanisms, UCN3 can have more long-lasting and far-reaching effects. To date, a modest number of studies have reported the presence of UCN3 or its receptor CRFR2 in the auditory system, particularly in the cochlea and the superior olivary complex, and have highlighted the importance of this stress neuropeptide for protecting auditory function. However, a comprehensive map of all neurons synthesizing UCN3 or CRFR2 within the auditory pathway is lacking. Here, we utilize two reporter mouse lines to elucidate the expression patterns of UCN3 and CRFR2 in the auditory system. Additional immunolabelling enables further characterization of the neurons that synthesize UCN3 or CRFR2. Surprisingly, our results indicate that within the auditory system, UCN3 is expressed predominantly in principal cells, whereas CRFR2 expression is strongest in non-principal, presumably multisensory, cell types. Based on the presence or absence of overlap between UCN3 and CRFR2 labeling, our data suggest unusual modes of neuromodulation by UCN3, involving volume transmission and autocrine signaling.

Nitric Oxide Signaling in the Auditory Pathway.

Nitric oxide (NO) is of fundamental importance in regulating immune, cardiovascular, reproductive, neuromuscular, and nervous system function. It is rapidly synthesized and cannot be confined, it is highly reactive, so its lifetime is measured in seconds. These distinctive properties (contrasting with classical neurotransmitters and neuromodulators) give rise to the concept of NO as a "volume transmitter," where it is generated from an active source, diffuses to interact with proteins and receptors within a sphere of influence or volume, but limited in distance and time by its short half-life. In the auditory system, the neuronal NO-synthetizing enzyme, nNOS, is highly expressed and tightly coupled to postsynaptic calcium influx at excitatory synapses. This provides a powerful activity-dependent control of postsynaptic intrinsic excitability via cGMP generation, protein kinase G activation and modulation of voltage-gated conductances. NO may also regulate vesicle mobility via retrograde signaling. This Mini Review focuses on the auditory system, but highlights general mechanisms by which NO mediates neuronal intrinsic plasticity and synaptic transmission. The dependence of NO generation on synaptic and sound-evoked activity has important local modulatory actions and NO serves as a "volume transmitter" in the auditory brainstem. It also has potentially destructive consequences during intense activity or on spill-over from other NO sources during pathological conditions, when aberrant signaling may interfere with the precisely timed and tonotopically organized auditory system.

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.

Kv3.1 and Kv3.3 subunits differentially contribute to Kv3 channels and action potential repolarization in principal neurons of the auditory brainstem.

KEY POINTS: Kv3.1 and Kv3.3 subunits are highly expressed in the auditory brainstem, with little or no mRNA for Kv3.2 or Kv3.4. Changes in Kv3 currents and action potential (AP) firing were analysed from wild-type, Kv3.1 and Kv3.3 knockout (KO) mice. Both Kv3.1 and Kv3.3 immunostaining was present and western blots confirmed loss of subunit protein in the respective KO. Medial nucleus of the trapezoid body (MNTB) AP repolarization utilized Kv3.1 and/or Kv3.3; while in the lateral superior olive (LSO) Kv3.3 was essential. Voltage-gated calcium currents were unchanged between the genotypes. But APs evoked higher [Ca2+ ]i in LSO than MNTB neurons; and were highest in the Kv3.3KO, consistent with longer AP durations. High frequency stimulation increased AP failure rates and AP latency in LSO neurons from the Kv3.3KO, underlining the physiological consequences for binaural integration. LSO neurons require Kv3.3 for functional Kv3 channels, while MNTB neurons can utilize either Kv3.1 or Kv3.3 subunits. ABSTRACT: Kv3 voltage-gated potassium channels mediate action potential (AP) repolarization. The relative importance of Kv3.1 and Kv3.3 subunits for assembly of functional channels in neurons of the auditory brainstem was examined from the physiological perspective that speed and precision of AP firing are crucial for sound source localization. High levels of Kv3.1 and Kv3.3 mRNA and protein were measured, with no evidence of compensation by Kv3.2 or Kv3.4 in the respective knockout (KO) mouse. Using the KOs, composition of Kv3 channels was constrained to either Kv3.1 or Kv3.3 subunits in principal neurons of the medial nucleus of the trapezoid body (MNTB) and lateral superior olive (LSO); while TEA (1 mm) was employed to block Kv3-mediated outward potassium currents in voltage- and current clamp experiments. MNTB neuron APs (half-width 0.31 ± 0.08 ms, n = 25) were fast, reliable, and showed no distinction between channels assembled from Kv3.1 or Kv3.3 subunits (in the respective KO). LSO AP half-widths were also fast, but absolutely required Kv3.3 subunits for fast repolarization (half-widths: 0.25 ± 0.08 ms, n = 19 wild-type, 0.60 ± 0.17 ms, n = 21 Kv3.3KO, p = 0.0001). The longer AP duration increased LSO calcium influx and AP failure rates, and increased AP latency and jitter during high frequency repetitive firing. Both Kv3.1 and Kv3.3 subunits contribute to Kv3 channels in the MNTB (and compensate for each other in each KO); in contrast, LSO neurons require Kv3.3 subunits for fast repolarization and to sustain AP firing during high frequency stimulation. In conclusion, Kv3 channels exhibit both redundancy and Kv3.3 dominance between the brainstem nuclei involved in sound localization.

Urocortin 3 signalling in the auditory brainstem aids recovery of hearing after reversible noise-induced threshold shift.

KEY POINTS: Ongoing, moderate noise exposure does not instantly damage the auditory system but may cause lasting deficits, such as elevated thresholds and accelerated ageing of the auditory system. The neuromodulatory peptide urocortin-3 (UCN3) is involved in the body's recovery from a stress response, and is also expressed in the cochlea and the auditory brainstem. Lack of UCN3 facilitates age-induced hearing loss and causes permanently elevated auditory thresholds following a single 2 h noise exposure at moderate intensities. Outer hair cell function in mice lacking UCN3 is unaffected, so that the observed auditory deficits are most likely due to inner hair cell function or central mechanisms. Highly specific, rather than ubiquitous, expression of UCN3 in the brain renders it a promising candidate for designing drugs to ameliorate stress-related auditory deficits, including recovery from acoustic trauma. ABSTRACT: Environmental acoustic noise is omnipresent in our modern society, with sound levels that are considered non-damaging still causing long-lasting or permanent changes in the auditory system. The small neuromodulatory peptide urocortin-3 (UCN3) is the endogenous ligand for corticotropin-releasing factor receptor type 2 and together they are known to play an important role in stress recovery. UCN3 expression has been observed in the auditory brainstem, but its role remains unclear. Here we describe the detailed distribution of UCN3 expression in the murine auditory brainstem and provide evidence that UCN3 is expressed in the synaptic region of inner hair cells in the cochlea. We also show that mice with deficient UCN3 signalling experience premature ageing of the auditory system starting at an age of 4.7 months with significantly elevated thresholds of auditory brainstem responses (ABRs) compared to age-matched wild-type mice. Following a single, 2 h exposure to moderate (84 or 94 dB SPL) noise, UCN3-deficient mice exhibited significantly larger shifts in ABR thresholds combined with maladaptive recovery. In wild-type mice, the same noise exposure did not cause lasting changes to auditory thresholds. The presence of UCN3-expressing neurons throughout the auditory brainstem and the predisposition to hearing loss caused by preventing its normal expression suggests UCN3 as an important neuromodulatory peptide in the auditory system's response to loud sounds.

Slow NMDA-Mediated Excitation Accelerates Offset-Response Latencies Generated via a Post-Inhibitory Rebound Mechanism.

In neural circuits, action potentials (spikes) are conventionally caused by excitatory inputs whereas inhibitory inputs reduce or modulate neuronal excitability. We previously showed that neurons in the superior paraolivary nucleus (SPN) require solely synaptic inhibition to generate their hallmark offset response, a burst of spikes at the end of a sound stimulus, via a post-inhibitory rebound mechanism. In addition SPN neurons receive excitatory inputs, but their functional significance is not yet known. Here we used mice of both sexes to demonstrate that in SPN neurons, the classical roles for excitation and inhibition are switched, with inhibitory inputs driving spike firing and excitatory inputs modulating this response. Hodgkin-Huxley modeling suggests that a slow, NMDA receptor (NMDAR)-mediated excitation would accelerate the offset response. We find corroborating evidence from in vitro and in vivo recordings that lack of excitation prolonged offset-response latencies and rendered them more variable to changing sound intensity levels. Our results reveal an unsuspected function for slow excitation in improving the timing of post-inhibitory rebound firing even when the firing itself does not depend on excitation. This shows the auditory system employs highly specialized mechanisms to encode timing-sensitive features of sound offsets which are crucial for sound-duration encoding and have profound biological importance for encoding the temporal structure of speech.

When Sound Stops: Offset Responses in the Auditory System.

The auditory modality is fundamentally a temporal sense that requires analysis of changes in sound signals on timescales ranging from microseconds to minutes. To generate a faithful representation of changes in sound intensity and frequency over time, sound offsets (disappearances) as well as sound onsets (appearances) must be encoded by the auditory system. We review here the computational significance, perceptual roles, anatomical locations, and cellular and network origins of sound-offset responses in the mammalian auditory brain. We show that sound-offset responses arise from mechanisms and pathways distinct from those producing sound-onset responses, and are likely to be essential for auditory processing of temporally discontinuous sounds such as speech.

Strain-specific differences in the development of neuronal excitability in the mouse ventral nucleus of the trapezoid body.

This investigation compared the development of neuronal excitability in the ventral nucleus of the trapezoid body (VNTB) between two strains of mice with differing progression rates for age-related hearing loss. In contrast to CBA/Ca (CBA) mice, the C57BL/6J (C57) strain are subject to hearing loss from a younger age and are more prone to damage from sound over-exposure. Higher firing rates in the medial olivocochlear system (MOC) are associated with protection from loud sounds and these cells are located in the VNTB. We postulated that reduced neuronal firing of the MOC in C57 mice could contribute to hearing loss in this strain by reducing efferent protection. Whole cell patch clamp was used to compare the electrical properties of VNTB neurons from the two strains initially in two age groups: before and after hearing onset at âˆ¼ P9 and ∼P16, respectively. Prior to hearing onset VNTB neurons electrophysiological properties were identical in both strains, but started to diverge after hearing onset. One week after hearing onset VNTB neurons of C57 mice had larger amplitude action potentials but in contrast to CBA mice, their waveform failed to accelerate with increasing age, consistent with the faster inactivation of voltage-gated potassium currents in C57 VNTB neurons. The lower frequency action potential firing of C57 VNTB neurons at P16 was maintained to P28, indicating that this change was not a developmental delay. We conclude that C57 VNTB neurons fire at lower frequencies than in the CBA strain, supporting the hypothesis that reduced MOC firing could contribute to the greater hearing loss of the C57 strain.

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.

Input timing for spatial processing is precisely tuned via constant synaptic delays and myelination patterns in the auditory brainstem.

Precise timing of synaptic inputs is a fundamental principle of neural circuit processing. The temporal precision of postsynaptic input integration is known to vary with the computational requirements of a circuit, yet how the timing of action potentials is tuned presynaptically to match these processing demands is not well understood. In particular, action potential timing is shaped by the axonal conduction velocity and the duration of synaptic transmission delays within a pathway. However, it is not known to what extent these factors are adapted to the functional constraints of the respective circuit. Here, we report the finding of activity-invariant synaptic transmission delays as a functional adaptation for input timing adjustment in a brainstem sound localization circuit. We compared axonal and synaptic properties of the same pathway between two species with dissimilar timing requirements (gerbil and mouse): In gerbils (like humans), neuronal processing of sound source location requires exceptionally high input precision in the range of microseconds, but not in mice. Activity-invariant synaptic transmission and conduction delays were present exclusively in fast conducting axons of gerbils that also exhibited unusual structural adaptations in axon myelination for increased conduction velocity. In contrast, synaptic transmission delays in mice varied depending on activity levels, and axonal myelination and conduction velocity exhibited no adaptations. Thus, the specializations in gerbils and their absence in mice suggest an optimization of axonal and synaptic properties to the specific demands of sound localization. These findings significantly advance our understanding of structural and functional adaptations for circuit processing.

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

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

Physiology and anatomy of neurons in the medial superior olive of the mouse.

In mammals with good low-frequency hearing, the medial superior olive (MSO) computes sound location by comparing differences in the arrival time of a sound at each ear, called interaural time disparities (ITDs). Low-frequency sounds are not reflected by the head, and therefore level differences and spectral cues are minimal or absent, leaving ITDs as the only cue for sound localization. Although mammals with high-frequency hearing and small heads (e.g., bats, mice) barely experience ITDs, the MSO is still present in these animals. Yet, aside from studies in specialized bats, in which the MSO appears to serve functions other than ITD processing, it has not been studied in small mammals that do not hear low frequencies. Here we describe neurons in the mouse brain stem that share prominent anatomical, morphological, and physiological properties with the MSO in species known to use ITDs for sound localization. However, these neurons also deviate in some important aspects from the typical MSO, including a less refined arrangement of cell bodies, dendrites, and synaptic inputs. In vitro, the vast majority of neurons exhibited a single, onset action potential in response to suprathreshold depolarization. This spiking pattern is typical of MSO neurons in other species and is generated from a complement of Kv1, Kv3, and IH currents. In vivo, mouse MSO neurons show bilateral excitatory and inhibitory tuning as well as an improvement in temporal acuity of spiking during bilateral acoustic stimulation. The combination of classical MSO features like those observed in gerbils with more unique features similar to those observed in bats and opossums make the mouse MSO an interesting model for exploiting genetic tools to test hypotheses about the molecular mechanisms and evolution of ITD processing.

Tuning of Ranvier node and internode properties in myelinated axons to adjust action potential timing.

Action potential timing is fundamental to information processing; however, its determinants are not fully understood. Here we report unexpected structural specializations in the Ranvier nodes and internodes of auditory brainstem axons involved in sound localization. Myelination properties deviated significantly from the traditionally assumed structure. Axons responding best to low-frequency sounds had a larger diameter than high-frequency axons but, surprisingly, shorter internodes. Simulations predicted that this geometry helps to adjust the conduction velocity and timing of action potentials within the circuit. Electrophysiological recordings in vitro and in vivo confirmed higher conduction velocities in low-frequency axons. Moreover, internode length decreased and Ranvier node diameter increased progressively along the distal axon segments, which simulations show was essential to ensure precisely timed depolarization of the giant calyx of Held presynaptic terminal. Thus, individual anatomical parameters of myelinated axons can be tuned to optimize pathways involved in temporal processing.

Decreased temporal precision of neuronal signaling as a candidate mechanism of auditory processing disorder.

The sense of hearing is the fastest of our senses and provides the first all-or-none action potential in the auditory nerve in less than four milliseconds. Short stimulus evoked latencies and their minimal variability are hallmarks of auditory processing from spiral ganglia to cortex. Here, we review how even small changes in first spike latencies (FSL) and their variability (jitter) impact auditory temporal processing. We discuss a number of mouse models with degraded FSL/jitter whose mutations occur exclusively in the central auditory system and therefore might serve as candidates to investigate the cellular mechanisms underlying auditory processing disorders (APD).