Inhibition shapes acoustic responsiveness in spherical bushy cells.

<div>Please find the peer-reviewed publisher version linked below.</div><div><br></div>Signal processing in the auditory brainstem is based on an interaction of neuronal excitation and inhibition. To date, we have incomplete knowledge of how the dynamic interplay of both contributes to the processing power and temporal characteristics of signal coding. The spherical bushy cells (SBCs) of the anteroventral cochlear nucleus (AVCN) receive their primary excitatory input through auditory nerve fibers via large, axosomatic synaptic terminals called the endbulbs of Held and by additional, acoustically driven inhibitory inputs. SBCs provide the input to downstream nuclei of the brainstem sound source localization circuitry, such as the medial and lateral superior olive, which rely on temporal precise inputs. In this study, we used juxtacellular recordings in anesthetized Mongolian gerbils to assess the effect of acoustically evoked inhibition on the SBCs input-output function and on temporal precision of SBC spiking. Acoustically evoked inhibition proved to be strong enough to suppress action potentials (APs) of SBCs in a stimulus-dependent manner. Inhibition shows slow onset and offset dynamics and increasing strength at higher sound intensities. In addition, inhibition decreases the rising slope of the EPSP and prolongs the EPSP-to-AP transition time. Both effects can be mimicked by iontophoretic application of glycine. Inhibition also improves phase locking of SBC APs to low-frequency tones by acting as a gain control to suppress poorly timed EPSPs from generating postsynaptic APs to maintain precise SBC spiking across sound intensities. The present data suggest that inhibition substantially contributes to the processing power of second-order neurons in the ascending auditory system

Early Postnatal Development of Spontaneous and Acoustically Evoked Discharge Activity of Principal Cells of the Medial Nucleus of the Trapezoid Body: An In Vivo Study in Mice

The calyx of Held synapse in the medial nucleus of the trapezoid body of the auditory brainstem has become an established in vitro model to study the development of fast glutamatergic transmission in the mammalian brain. However, we still lack in vivo data at this synapse on the maturation of spontaneous and sound-evoked discharge activity before and during the early phase of acoustically evoked signal processing (i.e., before and after hearing onset). Here we report in vivo single-unit recordings in mice from postnatal day 8 (P8) to P28 with a specific focus on developmental changes around hearing onset (P12). Data were obtained from two mouse strains commonly used in brain slice recordings: CBA/J and C57BL/6J. Spontaneous discharge rates progressively increased from P8 to P13, initially showing bursting patterns and large coefficients of variation (CVs), which changed to more continuous and random discharge activity accompanied by gradual decrease of CV around hearing onset. From P12 on, sound-evoked activity yielded phasic-tonic discharge patterns with discharge rates increasing up to P28. Response thresholds and shapes of tuning curves were adult-like by P14. A gradual shortening in response latencies was observed up to P18. The three-dimensional tonotopic organization of the medial nucleus of the trapezoid body yielded a high-to-low frequency gradient along the mediolateral and dorsoventral but not in the rostrocaudal axes. These data emphasize that models of signal transmission at the calyx of Held based on in vitro data have to take developmental changes in firing rates and response latencies up to the fourth postnatal week into account

Interaction of Excitation and Inhibition in Anteroventral Cochlear Nucleus Neurons That Receive Large Endbulb Synaptic Endings

Spherical bushy cells (SBCs) of the anteroventral cochlear nucleus (AVCN) receive their main excitatory input from auditory nerve fibers (ANFs) through large synapses, endbulbs of Held. These cells are also the target of inhibitory inputs whose function is not well understood. The present study examines the role of inhibition in the encoding of low-frequency sounds in the gerbil's AVCN. The presynaptic action potentials of endbulb terminals and postsynaptic action potentials of SBCs were monitored simultaneously in extracellular single-unit recordings in vivo. An input–output analysis of presynaptic and postsynaptic activity was performed for both spontaneous and acoustically driven activity. Two-tone stimulation and neuropharmacological experiments allowed the effects of neuronal inhibition and cochlear suppression on SBC activity to be distinguished. Ninety-one percent of SBCs showed significant neuronal inhibition. Inhibitory sidebands enclosed the high- or low-frequency, or both, sides of the excitatory areas of these units; this was reflected as a presynaptic to postsynaptic increase in frequency selectivity of up to one octave. Inhibition also affected the level-dependent responses at the characteristic frequency. Although in all units the presynaptic recordings showed monotonic rate-level functions, this was the case in only half of the postsynaptic recordings. In the other half of SBCs, postsynaptic inhibitory areas overlapped the excitatory areas, resulting in nonmonotonic rate-level functions. The results demonstrate that the sound-evoked spike activity of SBCs reflects the integration of acoustically driven excitatory and inhibitory input. The inhibition specifically affects the processing of the spectral, temporal, and intensity cues of acoustic signals

<i>Dep</i> recordings are not correlated with CF.

<p>(A) The distributions of CF for both nuclei differ: AVCN units (blue) are concentrated in the low frequency range reaching up to 3 kHz. The MNTB CFs (red) range over the whole spectrum, yet typically exceeding 3 kHz. The overlap of the MNTB with the AVCN range amounts to 16%. (B) Distribution of <i>Dep</i> and <i>No Dep</i> recordings in the AVCN with respect to CF for all recording conditions (see legend). If the classification as <i>Dep</i> was correlated with lower CFs (where stronger phase-locking occurs), the average CF of the <i>Dep</i> cases should be significantly lower than the average CF of the <i>No Dep</i> cases. The statistical comparison was not significant in any of the conditions (spontaneous, excitatory, single [black] and two-tone [gray] stimulations in the low- [LF] and high-frequency [HF] inhibitory/suppressive response regions) with all (Wilcoxon rank sum test for different medians of two groups).</p

IAP results and proportion of iPs for AVCN and MNTB for each recording condition.

<p>(A) For spontaneous activity the majority (56%) of the AVCN, but less than 5% of the MNTB recordings contained dependent iPs. (B) In the excitatory response condition the percentage of AVCN <i>Dep</i> recordings decreased to 50%. In the MNTB no <i>Dep</i> recordings were found. (C) In the inhibitory/suppressive response condition the percentage of AVCN <i>Dep</i> recordings increased to 80%, whereas again none of the MNTB recordings was classified as <i>Dep</i>. For AVCN <i>Dep</i> recordings the proportion of iPs with respect to the total number of (presynaptic) events estimates the insecurity of transmission (if iPs correspond to failures of transmission). (D) During spontaneous activity <i>No Dep</i> had a significantly smaller iP proportion than <i>Dep</i>, consistent with failures. (E) In the excitatory condition the iP proportion does not differ significantly between the <i>No Dep</i> and the <i>Dep</i> cases, although they both increase compared to the spontaneous condition. (F) In the inh./sup. condition the distribution of iP proportion for the <i>Dep</i> cases increases significantly compared to both previous conditions, whereas the corresponding distribution for the <i>No Dep</i> cases stays similar to the excitatory condition.</p

Examples of IAP for simulated, AVCN, and MNTB recordings of spontaneous activity.

<p>The left column shows voltage traces (black) and trigger levels for complex (red) and candidate waveforms (orange) for each unit. The corresponding average complex waveform (black) and its pointwise standard deviation (gray) is depicted in the middle column. The trigger potential (TP) (MNTB: presynaptic spike, AVCN: postsynaptic potential, probably the EPSC) is also indicated. The right column shows the histograms (orange) generated by triggering at the height of the TP (after aligned subtraction of the average complex waveform). Further the interspike interval histograms of the complex waveforms are shown, mainly for visual comparison. For the simulated data (A2, B2) the histograms reflect the failure containing condition by a decrease in CritWin (A2), and conversely the lack of decrease in the two unit condition (B2). Guided by the results from the known datasets, the AVCN data (C2, D2) can be interpreted: A substantial number of cells exhibited histograms similar to the cell in C2, suggesting failures of transmission, while the remaining cells showed histograms similar to the cell in D2. If a decrease occurred, its timing was predicted by the ISI histogram (blue). In the MNTB (E2, F2, G2) the most frequent finding was the absence of iP at the TPs height, leading to an empty histogram as in E2. Most of the units with iPs of sufficient height, exhibited no decrease of the histogram in CritWin, as in F2. In a small fraction of recordings a decrease was observed, yet, this could be accompanied by unusually high variability in timing from the presynaptic to the postsynaptic side as in G2 (see individual trace in middle column). IAP classified the recordings in A,B, and G as <i>Dep</i> and the remaining as <i>no Dep</i>.</p

Statistics and correlations for the number of iPs and different iP rates.

<p>(A1–3) #iPs in the two windows (RefWin,CritWin) for all recording conditions. In the AVCN iPs are generally more abundant, reflected in higher #iPs in the windows. In the MNTB the #iPs in the windows rises in the excited condition, probably due to activation of neighboring units. In the inh./sup. condition the #iPs in the windows is again reduced, probably due to correlated inh./sup. areas for neighboring units. (B1–3) The iP rate in RefWin equals or exceeds the overall iP rate, thus confirming that the IAP is not biased by considering only iPs close to the CWs. Especially for the MNTB, low iP rates in RefWin entail similarly low overall iP rates, hence correspond to very low #iPs rel. to #CWs. (C1–3) A comparison of SNR and the overall iP rate between the two nuclei shows that for the best SNR, in the AVCN the overall iP rate often remains substantial, whereas it drops to vanishingly low values in the MNTB. This indicates that in the MNTB virtually no iPs remain under conditions where dependent iPs should be best detectable.</p

Complex Waveforms (CW) in AVCN and MNTB and overview of the general task.

<p>(A) In the AVCN the CW usually has three components, P, A, and B <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007014#pone.0007014-Pfeiffer1" target="_blank">[11]</a>. Aside from this CW, also the combination of only P and A occurs, constituting candidates of failed transmission. Since A is usually larger than P, the height of A was used for triggering the P-A combinations. Hence, in the AVCN A is the trigger potential (TP). (B) In the MNTB the CW has two components, C1 and C2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007014#pone.0007014-Guinan1" target="_blank">[13]</a>. Here, the height of the presynaptic C1 serves to detect failure candidates, i.e. constitutes the TP here. (C) To check the reliability of transmission, three cases need to be distinguished (here illustrated without noise for a mean CW from an MNTB unit): (top) If the CW has a strong C1 component, but no potentials of a similar height (iPs) occur in the remaining trace, then the recording is from a single unit and there is no indication for failures. (middle) If iPs occur, but some of them are located too close (less than the refractory period, RP) to their counterpart (C1) in the CW, then the recording is from multiple units and the iPs do not stem from failures. (bottom) If iPs occur and they all respect the RP, then they likely correspond to failures (assuming other correlation factors, e.g. phase locking, are ruled out). (D) Under realistic recording conditions, iPs could be due to failures (only C1), other cells, or just noise fluctuations. Classical spike-sorting cannot reliably distinguish between these cases. If an iP is detected close to a C1, failures cannot necessarily be excluded since this could have been a noise fluctuation. To decide whether these violations are due to noise, a statistical test is required that compares the distribution of iPs at different distances from C1. In the AVCN, a corresponding argument would replace C1 with the P-A waveform.</p

Decreased Temporal Precision of Auditory Signaling in Kcna1-Null Mice: An Electrophysiological Study In Vivo

The voltage-gated potassium (Kv) channel subunit Kv1.1, encoded by the Kcna1 gene, is expressed strongly in the ventral cochlear nucleus (VCN) and the medial nucleus of the trapezoid body (MNTB) of the auditory pathway. To examine the contribution of the Kv1.1 subunit to the processing of auditory information, in vivo single-unit recordings were made from VCN neurons (bushy cells), axonal endings of bushy cells at MNTB cells (calyces of Held), and MNTB neurons of Kcna1-null (-/-) mice and littermate control (+/+) mice. Thresholds and spontaneous firing rates of VCN and MNTB neurons were not different between genotypes. At higher sound intensities, however, evoked firing rates of VCN and MNTB neurons were significantly lower in -/- mice than +/+ mice. The SD of the first-spike latency (jitter) was increased in VCN neurons, calyces, and MNTB neurons of -/- mice compared with +/+ controls. Comparison along the ascending pathway suggests that the increased jitter found in -/- MNTB responses arises mostly in the axons of VCN bushy cells and/or their calyceal terminals rather than in the MNTB neurons themselves. At high rates of sinusoidal amplitude modulations, -/- MNTB neurons maintained high vector strength values but discharged on significantly fewer cycles of the amplitude-modulated stimulus than +/+ MNTB neurons. These results indicate that in Kcna1-null mice the absence of the Kv1.1 subunit results in a loss of temporal fidelity (increased jitter) and the failure to follow high-frequency amplitude-modulated sound stimulation in vivo

Activity-dependent modulation of inhibitory synaptic kinetics in the cochlear nucleus

<div>Spherical bushy cells (SBCs) in the anteroventral cochlear nucleus respond to acoustic stimulation with discharges that precisely encode the phase of low-frequency sound. The accuracy of spiking is crucial for sound localization and speech perception. Compared to the auditory nerve input, temporal precision of SBC spiking is improved through the</div><div>engagement of acoustically evoked inhibition. Recently, the inhibition was shown to be less precise than previously understood. It shifts from predominantly glycinergic to synergistic GABA/glycine transmission in an activity-dependent manner. Concurrently, the inhibition attains a tonic character through temporal summation. The present study provides a comprehensive understanding of the mechanisms underlying this slow inhibitory input.</div><div>We performed whole-cell voltage clamp recordings on SBCs from juvenile Mongolian gerbils and recorded evoked inhibitory postsynaptic currents (IPSCs) at physiological rates. The data reveal activity-dependent IPSC kinetics, i.e., the decay is slowed with increased input rates or recruitment. Lowering the release probability yielded faster decay kinetics of the single- and short train-IPSCs at 100 Hz, suggesting that transmitter quantity plays an important role in controlling the decay. Slow transmitter clearance from the synaptic cleft caused prolonged receptor binding and, in the case of glycine, spillover to nearby synapses. The GABAergic component prolonged the decay by contributing to the asynchronous vesicle release depending on the input rate. Hence, the different factors controlling the amount of transmitters in the synapse jointly slow the inhibition during physiologically relevant activity. Taken together, the slow time course is predominantly determined by the receptor kinetics and transmitter clearance during short stimuli, whereas long duration or high frequency stimulation additionally engage asynchronous release to prolong IPSCs.</div><div><br></div><div>[This Document is Protected by copyright and was first published by Frontiers. All rights reserved, it is reproduced with permission.]</div