INHIBITORY FUNCTION IN AUDITORY PROCESSING EDITED BY : R. Michael Burger, Conny Kopp-Scheinpflug and Ian D. Forsythe PUBLISHED IN : Frontiers in Neural Circuits 1 October 2015 | Inhibitory Function in Auditory Processing Frontiers in Neural Circuits Frontiers Copyright Statement © Copyright 2007-2015 Frontiers Media SA. All rights reserved. All content included on this site, such as text, graphics, logos, button icons, images, video/audio clips, downloads, data compilations and software, is the property of or is licensed to Frontiers Media SA (“Frontiers”) or its licensees and/or subcontractors. The copyright in the text of individual articles is the property of their respective authors, subject to a license granted to Frontiers. The compilation of articles constituting this e-book, wherever published, as well as the compilation of all other content on this site, is the exclusive property of Frontiers. For the conditions for downloading and copying of e-books from Frontiers’ website, please see the Terms for Website Use. If purchasing Frontiers e-books from other websites or sources, the conditions of the website concerned apply. Images and graphics not forming part of user-contributed materials may not be downloaded or copied without permission. Individual articles may be downloaded and reproduced in accordance with the principles of the CC-BY licence subject to any copyright or other notices. They may not be re-sold as an e-book. As author or other contributor you grant a CC-BY licence to others to reproduce your articles, including any graphics and third-party materials supplied by you, in accordance with the Conditions for Website Use and subject to any copyright notices which you include in connection with your articles and materials. All copyright, and all rights therein, are protected by national and international copyright laws. The above represents a summary only. For the full conditions see the Conditions for Authors and the Conditions for Website Use. ISSN 1664-8714 ISBN 978-2-88919-667-8 DOI 10.3389/978-2-88919-667-8 About Frontiers Frontiers is more than just an open-access publisher of scholarly articles: it is a pioneering approach to the world of academia, radically improving the way scholarly research is managed. The grand vision of Frontiers is a world where all people have an equal opportunity to seek, share and generate knowledge. Frontiers provides immediate and permanent online open access to all its publications, but this alone is not enough to realize our grand goals. Frontiers Journal Series The Frontiers Journal Series is a multi-tier and interdisciplinary set of open-access, online journals, promising a paradigm shift from the current review, selection and dissemination processes in academic publishing. All Frontiers journals are driven by researchers for researchers; therefore, they constitute a service to the scholarly community. At the same time, the Frontiers Journal Series operates on a revolutionary invention, the tiered publishing system, initially addressing specific communities of scholars, and gradually climbing up to broader public understanding, thus serving the interests of the lay society, too. Dedication to Quality Each Frontiers article is a landmark of the highest quality, thanks to genuinely collaborative interactions between authors and review editors, who include some of the world’s best academicians. Research must be certified by peers before entering a stream of knowledge that may eventually reach the public - and shape society; therefore, Frontiers only applies the most rigorous and unbiased reviews. Frontiers revolutionizes research publishing by freely delivering the most outstanding research, evaluated with no bias from both the academic and social point of view. By applying the most advanced information technologies, Frontiers is catapulting scholarly publishing into a new generation. What are Frontiers Research Topics? Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: researchtopics@frontiersin.org 2 October 2015 | Inhibitory Function in Auditory Processing Frontiers in Neural Circuits There seems little doubt that from the earliest evolutionary beginnings, inhibition has been a fundamental feature of neuronal circuits. - even the simplest life forms sense and interact with their environment, orienting or approaching positive stimuli while avoiding aversive stimuli. This requires internal signals that both drive and suppress behavior. Traditional descriptions of inhibition sometimes limit its role to the suppression of action potential generation. This view fails to capture the vast breadth of inhibitory function now known to exist in neural circuits. INHIBITORY FUNCTION IN AUDITORY PROCESSING Image: The medial superior olive is a major binaural center in the sound localization pathway which receives prominant inhibitory input from the MNTB and LNTB. Here MAP2 (red) immunolabeled MSO principal cells are shown expressing glycine receptor (green). Source with Permission: R. Michael Burger, Ph.D., Lehigh University Topic Editors: R. Michael Burger, Ph.D. , Lehigh University, USA Conny Kopp-Scheinpflug, Ph.D., Ludwig-Maximilians University Munich, Germany Ian D. Forsythe, Ph.D., University of Leicester, UK 3 October 2015 | Inhibitory Function in Auditory Processing Frontiers in Neural Circuits A modern perspective on inhibitory signaling comprises a multitude of mechanisms; For exam- ple, inhibition can act via a shunting mechanism to speed the membrane time constant and reduce synaptic integration time. It can act via G-protein coupled receptors to initiate second messenger cascades that influence synaptic strength. Inhibition contributes to rhythm generation and can even activate ion channels that mediate inward currents to drive action potential generation. Inhibition also appears to play a role in shaping the properties of neural circuitry over longer time scales. Experience-dependent synaptic plasticity in developing and mature neural circuits underlies behavioral memory and has been intensively studied over the past decade. At excitatory synapses, adjustments of synaptic efficacy are regulated predominantly by changes in the number and function of postsynaptic glutamate receptors. There is, however, increasing evidence for inhibitory modulation of target neuron excitability playing key roles in experience-dependent plasticity. One reason for our limited knowledge about plasticity at inhibitory synapses is that in most circuits, neurons receive convergent inputs from disparate sources. This problem can be overcome by investigating inhibitory circuits in a system with well-defined inhibitory nuclei and projections, each with a known computational function. Compared to other sensory systems, the auditory system has evolved a large number of sub- thalamic nuclei each devoted to processing distinct features of sound stimuli. This information once extracted is then re-assembled to form the percept the acoustic world around us. The well-understood function of many of these auditory nuclei has enhanced our understanding of inhibition’s role in shaping their responses from easily distinguished inhibitory inputs. In particular, neurons devoted to processing the location of sound sources receive a complement of discrete inputs for which in vivo activity and function are well understood. Investigation of these areas has led to significant advances in understanding the development, physiology, and mechanistic underpinnings of inhibition that apply broadly to neuroscience. In this series of papers, we provide an authoritative resource for those interested in exploring the variety of inhibitory circuits and their function in auditory processing. We present original research and focused reviews touching on development, plasticity, anatomy, and evolution of inhibitory circuitry. We hope our readers will find these papers valuable and inspirational to their own research endeavors. Citation: Burger, R. M., Kopp-Scheinpflug, C., Forsythe, I. D., eds. (2015). Inhibitory Function in Auditory Processing. Lausanne: Frontiers Media. doi: 10.3389/978-2-88919-667-8 Cover image: While excitatory inputs to the medial nucleus of the trapezoid body (MNTB), mediated by the calyx of Held, are well established, it is much less known that MNTB neurons also receive inhibitory inputs, largely mediated by glycine. Inhibitory inputs to MNTB are mediated by several fibers, each of which makes several synaptic contacts along the principal cell body (shown in red, large image). These synapses produce mostly glycinergic currents that are large, have fast kinetics, and can sustain prolonged activity consisting of thousands of stimulations. Source with permission: Otto Albrecht, Ph.D. and Achim Klug, Ph.D., University of Colorado Medical School 4 October 2015 | Inhibitory Function in Auditory Processing Frontiers in Neural Circuits Table of Contents 06 Editorial: Inhibitory function in auditory processing R. M. Burger, Ian D. Forsythe and Conny Kopp-Scheinpflug 09 Linear coding of complex sound spectra by discharge rate in neurons of the medial nucleus of the trapezoid body (MNTB) and its inputs Kanthaiah Koka and Daniel J. Tollin 28 The relative contributions of MNTB and LNTB neurons to inhibition in the medial superior olive assessed through single and paired recordings Michael T. Roberts, Stephanie C. Seeman and Nace L. Golding 42 Inhibitory projections from the ventral nucleus of the trapezoid body to the medial nucleus of the trapezoid body in the mouse Otto Albrecht, Anna Dondzillo, Florian Mayer, John A. Thompson and Achim Klug 57 Distribution of glycine receptors on the surface of the mature calyx of Held nerve terminal Johana Trojanova, Akos Kulik, Jiri Janacek, Michaela Kralikova, Josef Syka and Rostislav Turecek 69 Development of glycinergic innervation to the murine LSO and SPN in the presence and absence of the MNTB Stefanie C. Altieri, Tianna Zhao, Walid Jalabi and Stephen M. Maricich 77 Cell-type specific short-term plasticity at auditory nerve synapses controls feed-forward inhibition in the dorsal cochlear nucleus Miloslav Sedlacek and Stephan D. Brenowitz 89 Superficial stellate cells of the dorsal cochlear nucleus Pierre F . Apostolides and Laurence O. Trussell 98 Inhibitory glycinergic neurotransmission in the mammalian auditory brainstem upon prolonged stimulation: short-term plasticity and synaptic reliability Florian Kramer, Désirée Griesemer, Dennis Bakker, Sina Brill, Jürgen Franke, Erik Frotscher and Eckhard Friauf 120 Developmental expression of inhibitory synaptic long-term potentiation in the lateral superior olive Vibhakar C. Kotak and Dan H. Sanes 128 Nitric oxide signaling modulates synaptic inhibition in the superior paraolivary nucleus (SPN) via cGMP-dependent suppression of KCC2 Lina Yassin, Susanne Radtke-Schuller, Hila Asraf, Benedikt Grothe, Michal Hershfinkel, Ian D. Forsythe and Cornelia Kopp-Scheinpflug 140 VGLUT3 does not synergize GABA/glycine release during functional refinement of an inhibitory auditory circuit Daniel T. Case, Javier Alamilla and Deda C. Gillespie 5 October 2015 | Inhibitory Function in Auditory Processing Frontiers in Neural Circuits 148 Glycinergic transmission modulates GABAergic inhibition in the avian auditory pathway Matthew J. Fischl and R. Michael Burger 161 Activity-dependent modulation of inhibitory synaptic kinetics in the cochlear nucleus Jana Nerlich, Christian Keine, Rudolf Rübsamen, R. Michael Burger and Ivan Milenkovic 175 GABAergic and glycinergic inhibitory synaptic transmission in the ventral cochlear nucleus studied in VGAT channelrhodopsin-2 mice Ruili Xie and Paul B. Manis 190 Interplay between low threshold voltage-gated K + channels and synaptic inhibition in neurons of the chicken nucleus laminaris along its frequency axis William R. Hamlet, Yu-Wei Liu, Zheng-Quan Tang and Yong Lu 205 Neuronal specializations for the processing of interaural difference cues in the chick Harunori Ohmori 213 The natural history of sound localization in mammals – a story of neuronal inhibition Benedikt Grothe and Michael Pecka EDITORIAL published: 01 September 2015 doi: 10.3389/fncir.2015.00045 Frontiers in Neural Circuits | www.frontiersin.org September 2015 | Volume 9 | Article 45 Edited and reviewed by: Robert C. Froemke, New York University School of Medicine, USA *Correspondence: R. M. Burger, rmb206@lehigh.edu Received: 29 May 2015 Accepted: 13 August 2015 Published: 01 September 2015 Citation: Burger RM, Forsythe ID and Kopp-Scheinpflug C (2015) Editorial: Inhibitory function in auditory processing. Front. Neural Circuits 9:45. doi: 10.3389/fncir.2015.00045 Editorial: Inhibitory function in auditory processing R. M. Burger 1 *, Ian D. Forsythe 2 and Conny Kopp-Scheinpflug 3 1 Department of Biological Sciences, Lehigh University, Bethlehem, PA, USA, 2 Department of Cell Physiology and Pharmacology, College of Medicine, Biological Sciences, Psychology, University of Leicester, Leicester, UK, 3 Division of Neurobiology, Department of Biology II, Ludwig-Maximilians-University Munich, Planegg-Martinsried, Germany Keywords: GABA, glycine, nitric oxide, plasticity, gap junctions, sound localization, MNTB, co-release In recent decades, with the convergence of high-resolution anatomical and physiological techniques, a perspective is emerging on inhibition in the nervous system that recognizes the vast diversity of functions it serves. These include roles in modulation, development, and plasticity, in addition to the common perception of inhibition as spike suppression. Progress toward this more nuanced understanding of inhibition has derived from many studies across the nervous system, but here we focus on part of the brainstem auditory system, where discrete inhibitory nuclei interact in unique and fascinating ways to integrate and compute binaural information in the circuitry for sound source localization. We have solicited studies for this special topic on inhibition in the auditory brainstem circuitry from laboratories around the world. The assembled manuscripts provide an authoritative collection of concepts across the breadth of neuroscience research on inhibitory function that focus on three major themes. Inhibition in the Superior Olive: The Medial Nucleus of the Trapezoid Body (MNTB) A major advantage of investigating inhibition in the auditory pathway is the distribution of inhibitory centers among its subthalamic nuclei. In general, these nuclei are involved in computing the azimuth location of a sound source, by integrating the acoustic stimulus from both ears. The superior olivary complex (SOC) is the first region of the brain to compute sound location by comparing the input to the two ears. The MNTB is central to this circuitry and is highly specialized; being driven by the Calyx of Held (one of the largest synapses in vertebrates) and providing a powerful glycinergic inhibition to its targets. Despite its pivotal role in this circuit and extensive investigation, our understanding remains incomplete, the details of its inputs, output, and sound encoding are still being explored. The MNTB projects to multiple targets in the SOC including those that process cues for sound localization. Two studies investigate what information is conveyed by these neurons, with regard to both temporal and spectral encoding. The first, by Koka and Tollin (2014) demonstrates that the MNTB accurately and linearly encodes spectral information. The spectral content represented in output spiking is crucial for understanding what binaural comparisons the MNTB’s targets can make. For example the medial superior olive (MSO), which receives contralateral ear-derived inhibition from the MNTB and which also receives an analogous inhibitory input from the ipsilateral ear via the LNTB. In an innovative in vitro study, Roberts et al. (2014) compared these two neighboring inhibitory inputs to the MSO. They demonstrated that the two inputs have similar latencies but do not share identical temporal encoding properties. The MNTB also receives inhibition, the origin of which has been a source of speculation for many years. Two studies help refine our understanding of inhibitory input to the MNTB. First, | 6 Burger et al. Editorial: Inhibitory function in auditory processing Albrecht et al. (2014) identify the ventral nucleus of the trapezoid body (VNTB) as a major source of glycinergic input to the MNTB. They also show that this input follows a similar developmental pattern to that of the MNTB itself, with mixed GABA/glycine release early in development followed by primarily glycine release later in development. Second, Trojanova et al. (2014) show that one target of this glycinergic input is targeting the presynaptic terminals of the glutamatergic Calyx of Held. This study shows a compelling pattern of glycine receptor expression on the terminals at locations neighboring putative glutamate release sites and apposed to inhibitory terminals. This study shows anatomical evidence suggesting that glycine receptors are poised to modulate release of excitatory transmitter directly via spillover of inhibitory transmitter. The MNTB appears to be so central to auditory circuitry, that it is difficult to imagine how the network could adapt to its absence, but genetic tools have allowed Altieri et al. (2014) to address this question. They investigated the development of markers for inhibition in the SOC in Engrailed − / − mice that fail to develop their MNTB nucleus. These mice are thus deprived of a major source of glycinergic inhibition to the LSO, MSO, and superior paraolivary nucleus (SPN). Surprisingly, development of immunohistochemical markers for glycinergic transmission, although delayed, reach typical levels in adulthood, demonstrating remarkable developmental plasticity in this system and provide evidence for alternative sources of inhibitory input. Short-term, Long-term, and Novel Mechanisms of Inhibitory Plasticity Synaptic plasticity allows developmental change and activity- dependent adaptation of information transmission throughout the nervous system. In the auditory pathway, myriad examples of plasticity of inhibitory signaling are demonstrated. They include classical forms such as LTP, LTD, depression and facilitation, as well as novel forms described below. Plasticity is a prominent feature of processing in another region of the auditory brainstem, the dorsal cochlear nucleus (DCN), which includes complex intra-CN inhibitory circuitry driven by the auditory nerve via the tuberculoventral interneuron. Sedlacek and Brenowitz (2014) carefully dissects this circuit to reveal how different contributions of short term synaptic plasticity among direct and disynaptic pathways in the DCN strongly influence its primary output neuron, the fusiform cells. Indeed, the circuitry of the fusiform cell is complex and involves several cell types intrinsic to the DCN. Apostolides and Trussell (2014) explores a poorly understood component of this circuitry, called the superficial stellate cell (SSC). SSCs not only form inhibitory synapses but also are electrically coupled to fusiform cells as well as one another. SSCs appear well positioned to mediate a coordinated non-auditory derived modulation of DCN output. In the SOC, Kramer et al. (2014) investigated short-term synaptic plasticity using a novel “marathon” stimulation protocol to reveal components of synaptic plasticity rarely analyzed in previous works. They demonstrate that the inhibition to the LSO via MNTB is very robust with respect to reliability, but more prone to depression than previously reported in studies using less demanding stimuli. Long-term synaptic plasticity is a hallmark of excitatory synaptic coupling, particularly during development. In contrast, long-term plasticity at inhibitory synapses is less commonly studied. One exception is at the MNTB-LSO synapse where Kotak and Sanes (2014) have previously demonstrated GABA B receptor-dependent long term depression, especially early in development. In their current paper, they add to this body of work by demonstrating that this synapse also expresses long-term potentiation, but somewhat later in development. This potentiation surprisingly also depends on GABA B receptor function. Plasticity of inhibition can also occur indirectly, as demonstrated in the SPN, a synaptic target of the MNTB. Yassin et al. (2014) in a comparative study across species and between the SOC nuclei reveal that nitric oxide (NO) signaling dynamically modulates inhibitory strength. Interestingly, NO acts postsynaptically through a cGMP dependent pathway to suppress KCC2. This outwardly directed potassium chloride co-transporter is crucially involved in setting the neuronal Cl − reversal potential. The NO-dependent depolarizing shift in reversal potential demonstrates a possible means to modulate inhibition in SPN neurons, without influencing inhibition in other collateral targets of the MNTB. Diversity of Inhibition in Monaural and Binaural Nuclei Inhibitory neurons of the SOC typically release two or more transmitters in early development, but revert to a single dominant transmitter following hearing onset. This general principle has been refined in the last two decades. Recently however, it has become apparent that transmitter release in mature auditory circuitry may be more complex than previously appreciated. In this issue, Case et al. (2014) extends these studies to investigate the role of vesicular glutamate transporter expression in “glycinergic” MNTB neurons. Two other studies, by Fischl and Burger (2014) and Nerlich et al. (2014) extend recent findings that GABAergic and glycinergic inputs to the cochlear nucleus are dominated by a single neurotransmitter at low stimulus rates, but surprisingly become multi-transmitter release synapses at high stimulus rates. This principle applies to both birds and mammals and at multiple synapses. In a complementary study Xie and Manis (2014) use optical tools to finely dissect the properties of both GABAergic and glycinergic transmission in two types of cochlear nucleus neurons, showing that kinetics and short-term plasticity are heavily dependent on the synaptic target. One underappreciated aspect of inhibition that is emerging in the literature is that classical synaptic inhibition can engage voltage gated ion channels and signaling pathways beyond their classical receptors. Hamlet et al. (2014) investigate functional coupling between GABA receptors and low voltage gated potassium channels in nucleus laminaris (NL) of the chick. In NL, Frontiers in Neural Circuits | www.frontiersin.org September 2015 | Volume 9 | Article 45 | 7 Burger et al. Editorial: Inhibitory function in auditory processing GABA is depolarizing and activates this potassium current. The present study demonstrates the precise and profound interplay between the Cl − and K + conductances occurring in both pre and postsynaptic compartments of this circuitry. A Broader View of Inhibition Finally, this Research Topic presents two review offerings. The first, from Ohmori (2014) focuses on specializations of the sound localization pathway in the chick. This is a major model system that shares many features with mammalian circuitry. The second from Grothe and Pecka (2014) presents a novel hypothesis concerning the evolutionary origins of the role of inhibition in the superior olive by synthesizing what is known about the origin of the tympanic ear, the fossil record, and inhibitory circuitry in extant animals. Together these studies and perspectives provide a taste of current concepts, with promises of more exciting insights into auditory function around the corner. Inhibition is nonetheless important for its suppression of mere excitation, and as we see here this field is vibrant and forward-looking. We hope that neuroscientists investigating the physiology of inhibition beyond the auditory system will find this work equally exciting and we thank all of our contributing authors for their excellent work. And to you, our readers, we hope you find some inspiration for your own research. References Albrecht, O., Dondzillo, A., Mayer, F., Thompson, J. A., and Klug, A. (2014). Inhibitory projections from the ventral nucleus of the trapezoid body to the medial nucleus of the trapezoid body in the mouse. Front. Neural Circuits 8:83. doi: 10.3389/fncir.2014.00083 Altieri, S. C., Zhao, T., Jalabi, W., and Maricich, S. M. (2014). Development of glycinergic innervation to the murine LSO and SPN in the presence and absence of the MNTB. Front. Neural Circuits 8:109. doi: 10.3389/fncir.2014.00109 Apostolides, P. F., and Trussell, L. O. (2014). Superficial stellate cells of the dorsal cochlear nucleus. Front. Neural Circuits 8:63. doi: 10.3389/fncir. 2014.00063 Case, D. T., Alamilla, J., and Gillespie, D. C. (2014). VGLUT3 does not synergize GABA/glycine release during functional refinement of an inhibitory auditory circuit. Front. Neural Circuits 8:140. doi: 10.3389/fncir.2014.00140 Fischl, M. J., and Burger, R. M. (2014). Glycinergic transmission modulates GABAergic inhibition in the avian auditory pathway. Front. Neural Circuits 8:19. doi: 10.3389/fncir.2014.00019 Grothe, B., and Pecka, M. (2014). The natural history of sound localization in mammals – a story of neuronal inhibition. Front. Neural Circuits 8:116. doi: 10.3389/fncir.2014.00116 Hamlet, W. R., Liu, Y.-W., Tang, Z.-Q., and Lu, Y. (2014). Interplay between low threshold voltage-gated K+ channels and synaptic inhibition in neurons of the chicken nucleus laminaris along its frequency axis. Front. Neural Circui 8:51. doi: 10.3389/fncir.2014.00051 Koka, K., and Tollin, D. J. (2014). Linear coding of complex sound spectra by discharge rate in neurons of the medial nucleus of the trapezoid body (MNTB) and its inputs. Front. Neural Circuits 8:144. doi: 10.3389/fncir.2014.00144 Kotak, V. C., and Sanes, D. H. (2014). Developmental expression of inhibitory synaptic long-term potentiation in the lateral superior olive. Front. Neural Circuits 8:67. doi: 10.3389/fncir.2014.00067 Kramer, F., Griesemer, D., Bakker, D., Brill, S., Franke, J., Frotscher, E., et al. (2014). Inhibitory glycinergic neurotransmission in the mammalian auditory brainstem upon prolonged stimulation: short-term plasticity and synaptic reliability. Front. Neural Circuits 8:14. doi: 10.3389/fncir.2014.00014 Nerlich, J., Keine, C., Rübsamen, R., Burger, R. M., and Milenkovic, I. (2014). Activity-dependent modulation of inhibitory synaptic kinetics in the cochlear nucleus. Front. Neural Circuits 8:145. doi: 10.3389/fncir.2014.00145 Ohmori, H. (2014). Neuronal specializations for the processing of interaural difference cues in the chick. Front. Neural Circuits 8:47. doi: 10.3389/fncir.2014.00047 Roberts, M. T., Seeman, S. C., and Golding, N. L. (2014). The relative contributions of MNTB and LNTB neurons to inhibition in the medial superior olive assessed through single and paired recordings. Front. Neural Circuits 8:49. doi: 10.3389/fncir.2014.00049 Sedlacek, M., and Brenowitz, S. D. (2014). Cell-type specific short-term plasticity at auditory nerve synapses controls feed-forward inhibition in the dorsal cochlear nucleus. Front. Neural Circuits 8:78. doi: 10.3389/fncir.2014.00078 Trojanova, J., Kulik, A., Janacek, J., Kralikova, M., Syka, J., and Turecek, R. (2014). Distribution of glycine receptors on the surface of the mature calyx of Held nerve terminal. Front. Neural Circuits 8:120. doi: 10.3389/fncir.2014.00120 Xie, R., and Manis, P. B. (2014). GABAergic and glycinergic inhibitory synaptic transmission in the ventral cochlear nucleus studied in VGAT channelrhodopsin-2 mice. Front. Neural Circuits 8:84. doi: 10.3389/fncir.2014.00084 Yassin, L., Radtke-Schuller, S., Asraf, H., Grothe, B., Hershfinkel, M., Forsythe, I. D., et al. (2014). Nitric oxide signaling modulates synaptic inhibition in the superior paraolivary nucleus (SPN) via cGMP-dependent suppression of KCC2. Front. Neural Circuits 8:65. doi: 10.3389/fncir.2014.00065 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2015 Burger, Forsythe and Kopp-Scheinpflug. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Neural Circuits | www.frontiersin.org September 2015 | Volume 9 | Article 45 | 8 ORIGINAL RESEARCH ARTICLE published: 16 December 2014 doi: 10.3389/fncir.2014.00144 Linear coding of complex sound spectra by discharge rate in neurons of the medial nucleus of the trapezoid body (MNTB) and its inputs Kanthaiah Koka and Daniel J. Tollin * Department of Physiology and Biophysics, University of Colorado School of Medicine, Aurora, CO, USA Edited by: Conny Kopp-Scheinpflug, Ludwig-Maximilians-University Munich, Germany Reviewed by: Rudolf Rübsamen, University of Leipzig, Germany Michael Pecka, Ludwig-Maximilians-University Munich, Germany *Correspondence: Daniel J. Tollin, Department of Physiology and Biophysics, University of Colorado School of Medicine, RC1-N, Stop 8307 , 12800 E. 19th Avenue, Aurora, CO 80045, USA e-mail: daniel.tollin@ucdenver.edu The interaural level difference (ILD) cue to sound location is first encoded in the lateral superior olive (LSO). ILD sensitivity results because the LSO receives excitatory input from the ipsilateral cochlear nucleus and inhibitory input indirectly from the contralateral cochlear nucleus via glycinergic neurons of the ipsilateral medial nucleus of the trapezoid body (MNTB). It is hypothesized that in order for LSO neurons to encode ILDs, the sound spectra at both ears must be accurately encoded via spike rate by their afferents. This spectral-coding hypothesis has not been directly tested in MNTB, likely because MNTB neurons have been mostly described and studied recently in regards to their abilities to encode temporal aspects of sounds, not spectral. Here, we test the hypothesis that MNTB neurons and their inputs from the cochlear nucleus and auditory nerve code sound spectra via discharge rate. The Random Spectral Shape (RSS) method was used to estimate how the levels of 100-ms duration spectrally stationary stimuli were weighted, both linearly and non-linearly, across a wide band of frequencies. In general, MNTB neurons, and their globular bushy cell inputs, were found to be well-modeled by a linear weighting of spectra demonstrating that the pathways through the MNTB can accurately encode sound spectra including those resulting from the acoustical cues to sound location provided by head-related directional transfer functions (DTFs). Together with the anatomical and biophysical specializations for timing in the MNTB-LSO complex, these mechanisms may allow ILDs to be computed for complex stimuli with rapid spectrotemporally-modulated envelopes such as speech and animal vocalizations and moving sound sources. Keywords: calyx of held, medial nucleus of the trapezoid body, lateral superior olive, spectrotemporal receptive field, sound localization, temporal processing INTRODUCTION The interaural level difference (ILD) cue to sound location requires neural encoding of the shapes and magnitudes of sound spectra. ILDs result from frequency- and direction-dependent modifications of sound by the head and pinnae and are defined as the difference in spectra of the signals at the two ears (Tollin and Koka, 2009a,b). In the mammalian brainstem, the superior oli- vary complex contains a circuit comprising the ipsilateral medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO) that is essential for ILD encoding (Tollin, 2003). LSO neurons receive excitatory input from spherical bushy cells (SBCs) of the ipsilateral cochlear nucleus (CN) and inhibitory input from the contralateral ear via the MNTB; the MNTB receives excita- tory input from globular bushy cells (GBCs) of the contralateral CN. SBCs and GBCs receive excitatory inputs from the auditory nerve. These inputs confer upon single LSO neurons the ability to compute a neural correlate of ILDs (Boudreau and Tsuchitani, 1968). Although there is consensus that the LSO initially encodes ILDs and that the inhibitory input to the LSO from the MNTB is essential, the mechanisms are still not well understood. The accurate and precise encoding of ILD observed in LSO neurons (e.g., Tollin et al., 2008) would seem to imply that the neu- rons comprising the ascending inputs to LSO must be accurately encoding sound spectra at the two ears. However, this hypoth- esis has not been explicitly tested. One reason for this may be that the MNTB and bushy cells are mostly described, and thus studied, in regards to their exquisite abilities to encode tempo- ral aspects of sounds (Wu and Kelly, 1993; Taschenberger and Von Gersdorff, 2000; Futai et al., 2001; Joshi et al., 2004; Lorteije et al., 2009) but not spectral. The exquisite temporal processing capabilities of MNTB result from several specializations. First, the input from GBCs onto MNTB neurons forms the largest, most secure synapses in the CNS, the calyx of Held (Jean-Baptiste and Morest, 1975; McLaughlin et al., 2008). Each MNTB neuron receives only a single calyx, which can envelop up to half the soma surface, and large pre-synaptic terminals that produce large post- synaptic currents (Banks and Smith, 1992; Smith et al., 1998), facts that have made this synapse a model for synaptic trans- mission (Forsythe, 1994; Borst et al., 1995; Schneggenburger and Forsythe, 2006). MNTB neurons have short membrane time con- stants, receptors with fast kinetics, and specialized ion channels Frontiers in Neural Circuits www.frontiersin.org December 2014 | Volume 8 | Article 144 | NEURAL CIRCUITS 9 Koka and Tollin Spectral coding by MNTB neurons that together with specializations in the calyx result in large, rapid EPSPs that excite MNTB neurons with nearly invariant synap- tic delays (Wu and Kelly, 1991; Banks and Smith, 1992; von Gersdorff and Borst, 2002; Trussell, 2002; although see Tolnai et al., 2009) making them indeed well suited to preserve temporal information that is important for the encoding of the binau- ral cues to sound location (Joris and Yin, 1998; Tollin and Yin, 2005). Despite these extraordinary specializations for temporal fidelity, we hypothesize that MNTB neurons must also accurately code the shapes of the sound spectra at the ears over short time intervals in order to account for the abilities of LSO neurons to encode the frequency-dependent acoustic ILDs (Tollin and Yin, 2002a,b; Tollin et al., 2008; Tsai et al., 2010) and for animals such as cats to use these ILD cues to accurately and precisely localize high-frequency sound sources (Tollin et al., 2005, 2013; Moore et al., 2008; Gai et al., 2013; Ruhland et al., 2013). Here, a systems identification method, the Random Spectral Shape (RSS) technique (Yu and Young, 2000), was used to test the hypothesis that MNTB neurons and their inputs, the GBCs and auditory nerve fibers, encode stationary sound spectra linearly via their discharge rate. The RSS technique estimates the spectral weighting function that describes how spectra are linearly and non-linearly weighted to produce a discharge rate. Both GBC and MNTB neurons were well modeled by a linear weighting of sound spectra, consistent with previous reports in auditory nerve and other CN neurons (Yu and Young, 2000, 2013; Young and Calhoun, 2005). Together with the anatomical and biophysical specializations for timing in the neural circuits comprising the GBC, MNTB, and LSO, the mechanisms that produce accurate linear coding of spectral levels in these neurons may allow ILD cues to be coded for complex biologically-relevant stimuli with rapid spectrotemporally-modulated envelopes such as speech and animal vocalizations and moving sound sources. MATERIALS AND METHODS ANIMALS, APPARATUS, AND EXPERIMENTAL PROCEDURES All surgical and experimental procedures complied with the guidelines of the University of Colorado Anschutz Medical Campus Animal Care and Use Committee and the National Institutes of Health. Methods are based on those described in Tollin et al. (2008) and Tsai et al. (2010). Adult cats with clean external ears were initially anesthetized with ketamine hydrochlo- ride (20 mg/kg) along with acepromazine (0.1 mg/kg). Atropine sulfate (0.05 mg/kg) was also given to reduce mucous secre- tions, and a tracheal cannula was inserted. Supplemental doses of sodium pentobarbital (3–5 mg/kg) were administered intra- venously into the femoral vein as needed to maintain areflexia. Heart rate was continuously monitored as was core body temper- ature (with a rectal probe), the latter maintained with a heating pad at 37 ◦ C (Model TC 100, CWE, Inc., Ardmore, PA). Blood- oxygen levels, respiratory rate, and end-tidal CO 2 were measured continuously via a capnograph (Surgivet V90040, Waukesha, WI) and mean arterial blood pressure (femoral artery) was moni- tored with a pressure transducer (Harvard Apparatus research blood pressure transducer, Holliston, MA). Both pinnae were cut transversely, removed, and tight-fitting custom built hollow earpieces were fitted tightly into the external auditory meati. Polyethylene tubing (Intramedic, PE-90, 30 cm, 0.9 mm ID) was glued into a small hole made in each bulla to maintain normal middle ear pressure. The trapezoid body and the MNTB was approached ven- trally by drilling small holes into the basioccipital bone. Parylene-coated tungsten microelectrodes (1–2 M , Microprobe, Clarksburg, MD) were advanced ventromedially to dorsolater- ally at an angle of 26–30 ◦ into the brainstem by a microdrive (Kopf Model 662, Tujunga, CA) affixed to a micromanipulator that could be remotely advanced from outside the double-walled sound-attenuating chamber (Industrial Acoustics, Bronx, NY). Electrical activity was amplified (ISO-80, WPI, Sarasota, FL) and filtered (300–3000 Hz; Stanford Research Systems SRS 560, Sunnyvale, CA). Unit responses were discriminated with a BAK amplitude-time window discriminator (Model DDIS-1, Mount Airy, MD) and spike times were stored at a precision of 1 μ s via a Tucker-Davis Technologies (TDT, Alachua, FL) RV8. Stimuli: general All stimuli were generated digitally at 24-bit resolution and con- verted to analog at a nominal rate of 100 kHz by a TDT RX-6. Overall stimulus level to each ear was independently controlled in 1 dB steps using a pair of TDT PA-5s. The conditioned output of the D/A converter was sent to an acoustic assembly (one for each ear) comprising a TDT EC1 electrostatic speaker, a calibrated probe-tube microphone (Bruel and Kjaer Type 4182, Norcross, GA), and a hollow earpiece that was fit tightly into the cut end of the auditory meatus and sealed with petroleum jelly. The hol- low earpiece accommodated the small probe-tube microphone by which the sound delivery system to each ear was calibrated for tones between 50 Hz and 40 kHz in 50–100 Hz steps. The cal