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The physiological diversity of inhibitory neurons provides ample opportunity to influence a wide range of computational roles through their varied activity patterns, especially via feedback loops. In the avian auditory brain stem, inhibition originates primarily from the superior olivary nucleus (SON), and so it is critical to understand the intrinsic physiological properties and processing capabilities of these neurons. Neurons in the SON receive ascending input via the cochlear nuclei: directly from the intensity-coding cochlear nucleus angularis (NA) and indirectly via the interaural timing nucleus laminaris (NL), which itself receives input from cochlear nucleus magnocellularis (NM). Two distinct populations of SON neurons provide inhibitory feedback either to ipsilateral NA, NL, and the timing cochlear nucleus NM or to the contralateral SON. To determine whether these populations correspond to distinct response types, we investigated their electrophysiology in brain stem slices, using patch-clamp electrophysiology. We identified three phenotypes: single-spiking, chattering tonic, and regular tonic neurons. The two tonic phenotypes displayed distinct firing patterns and different membrane properties. Fluctuating "noisy" currents used to probe the capability of SON neurons to encode temporal features showed that each phenotype differed in sensitivity to temporally modulated input. By using cell fills and anatomical reconstructions, we could correlate the firing phenotypes with their axonal projection patterns. We found that SON axons exited via three fiber tracts, with each tract composed of specific phenotypes. These results provide a basis for understanding the role of specific inhibitory cell types in auditory function and elucidate the organization of the SON outputs. NEW & NOTEWORTHY Inhibitory inputs for the avian brain stem originate primarily from the superior olivary nucleus (SON). We describe three intrinsic phenotypes of SON neurons and show how they differ in their temporal processing and projection patterns. We propose that the two types of tonic firing neurons (including one novel type) and the single-spiking neurons in SON comprise separate feedback circuits that may differentially influence the auditory information flowing via the cochlear nuclei and nucleus laminaris.

Sound localization relies on minute differences in the timing and intensity of sound arriving at both ears. Neurons of the lateral superior olive (LSO) in the brainstem process these interaural disparities by precisely detecting excitatory and inhibitory synaptic inputs. Aging generally induces selective loss of inhibitory synaptic transmission along the entire auditory pathways, including the reduction of inhibitory afferents to LSO. Electrophysiological recordings in animals, however, reported only minor functional changes in aged LSO. The perplexing discrepancy between anatomical and physiological observations suggests a role for activity-dependent plasticity that would help neurons retain their binaural tuning function despite loss of inhibitory inputs. To explore this hypothesis, we use a computational model of LSO to investigate mechanisms underlying the observed functional robustness against age-related loss of inhibitory inputs. The LSO model is an integrate-and-fire type enhanced with a small amount of low-voltage activated potassium conductance and driven with (in)homogeneous Poissonian inputs. Without synaptic input loss, model spike rates varied smoothly with interaural time and level differences, replicating empirical tuning properties of LSO. By reducing the number of inhibitory afferents to mimic age-related loss of inhibition, overall spike rates increased, which negatively impacted binaural tuning performance, measured as modulation depth and neuronal discriminability. To simulate a recovery process compensating for the loss of inhibitory fibers, the strength of remaining inhibitory inputs was increased. By this modification, effects of inhibition loss on binaural tuning were considerably weakened, leading to an improvement of functional performance. These neuron-level observations were further confirmed by population modeling, in which binaural tuning properties of multiple LSO neurons were varied according to empirical measurements. These results demonstrate the plausibility that homeostatic plasticity could effectively counteract known age-dependent loss of inhibitory fibers in LSO and suggest that behavioral degradation of sound localization might originate from changes occurring more centrally., Competing Interests: The authors have declared that no competing interests exist.

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.

"Growing old" is the most common cause of hearing loss. Age-related hearing loss (ARHL) (presbycusis) first affects the ability to understand speech in background noise, even when auditory thresholds in quiet are normal. It has been suggested that cochlear denervation ("synaptopathy") is an early contributor to age-related auditory decline. In the present work, we characterized age-related cochlear synaptic degeneration and hair cell loss in mice with enhanced α9α10 cholinergic nicotinic receptors gating kinetics ("gain of function" nAChRs). These mediate inhibitory olivocochlear feedback through the activation of associated calcium-gated potassium channels. Cochlear function was assessed via distortion product otoacoustic emissions and auditory brainstem responses. Cochlear structure was characterized in immunolabeled organ of Corti whole mounts using confocal microscopy to quantify hair cells, auditory neurons, presynaptic ribbons, and postsynaptic glutamate receptors. Aged wild-type mice had elevated acoustic thresholds and synaptic loss. Afferent synapses were lost from inner hair cells throughout the aged cochlea, together with some loss of outer hair cells. In contrast, cochlear structure and function were preserved in aged mice with gain-of-function nAChRs that provide enhanced olivocochlear inhibition, suggesting that efferent feedback is important for long-term maintenance of inner ear function. Our work provides evidence that olivocochlear-mediated resistance to presbycusis-ARHL occurs via the α9α10 nAChR complexes on outer hair cells. Thus, enhancement of the medial olivocochlear system could be a viable strategy to prevent age-related hearing loss., Competing Interests: The authors declare no competing interest.

The auditory part of the brainstem is composed of several nuclei specialized in the computation of the different spectral and temporal features of the sound before it reaches the higher auditory regions. There are a high diversity of neuronal types in these nuclei, many with remarkable electrophysiological and synaptic properties unique to these structures. This diversity reflects specializations necessary to process the different auditory signals in order to extract precisely the acoustic information necessary for the auditory perception by the animal. Low threshold Kv1 channels and HCN channels are expressed in neurons that use timing clues for auditory processing, like bushy and octopus cells, in order to restrict action potential firing and reduce input resistance and membrane time constant. Kv3 channels allow principal neurons of the MNTB and pyramidal DCN neurons to fire fast trains of action potentials. Calcium channels on cartwheel DCN neurons produce complex spikes characteristic of these neurons. Calyceal synapses compensate the low input resistance of bushy and principal neurons of the MNTB by releasing hundreds of glutamate vesicles resulting in large EPSCs acting in fast ionotropic glutamate receptors, in order to reduce temporal summation of synaptic potentials, allowing more precise correspondence of pre- and post-synaptic potentials, and phase-locking. Pre-synaptic calyceal sodium channels have fast recovery from inactivation allowing extremely fast trains of action potential firing, and persistent sodium channels produce spontaneous activity of fusiform neurons at rest, which expands the dynamic range of these neurons. The unique combinations of different ion channels, ionotropic receptors and synaptic structures create a unique functional diversity of neurons extremely adapted to their complex functions in the auditory processing., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Neural morphology and membrane properties vary greatly between cell types in the nervous system. The computations and local circuit connectivity that neurons support are thought to be the key factors constraining the cells' biophysical properties. Nevertheless, additional constraints can be expected to further shape neuronal design. Here, we focus on a particularly energy-intense system (as indicated by metabolic markers): principal neurons in the medial superior olive (MSO) nucleus of the auditory brainstem. Based on a modeling approach, we show that a trade-off between the level of performance of a functionally relevant computation and energy consumption predicts optimal ranges for cell morphology and membrane properties. The biophysical parameters appear most strongly constrained by functional needs, while energy use is minimized as long as function can be maintained. The key factors that determine model performance and energy consumption are 1) the saturation of the synaptic conductance input and 2) the temporal resolution of the postsynaptic signals as they reach the soma, which is largely determined by active membrane properties. MSO cells seem to operate close to pareto optimality, i.e., the trade-off boundary between performance and energy consumption that is formed by the set of optimal models. Good performance for drastically lower costs could in theory be achieved by small neurons without dendrites, as seen in the avian auditory system, pointing to additional constraints for mammalian MSO cells, including their circuit connectivity., Competing Interests: The authors have declared that no competing interests exist.

Key Points: During the computation of sound localization, neurons of the lateral superior olive (LSO) integrate synaptic excitation arising from the ipsilateral ear with inhibition from the contralateral ear. We characterized the functional connectivity of the inhibitory and excitatory inputs onto LSO neurons in terms of unitary synaptic strength and convergence. Unitary IPSCs can generate large conductances, although their strength varies over a 10-fold range in a given recording. By contrast, excitatory inputs are relatively weak. The conductance associated with IPSPs needs to be at least 2-fold stronger than the excitatory one to guarantee effective inhibition of action potential (AP) firing. Computational modelling showed that strong unitary inhibition ensures an appropriate slope and midpoint of the tuning curve of LSO neurons. Conversely, weak but numerous excitatory inputs filter out spontaneous AP firing from upstream auditory neurons., Abstract: The lateral superior olive (LSO) is a binaural nucleus in the auditory brainstem in which excitation from the ipsilateral ear is integrated with inhibition from the contralateral ear. It is unknown whether the strength of the unitary inhibitory and excitatory inputs is adapted to allow for optimal tuning curves of LSO neuron action potential (AP) firing. Using electrical and optogenetic stimulation of afferent synapses, we found that the strength of unitary inhibitory inputs to a given LSO neuron can vary over a ∼10-fold range, follows a roughly log-normal distribution, and, on average, causes a large conductance (9 nS). Conversely, unitary excitatory inputs, stimulated optogenetically under the bushy-cell specific promoter Math5, were numerous, and each caused a small conductance change (0.7 nS). Approximately five to seven bushy cell inputs had to be active simultaneously to bring an LSO neuron to fire. In double stimulation experiments, the effective inhibition window caused by IPSPs was short (1-3 ms) and its length depended on the inhibitory conductance; an ∼2-fold stronger inhibition than excitation was needed to suppress AP firing. Computational modelling suggests that few, but strong, unitary IPSPs create a tuning curve of LSO neuron firing with an appropriate slope and midpoint. Furthermore, weak but numerous excitatory inputs reduce the spontaneous AP firing that LSO neurons would otherwise inherit from their upstream auditory neurons. Thus, the specific connectivity and strength of unitary excitatory and inhibitory inputs to LSO neurons is optimized for the computations performed by these binaural neurons., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

The integration of excitatory and inhibitory synaptic inputs is fundamental to neuronal processing. In the mammalian auditory brainstem, neurons compare excitatory and inhibitory inputs from the ipsilateral and contralateral ear, respectively, for sound localization. However, the temporal precision and functional roles of inhibition in this integration process are unclear. Here, we demonstrate by in vivo recordings from the lateral superior olive (LSO) that inhibition controls spiking with microsecond precision throughout high frequency click trains. Depending on the relative timing of excitation and inhibition, neuronal spike probability is either suppressed or-unexpectedly-facilitated. In vitro conductance-clamp LSO recordings establish that a reduction in the voltage threshold for spike initiation due to a prior hyperpolarization results in post-inhibitory facilitation of otherwise sub-threshold synaptic events. Thus, microsecond-precise differences in the arrival of inhibition relative to excitation can facilitate spiking in the LSO, thereby promoting spatial sensitivity during the processing of faint sounds.

During auditory development, changes in membrane properties promote the ability of excitatory neurons in the brain stem to code aspects of sound, including the level and timing of a stimulus. Some of these changes coincide with hearing onset, suggesting that sound-driven neural activity produces developmental plasticity of ion channel expression. While it is known that the coding properties of excitatory neurons are modulated by inhibition in the mature system, it is unknown whether there are also developmental changes in the membrane properties of brain stem inhibitory neurons. We investigated the primary source of inhibition in the avian auditory brain stem, the superior olivary nucleus (SON). The present studies test the hypothesis that, as in excitatory neurons, the membrane properties of these inhibitory neurons change after hearing onset. We examined SON neurons at different stages of auditory development: embryonic days 14-16 (E14-E16), a time at which cochlear ganglion neurons are just beginning to respond to sound; later embryonic stages (E18-E19); and after hatching (P0-P2). We used in vitro whole cell patch electrophysiology to explore physiological changes in SON. Age-related changes were observed at the level of a single spike and in multispiking behavior. In particular, tonic behavior, measured as a neuron's ability to sustain tonic firing over a range of current steps, became more common later in development. Voltage-clamp recordings and biophysical models were employed to examine how age-related increases in ion currents enhance excitability in SON. Our findings suggest that concurrent increases in sodium and potassium currents underlie the emergence of tonic behavior. NEW & NOTEWORTHY This article is the first to examine heterogeneity of neuronal physiology in the inhibitory nucleus of the avian auditory system and demonstrate that tonic firing here emerges over development. By pairing computer simulations with physiological data, we show that increases in both sodium and potassium channels over development are necessary for the emergence of tonic firing.

Background: In the mammalian superior olivary complex (SOC), synaptic inhibition contributes to the processing of binaural sound cues important for sound localization. Previous analyses demonstrated a tonotopic gradient for postsynaptic proteins mediating inhibitory neurotransmission in the lateral superior olive (LSO), a major nucleus of the SOC. To probe, whether a presynaptic molecular gradient exists as well, we investigated immunoreactivity against the vesicular inhibitory amino acid transporter (VIAAT) in the mouse auditory brainstem., Results: Immunoreactivity against VIAAT revealed a gradient in the LSO and the superior paraolivary nucleus (SPN) of NMRI mice, with high expression in the lateral, low frequency processing limb and low expression in the medial, high frequency processing limb of both nuclei. This orientation is opposite to the previously reported gradient of glycine receptors in the LSO. Other nuclei of the SOC showed a uniform distribution of VIAAT-immunoreactivity. No gradient was observed for the glycine transporter GlyT2 and the neuronal protein NeuN. Formation of the VIAAT gradient was developmentally regulated and occurred around hearing-onset between postnatal days 8 and 16. Congenital deaf Claudin14 -/- mice bred on an NMRI background showed a uniform VIAAT-immunoreactivity in the LSO, whereas cochlear ablation in NMRI mice after hearing-onset did not affect the gradient. Additional analysis of C57Bl6/J, 129/SvJ and CBA/J mice revealed a strain-specific formation of the gradient., Conclusions: Our results identify an activity-regulated gradient of VIAAT in the SOC of NRMI mice. Its absence in other mouse strains adds a novel layer of strain-specific features in the auditory system, i.e. tonotopic organization of molecular gradients. This calls for caution when comparing data from different mouse strains frequently used in studies involving transgenic animals. The presence of strain-specific differences offers the possibility of genetic mapping to identify molecular factors involved in activity-dependent developmental processes in the auditory system. This would provide an important step forward concerning improved auditory rehabilitation in cases of congenital deafness.

The trapezoid body (TB) contains axons of neurons in the anteroventral cochlear nucleus projecting to monaural and binaural nuclei in the superior olivary complex (SOC). Characterization of these monaural inputs is important for the interpretation of response properties of SOC neurons. In particular, understanding of the sensitivity to interaural time differences (ITDs) in neurons of the medial and lateral superior olive requires knowledge of the temporal firing properties of the monaural excitatory and inhibitory inputs to these neurons. In recent years, studies of ITD sensitivity of SOC neurons have made increasing use of small animal models with good low-frequency hearing, particularly the gerbil. We presented stimuli as used in binaural studies to monaural neurons in the TB and studied their temporal coding. We found that general trends as have been described in the cat are present in gerbil, but with some important differences. Phase-locking to pure tones tends to be higher in TB axons and in neurons of the medial nucleus of the TB (MNTB) than in the auditory nerve for neurons with characteristic frequencies (CFs) below 1 kHz, but this enhancement is quantitatively more modest than in cat. Stronger enhancement is common when TB neurons are stimulated at low frequencies below CF. It is rare for TB neurons in gerbil to entrain to low-frequency stimuli, i.e., to discharge a well-timed spike on every stimulus cycle. Also, complex phase-locking behavior, with multiple modes of increased firing probability per stimulus cycle, is common in response to low frequencies below CF. NEW & NOTEWORTHY Phase-locking is an important property of neurons in the early auditory pathway: it is critical for the sensitivity to time differences between the two ears enabling spatial hearing. Studies in cat have shown an improvement in phase-locking from the peripheral to the central auditory nervous system. We recorded from axons in an output tract of the cochlear nucleus and show that a similar but more limited form of temporal enhancement is present in gerbil., (Copyright © 2017 the American Physiological Society.)

Evidence indicates that CO 2 /H + -evoked ATP released from retrotrapezoid nucleus (RTN) astrocytes modulates the activity of CO 2 -sensitive neurons. RTN astrocytes also sense H + by inhibition of Kir4.1 channels; however, the relevance of this pH-sensitive current remains unclear since ATP release appears to involve CO 2 -dependent gating of connexin 26 hemichannels. Considering that depolarization mediated by H + inhibition of Kir4.1 channels is expected to increase sodium bicarbonate cotransporter (NBC) conductance and favor Ca 2+ influx via the sodium calcium exchanger (NCX), we hypothesize that depolarization in the presence of CO 2 is sufficient to facilitate ATP release and enhance respiratory output. Here, we confirmed that acute exposure to fluorocitrate (FCt) reversibly depolarizes RTN astrocytes and increased activity of RTN neurons by a purinergic-dependent mechanism. We then made unilateral injections of FCt into the RTN or two other putative chemoreceptor regions (NTS and medullary raphe) to depolarize astrocytes under control conditions and during P2-recepetor blockade while measuring cardiorespiratory activities in urethane-anesthetized, vagotomized, artificially ventilated male Wistar rats. Unilateral injection of FCt into the RTN increased phrenic (PNA) amplitude and frequency without changes in arterial pressure. Unilateral injection of pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS, a P2-receptor antagonist) into the RTN dampened both PNA amplitude and frequency responses to FCt. Injection of MRS2179 (P2Y1-receptor antagonist) into the RTN did not affect the FCt-induced respiratory responses. Fluorocitrate had no effect on breathing when injected into the NTS or raphe. These results suggest that depolarization can facilitate purinergic enhancement of respiratory drive from the RTN. NEW & NOTEWORTHY Astrocytes in the retrotrapezoid nucleus (RTN) are known to function as respiratory chemoreceptors; however, it is not clear whether changes in voltage contribute to astrocyte chemoreception. We showed that depolarization of RTN astrocytes at constant CO 2 levels is sufficient to modulate RTN chemoreception by a purinergic-dependent mechanism. These results support the possibility that astrocyte depolarization can facilitate purinergic enhancement of respiratory drive from the RTN., (Copyright © 2017 the American Physiological Society.)

The superior olivary complex (SOC) is a collection of brainstem auditory nuclei which play essential roles in the localization of sound sources, temporal coding of vocalizations and descending modulation of the cochlea. Notwithstanding, the SOC nuclei vary considerably between species in accordance with the auditory needs of the animal. The canine SOC was subjected to anatomical and physiological examination nearly 50 years ago and was then virtually forgotten. Herein, we aimed to characterize the nuclei of the canine SOC using quantitative morphometrics, estimation of neuronal number, histochemistry for perineuronal nets and immunofluorescence for the calcium binding proteins calbindin and calretinin. We found the principal nuclei to be extremely well developed: the lateral superior olive (LSO) contained over 20,000 neurons and the medial superior olive (MSO) contained over 15,000 neurons. In nearly all non-chiropterian terrestrial mammals, the MSO exists as a thin, vertical column of neurons. The canine MSO was folded into a U-shaped contour and had associated with the ventromedial tip a small, round collection of neurons we termed the tail nucleus of the MSO. Further, we found evidence within the LSO, MSO and medial nucleus of the trapezoid body (MNTB) for significant morphological variations along the mediolateral or rostrocaudal axes. Finally, the majority of MNTB neurons were calbindin-immunopositive and associated with calretinin-immunopositive calyceal terminals. Together, these observations suggest the canine SOC complies with the basic plan of the mammalian SOC but possesses a number of unique anatomical features., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Auditory streaming enables perception and interpretation of complex acoustic environments that contain competing sound sources. At early stages of central processing, sounds are segregated into separate streams representing attributes that later merge into acoustic objects. Streaming of temporal cues is critical for perceiving vocal communication, such as human speech, but our understanding of circuits that underlie this process is lacking, particularly at subcortical levels. The superior paraolivary nucleus (SPON), a prominent group of inhibitory neurons in the mammalian brainstem, has been implicated in processing temporal information needed for the segmentation of ongoing complex sounds into discrete events. The SPON requires temporally precise and robust excitatory input(s) to convey information about the steep rise in sound amplitude that marks the onset of voiced sound elements. Unfortunately, the sources of excitation to the SPON and the impact of these inputs on the behavior of SPON neurons have yet to be resolved. Using anatomical tract tracing and immunohistochemistry, we identified octopus cells in the contralateral cochlear nucleus (CN) as the primary source of excitatory input to the SPON. Cluster analysis of miniature excitatory events also indicated that the majority of SPON neurons receive one type of excitatory input. Precise octopus cell-driven onset spiking coupled with transient offset spiking make SPON responses well-suited to signal transitions in sound energy contained in vocalizations. Targets of octopus cell projections, including the SPON, are strongly implicated in the processing of temporal sound features, which suggests a common pathway that conveys information critical for perception of complex natural sounds.

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 K v 1, K v 3, and I H 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., (Copyright © 2016 the American Physiological Society.)

Pb exposure is associated with cognitive deficits including Attention Deficit Hyperactivity Disorder (ADHD) in children and alters auditory temporal processing in humans and animals. Serotonin has been implicated in auditory temporal processing and previous studies from our laboratory have demonstrated that developmental Pb decreases expression of serotonin (5-HT) in the adult murine lateral superior olive (LSO). During development, certain non-serotonergic sensory neurons, including auditory LSO neurons, transiently take up 5-HT through the serotonin reuptake transporter (SERT). The uptake of 5-HT is important for development of sensory systems. This study examines the effect of Pb on the serotonergic system in the LSO of the early postnatal mouse. Mice were exposed to moderate Pb (0.01mM) or high Pb (0.1mM) throughout gestation and postnatal day 4 (P4) and P8. We found that Pb exposure prolongs the normal developmental expression of 5-HT by LSO neurons and this is correlated with expression of SERT on LSO cell bodies. The prolonged expression of 5-HT by postnatal LSO neurons is correlated with decreased synaptic immunolabeling within the LSO. This Pb-associated decrease in synaptic density within the LSO could contribute to the auditory temporal processing deficits and cognitive deficits associated with developmental Pb exposure., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In mammals, the superior olivary complex (SOC) of the brainstem is composed of nuclei that integrate afferent auditory originating from both ears. Here, the expression of different calcium-binding proteins in subnuclei of the SOC was studied in distantly related mammals, the Mongolian gerbil (Meriones unguiculatus) and the gray short-tailed opossum (Monodelphis domestica) to get a better understanding of the basal nuclear organization of the SOC. Combined immunofluorescence labeling of the calcium-binding proteins (CaBPs) parvalbumin, calbindin-D28k, and calretinin as well as pan-neuronal markers displayed characteristic distribution patterns highlighting details of neuronal architecture of SOC nuclei. Parvalbumin was found in almost all neurons of SOC nuclei in both species, while calbindin and calretinin were restricted to specific cell types and axonal terminal fields. In both species, calbindin displayed a ubiquitous and mostly selective distribution in neurons of the medial nucleus of trapezoid body (MNTB) including their terminal axonal fields in different SOC targets. In Meriones, calretinin and calbindin showed non-overlapping expression patterns in neuron somata and terminal fields throughout the SOC. In Monodelphis, co-expression of calbindin and calretinin was observed in the MNTB, and hence both CaBPs were also co-localized in terminal fields within the adjacent SOC nuclei. The distribution patterns of CaBPs in both species are discussed with respect to the intrinsic neuronal SOC circuits as part of the auditory brainstem system that underlie the binaural integrative processing of acoustic signals as the basis for localization and discrimination of auditory objects.

Astrocytes form large gap junctional networks that contribute to ion and neurotransmitter homeostasis. Astrocytes concentrate in the lateral superior olive (LSO), a prominent auditory brainstem center. Compared to the LSO, astrocyte density is lower in the region dorsal to the LSO (dLSO) and in the internuclear space between the LSO, the superior paraolivary nucleus (SPN). We questioned whether astrocyte networks exhibit certain properties that reflect the precise neuronal arrangement. Employing whole-cell patch-clamp and concomitant injection of a gap junction-permeable tracer, we analyzed size and orientation of astrocyte networks in LSO, dLSO, and SPN-LSO in acute brainstem slices of mice at postnatal days 10-20. The majority of LSO networks exhibited an oval topography oriented orthogonally to the tonotopic axis, whereas dLSO networks showed no preferred orientation. This correlated with the overall astrocyte morphology in both regions, i.e. LSO astrocyte processes were oriented mainly orthogonally to the tonotopic axis. To assess the spread of small ions within LSO networks, we analyzed the diffusion of Na(+) signals between cells using Na(+) imaging. We found that Na(+) not only diffused between SR101(+) astrocytes, but also from astrocytes into SR101(-) cells. Using PLP-GFP mice for tracing, we could show that LSO networks contained astrocytes and oligodendrocytes. Together, our results demonstrate that LSO astrocytes and LSO oligodendrocytes form functional anisotropic panglial networks that are oriented predominantly orthogonally to the tonotopic axis. Thus, our results point toward an anisotropic ion and metabolite diffusion and a limited glial crosstalk between neighboring isofrequency bands in the LSO. GLIA 2016;64:1892-1911., (© 2016 Wiley Periodicals, Inc.)

The mammalian lateral superior olive (LSO) codes disparities in the intensity of the sound that reaches the two ears by integrating ipsilateral excitation and contralateral inhibition, but it remains unclear what particular neuron types convey acoustic information to the nucleus. It is also uncertain whether the known conspicuous morphofunctional differences and gradients along the tonotopic axis of the LSO relate to qualitative and/or quantitative regional differences in its afferents. To clarify these issues, we made small, single injections of the neuroanatomical tracer biotinylated dextran amine (BDA) into different tonotopic regions of the LSO of albino rats and analyzed the neurons labeled retrogradely in brainstem auditory nuclei. We demonstrate that the LSO is innervated tonotopically by four brainstem neuron types: spherical bushy cells and planar multipolar neurons of the ipsilateral ventral cochlear nucleus, principal neurons of the ipsilateral medial nucleus of the trapezoid body, and small multipolar neurons of the contralateral ventral nucleus of the trapezoid body. Unexpectedly, the proportion of labeled neurons of each type was virtually identical in all cases, thus indicating that all tonotopic regions of the LSO receive a similar combination of inputs. Even more surprisingly, our data also suggest that the representation of frequencies in the LSO differs from that of the nuclei that innervate it: compared to the latter nuclei, the LSO seems to possess a relatively larger portion of its volume devoted to processing frequencies in the lower-middle part of the spectrum, and a relative smaller portion devoted to higher frequencies. J. Comp. Neurol. 524:2230-2250, 2016. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

The ongoing activity of neurons generates a spatially and time-varying field of extracellular voltage (Ve). This Ve field reflects population-level neural activity, but does it modulate neural dynamics and the function of neural circuits? We provide a cable theory framework to study how a bundle of model neurons generates Ve and how this Ve feeds back and influences membrane potential (Vm). We find that these "ephaptic interactions" are small but not negligible. The model neural population can generate Ve with millivolt-scale amplitude, and this Ve perturbs the Vm of "nearby" cables and effectively increases their electrotonic length. After using passive cable theory to systematically study ephaptic coupling, we explore a test case: the medial superior olive (MSO) in the auditory brain stem. The MSO is a possible locus of ephaptic interactions: sounds evoke large (millivolt scale)Vein vivo in this nucleus. The Ve response is thought to be generated by MSO neurons that perform a known neuronal computation with submillisecond temporal precision (coincidence detection to encode sound source location). Using a biophysically based model of MSO neurons, we find millivolt-scale ephaptic interactions consistent with the passive cable theory results. These subtle membrane potential perturbations induce changes in spike initiation threshold, spike time synchrony, and time difference sensitivity. These results suggest that ephaptic coupling may influence MSO function., (Copyright © 2016 the American Physiological Society.)

During development GABA and glycine synapses are initially excitatory before they gradually become inhibitory. This transition is due to a developmental increase in the activity of neuronal potassium-chloride cotransporter 2 (KCC2), which shifts the chloride equilibrium potential (ECl) to values more negative than the resting membrane potential. While the role of early GABA and glycine depolarizations in neuronal development has become increasingly clear, the role of the transition to hyperpolarization in synapse maturation and circuit refinement has remained an open question. Here we investigated this question by examining the maturation and developmental refinement of GABA/glycinergic and glutamatergic synapses in the lateral superior olive (LSO), a binaural auditory brain stem nucleus, in KCC2-knockdown mice, in which GABA and glycine remain depolarizing. We found that many key events in the development of synaptic inputs to the LSO, such as changes in neurotransmitter phenotype, strengthening and elimination of GABA/glycinergic connection, and maturation of glutamatergic synapses, occur undisturbed in KCC2-knockdown mice compared with wild-type mice. These results indicate that maturation of inhibitory and excitatory synapses in the LSO is independent of the GABA and glycine depolarization-to-hyperpolarization transition., (Copyright © 2016 the American Physiological Society.)

Little is known about the genetic pathways and transcription factors that control development and maturation of central auditory neurons. En1, a gene expressed by a subset of developing and mature superior olivary complex (SOC) cells, encodes a homeodomain transcription factor important for neuronal development in the midbrain, cerebellum, hindbrain and spinal cord. Using genetic fate-mapping techniques, we show that all En1-lineal cells in the SOC are neurons and that these neurons are glycinergic, cholinergic and GABAergic in neurotransmitter phenotype. En1 deletion does not interfere with specification or neural fate of these cells, but does cause aberrant positioning and subsequent death of all En1-lineal SOC neurons by early postnatal ages. En1-null cells also fail to express the transcription factor FoxP1, suggesting that FoxP1 lies downstream of En1. Our data define important roles for En1 in the development and maturation of a diverse group of brainstem auditory neurons., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Electrical synapses formed by gap junctions composed of connexin36 (Cx36) are widely distributed in the mammalian central nervous system (CNS). Here, we used immunofluorescence methods to document the expression of Cx36 in the cochlear nucleus and in various structures of the auditory pathway of rat and mouse. Labeling of Cx36 visualized exclusively as Cx36-puncta was densely distributed primarily on the somata and initial dendrites of neuronal populations in the ventral cochlear nucleus, and was abundant in superficial layers of the dorsal cochlear nucleus. Other auditory centers displaying Cx36-puncta included the medial nucleus of the trapezoid body (MNTB), regions surrounding the lateral superior olivary nucleus, the dorsal nucleus of the medial lemniscus, the nucleus sagulum, all subnuclei of the inferior colliculus, and the auditory cerebral cortex. In EGFP-Cx36 transgenic mice, EGFP reporter was detected in neurons located in each of auditory centers that harbored Cx36-puncta. In the ventral cochlear nuclei and the MNTB, many neuronal somata were heavily innervated by nerve terminals containing vesicular glutamate transporter-1 (vglut1) and Cx36 was frequently localized at these terminals. Cochlear ablation caused a near total depletion of vglut1-positive terminals in the ventral cochlear nuclei, with a commensurate loss of labeling for Cx36 around most neuronal somata, but preserved Cx36-puncta at somatic neuronal appositions. The results suggest that electrical synapses formed by Cx36-containing gap junctions occur in most of the widely distributed centers of the auditory system. Further, it appears that morphologically mixed chemical/electrical synapses formed by nerve terminals are abundant in the ventral cochlear nucleus, including those at endbulbs of Held formed by cochlear primary afferent fibers, and those at calyx of Held synapses on MNTB neurons., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neurons in the superior olivary complex (SOC) integrate excitatory and inhibitory inputs to localize sounds in space. The majority of these inhibitory inputs have been thought to arise within the SOC from the medial nucleus of the trapezoid body (MNTB). However, recent work demonstrates that glycinergic innervation of the SOC persists in Egr2; En1(CKO) mice that lack MNTB neurons, suggesting that there are other sources of this innervation (Jalabi et al., 2013). To study the development of MNTB- and non-MNTB-derived glycinergic SOC innervation, we compared immunostaining patterns of glycine transporter 2 (GlyT2) at several postnatal ages in control and Egr2; En1(CKO) mice. GlyT2 immunostaining was present at birth (P0) in controls and reached adult levels by P7 in the superior paraolivary nucleus (SPN) and by P12 in the lateral superior olive (LSO). In Egr2; En1(CKO) mice, glycinergic innervation of the LSO developed at a similar rate but was delayed by one week in the SPN. Conversely, consistent reductions in the number of GlyT2(+) boutons located on LSO somata were seen at all ages in Egr2; En1(CKO) mice, while these numbers reached control levels in the SPN by adulthood. Dendritic localization of GlyT2+ boutons was unaltered in both the LSO and SPN of adult Egr2; En1(CKO) mice. On the postsynaptic side, adult Egr2; En1(CKO) mice had reduced glycine receptor α1 (GlyRα1) expression in the LSO but normal levels in the SPN. GlyRα2 was not expressed by LSO or SPN neurons in either genotype. These findings contribute important information for understanding the development of MNTB- and non-MNTB-derived glycinergic pathways to the mouse SOC.

Behavioral and neural findings demonstrate that animals can locate low-frequency sounds along the azimuth by detecting microsecond interaural time differences (ITDs). Information about ITDs is also available in the amplitude modulations (i.e., envelope) of high-frequency sounds. Since medial superior olivary (MSO) neurons encode low-frequency ITDs, we asked whether they employ a similar mechanism to process envelope ITDs with high-frequency carriers, and the effectiveness of this mechanism compared with the process of low-frequency sound. We developed a novel hybrid in vitro dynamic-clamp approach, which enabled us to mimic synaptic input to brain-slice neurons in response to virtual sound and to create conditions that cannot be achieved naturally but are useful for testing our hypotheses. For each simulated ear, a virtual sound, computer generated, was used as input to a computational auditory-nerve model. Model spike times were converted into synaptic input for MSO neurons, and ITD tuning curves were derived for several virtual-sound conditions: low-frequency pure tones, high-frequency tones modulated with two types of envelope, and speech sequences. Computational models were used to verify the physiological findings and explain the biophysical mechanism underlying the observed ITD coding. Both recordings and simulations indicate that MSO neurons are sensitive to ITDs carried by spectrotemporally complex virtual sounds, including speech tokens. Our findings strongly suggest that MSO neurons can encode ITDs across a broad-frequency spectrum using an input-slope-based coincidence-detection mechanism. Our data also provide an explanation at the cellular level for human localization performance involving high-frequency sound described by previous investigators., (Copyright © 2014 the American Physiological Society.)