Molecular and functional profiling of cell diversity and identity in the lateral superior olive, an auditory brainstem center with ascending and descending projections.

The lateral superior olive (LSO), a prominent integration center in the auditory brainstem, contains a remarkably heterogeneous population of neurons. Ascending neurons, predominantly principal neurons (pLSOs), process interaural level differences for sound localization. Descending neurons (lateral olivocochlear neurons, LOCs) provide feedback into the cochlea and are thought to protect against acoustic overload. The molecular determinants of the neuronal diversity in the LSO are largely unknown. Here, we used patch-seq analysis in mice at postnatal days P10-12 to classify developing LSO neurons according to their functional and molecular profiles. Across the entire sample (n = 86 neurons), genes involved in ATP synthesis were particularly highly expressed, confirming the energy expenditure of auditory neurons. Two clusters were identified, pLSOs and LOCs. They were distinguished by 353 differentially expressed genes (DEGs), most of which were novel for the LSO. Electrophysiological analysis confirmed the transcriptomic clustering. We focused on genes affecting neuronal input-output properties and validated some of them by immunohistochemistry, electrophysiology, and pharmacology. These genes encode proteins such as osteopontin, Kv11.3, and Kvß3 (pLSO-specific), calcitonin-gene-related peptide (LOC-specific), or Kv7.2 and Kv7.3 (no DEGs). We identified 12 "Super DEGs" and 12 genes showing "Cluster similarity." Collectively, we provide fundamental and comprehensive insights into the molecular composition of individual ascending and descending neurons in the juvenile auditory brainstem and how this may relate to their specific functions, including developmental aspects.

Tonotopic distribution and inferior colliculus projection pattern of inhibitory and excitatory cell types in the lateral superior olive of mice.

The principal neurons (PNs) of the lateral superior olive nucleus (LSO) are an important component of mammalian brainstem circuits that compare activity between the two ears and extract intensity and timing differences used for sound localization. There are two LSO PN transmitter types, glycinergic and glutamatergic, which also have different ascending projection patterns to the inferior colliculus (IC). Glycinergic LSO PNs project ipsilaterally while glutamatergic one's projections vary in laterality by species. In animals with good low-frequency hearing (<3 kHz) such as cats and gerbils, glutamatergic LSO PNs have both ipsilateral and contralateral projections; however, rats that lack this ability only have the contralateral pathway. Additionally, in gerbils, the glutamatergic ipsilateral projecting LSO PNs are biased to the low-frequency limb of the LSO suggesting this pathway may be an adaptation for low-frequency hearing. To further test this premise, we examined the distribution and IC projection pattern of LSO PNs in another high-frequency specialized species using mice by combining in situ hybridization and retrograde tracer injections. We observed no overlap between glycinergic and glutamatergic LSO PNs confirming they are distinct cell populations in mice as well. We found that mice also lack the ipsilateral glutamatergic projection from LSO to IC and that their LSO PN types do not exhibit pronounced tonotopic biases. These data provide insights into the cellular organization of the superior olivary complex and its output to higher processing centers that may underlie functional segregation of information.

Interaural frequency mismatch jointly modulates neural brainstem binaural interaction and behavioral interaural time difference sensitivity in humans.

The binaural interaction component (BIC) of the auditory brainstem response (ABR) is the difference obtained after subtracting the sum of right and left ear ABRs from binaurally evoked ABRs. The BIC has attracted interest as a biomarker of binaural processing abilities. Best binaural processing is presumed to require spectrally-matched inputs at the two ears, but peripheral pathology and/or impacts of hearing devices can lead to mismatched inputs. Such mismatching can degrade behavioral sensitivity to interaural time difference (ITD) cues, but might be detected using the BIC. Here, we examine the effect of interaural frequency mismatch (IFM) on BIC and behavioral ITD sensitivity in audiometrically normal adult human subjects (both sexes). Binaural and monaural ABRs were recorded and BICs computed from subjects in response to narrowband tones. Left ear stimuli were fixed at 4000 Hz while right ear stimuli varied over a ∼2-octave range (re: 4000 Hz). Separately, subjects performed psychophysical lateralization tasks using the same stimuli to determine ITD discrimination thresholds jointly as a function of IFM and sound level. Results demonstrated significant effects of IFM on BIC amplitudes, with lower amplitudes in mismatched conditions than frequency-matched. Behavioral ITD discrimination thresholds were elevated at mismatched frequencies and lower sound levels, but also more sharply modulated by IFM at lower sound levels. Combinations of ITD, IFM and overall sound level that resulted in fused and lateralized percepts were bound by the empirically-measured BIC, and also by model predictions simulated using an established computational model of the brainstem circuit thought to generate the BIC.

Listen to the Brain-Auditory Sound Source Localization in Neuromorphic Computing Architectures.

Conventional processing of sensory input often relies on uniform sampling leading to redundant information and unnecessary resource consumption throughout the entire processing pipeline. Neuromorphic computing challenges these conventions by mimicking biology and employing distributed event-based hardware. Based on the task of lateral auditory sound source localization (SSL), we propose a generic approach to map biologically inspired neural networks to neuromorphic hardware. First, we model the neural mechanisms of SSL based on the interaural level difference (ILD). Afterward, we identify generic computational motifs within the model and transform them into spike-based components. A hardware-specific step then implements them on neuromorphic hardware. We exemplify our approach by mapping the neural SSL model onto two platforms, namely the IBM TrueNorth Neurosynaptic System and SpiNNaker. Both implementations have been tested on synthetic and real-world data in terms of neural tunings and readout characteristics. For synthetic stimuli, both implementations provide a perfect readout (100% accuracy). Preliminary real-world experiments yield accuracies of 78% (TrueNorth) and 13% (SpiNNaker), RMSEs of 41∘ and 39∘, and MAEs of 18∘ and 29∘, respectively. Overall, the proposed mapping approach allows for the successful implementation of the same SSL model on two different neuromorphic architectures paving the way toward more hardware-independent neural SSL.

The lateral superior olive in the mouse: Two systems of projecting neurons.

The lateral superior olive (LSO) is a key structure in the central auditory system of mammals that exerts efferent control on cochlear sensitivity and is involved in the processing of binaural level differences for sound localization. Understanding how the LSO contributes to these processes requires knowledge about the resident cells and their connections with other auditory structures. We used standard histological stains and retrograde tracer injections into the inferior colliculus (IC) and cochlea in order to characterize two basic groups of neurons: (1) Principal and periolivary (PO) neurons have projections to the IC as part of the ascending auditory pathway; and (2) lateral olivocochlear (LOC) intrinsic and shell efferents have descending projections to the cochlea. Principal and intrinsic neurons are intermixed within the LSO, exhibit fusiform somata, and have disk-shaped dendritic arborizations. The principal neurons have bilateral, symmetric, and tonotopic projections to the IC. The intrinsic efferents have strictly ipsilateral projections, known to be tonotopic from previous publications. PO and shell neurons represent much smaller populations (<10% of principal and intrinsic neurons, respectively), have multipolar somata, reside outside the LSO, and have non-topographic, bilateral projections. PO and shell neurons appear to have widespread projections to their targets that imply a more diffuse modulatory function. The somata and dendrites of principal and intrinsic neurons form a laminar matrix within the LSO and share quantifiably similar alignment to the tonotopic axis. Their restricted projections emphasize the importance of frequency in binaural processing and efferent control for auditory perception. This study addressed and expanded on previous findings of cell types, circuit laterality, and projection tonotopy in the LSO of the mouse.

Tonotopic distribution and inferior colliculus projection pattern of inhibitory and excitatory cell types in the lateral superior olive of Mongolian gerbils.

Sound localization critically relies on brainstem neurons that compare information from the two ears. The conventional role of the lateral superior olive (LSO) is extraction of intensity differences; however, it is increasingly clear that relative timing, especially of transients, is also an important function. Cellular diversity within the LSO that is not well understood may underlie its multiple roles. There are glycinergic inhibitory and glutamatergic excitatory principal neurons in the LSO, however, there is some disagreement regarding their relative distribution and projection pattern. Here we employ in situ hybridization to definitively identify transmitter types combined with retrograde labeling of projections to the inferior colliculus (IC) to address these questions. Excitatory LSO neurons were more numerous (76%) than inhibitory ones. A smaller proportion of inhibitory neurons were IC-projecting (45% vs. 64% for excitatory) suggesting that inhibitory LSO neurons may have more projections to other regions such the lateral lemniscus or more distributed IC projections. Inhibitory LSO neurons almost exclusively projected ipsilaterally making up a sizeable proportion (41%) of the transmitter type-labeled ipsilateral IC projection from LSO and exhibited a moderate low frequency bias (10% difference H-L). Two thirds of excitatory neurons projected contralaterally and had a slight high frequency bias (4%). One third of excitatory LSO neurons projected ipsilaterally to the IC and these cells were strongly biased toward the low frequency limb of the LSO (37%). This projection appears to be species specific in animals with good low frequency hearing suggesting that it may be a specialization for such ability.

Corrigendum: The lateral superior olive in the mouse: Two systems of projecting neurons.

[This corrects the article DOI: 10.3389/fncir.2022.1038500.].

Ketamine-xylazine anesthesia depth affects auditory neuronal responses in the lateral superior olive complex of the gerbil.

Studies of in vivo neuronal responses to auditory inputs in the superior olive complex (SOC) are usually done under anesthesia. However, little attention has been paid to the effect of anesthesia itself on response properties. Here, we assessed the effect of anesthesia depth under ketamine-xylazine anesthetics on auditory evoked response properties of lateral SOC neurons. Anesthesia depth was tracked by monitoring EEG spectral peak frequencies. An increase in anesthesia depth led to a decrease of spontaneous discharge activities and an elevated response threshold. The temporal responses to suprathreshold tones were also affected, with adapted responses reduced but peak responses unaffected. Deepening the anesthesia depth also increased first spike latency. However, spike jitter was not affected. Auditory brainstem responses to clicks confirmed that ketamine-xylazine anesthesia depth affects auditory neuronal activities and the effect on spike rate and spike timing persists through the auditory pathway. We concluded from those observations that ketamine-xylazine affects lateral SOC response properties depending on the anesthesia depth.NEW & NOTEWORTHY We studied how the depth of ketamine-xylazine anesthesia altered response properties of lateral superior olive complex neurons, and auditory brainstem evoked responses. Our results provide direct evidence that anesthesia depth affects auditory neuronal responses and reinforce the notion that both the anesthetics and the anesthesia depth should be considered when interpreting/comparing in vivo neuronal recordings.

Altered Gap Junction Network Topography in Mouse Models for Human Hereditary Deafness.

Anisotropic gap junctional coupling is a distinct feature of astrocytes in many brain regions. In the lateral superior olive (LSO), astrocytic networks are anisotropic and oriented orthogonally to the tonotopic axis. In CaV1.3 knock-out (KO) and otoferlin KO mice, where auditory brainstem nuclei are deprived from spontaneous cochlea-driven neuronal activity, neuronal circuitry is disturbed. So far it was unknown if this disturbance is also accompanied by an impaired topography of LSO astrocyte networks. To answer this question, we immunohistochemically analyzed the expression of astrocytic connexin (Cx) 43 and Cx30 in auditory brainstem nuclei. Furthermore, we loaded LSO astrocytes with the gap junction-permeable tracer neurobiotin and assessed the network shape and orientation. We found a strong elevation of Cx30 immunoreactivity in the LSO of CaV1.3 KO mice, while Cx43 levels were only slightly increased. In otoferlin KO mice, LSO showed a slight increase in Cx43 as well, whereas Cx30 levels were unchanged. The total number of tracer-coupled cells was unaltered and most networks were anisotropic in both KO strains. In contrast to the WTs, however, LSO networks were predominantly oriented parallel to the tonotopic axis and not orthogonal to it. Taken together, our data demonstrate that spontaneous cochlea-driven neuronal activity is not required per se for the formation of anisotropic LSO astrocyte networks. However, neuronal activity is required to establish the proper orientation of networks. Proper formation of LSO astrocyte networks thus necessitates neuronal input from the periphery, indicating a critical role of neuron-glia interaction during early postnatal development in the auditory brainstem.

Role of GluA3 AMPA Receptor Subunits in the Presynaptic and Postsynaptic Maturation of Synaptic Transmission and Plasticity of Endbulb-Bushy Cell Synapses in the Cochlear Nucleus.

The AMPA receptor (AMPAR) subunit GluA3 has been suggested to shape synaptic transmission and activity-dependent plasticity in endbulb-bushy cell synapses (endbulb synapses) in the anteroventral cochlear nucleus, yet the specific roles of GluA3 in the synaptic transmission at endbulb synapses remains unexplored. Here, we compared WT and GluA3 KO mice of both sexes and identified several important roles of GluA3 in the maturation of synaptic transmission and short-term plasticity in endbulb synapses. We show that GluA3 largely determines the ultrafast kinetics of endbulb synapses glutamatergic currents by promoting the insertion of postsynaptic AMPARs that contain fast desensitizing flop subunits. In addition, GluA3 is also required for the normal function, structure, and development of the presynaptic terminal which leads to altered short term-depression in GluA3 KO mice. The presence of GluA3 reduces and slows synaptic depression, which is achieved by lowering the probability of vesicle release, promoting efficient vesicle replenishment, and increasing the readily releasable pool of synaptic vesicles. Surprisingly, GluA3 also makes the speed of synaptic depression rate-invariant. We propose that the slower and rate-invariant speed of depression allows an initial response window that still contains presynaptic firing rate information before the synapse is depressed. Because this response window is rate-invariant, GluA3 extends the range of presynaptic firing rates over which rate information in bushy cells can be preserved. This novel role of GluA3 may be important to allowing the postsynaptic targets of spherical bushy cells in mice use rate information for encoding sound intensity and sound localization.SIGNIFICANCE STATEMENT We report novel roles of the glutamate receptor subunit GluA3 in synaptic transmission in synapses between auditory nerve fibers and spherical bushy cells (BCs) in the cochlear nucleus. We show that GluA3 contributes to the generation of ultrafast glutamatergic currents at these synapses, which is important to preserve temporal information about the sound. Furthermore, we demonstrate that GluA3 contributes to the normal function and development of the presynaptic terminal, whose properties shape short-term plasticity. GluA3 slows and attenuates synaptic depression, and makes it less dependent on the presynaptic firing rates. This may help BCs to transfer information about the high rates of activity that occur at the synapse in vivo to postsynaptic targets that use rate information for sound localization.

Cytoarchitecture of the superior olivary complex of three neotropical species of bats (Noctilio leporinus, Phyllostomus hastatus and Carollia perspicillata) with different foraging behavior / Citoarquitetura do complexo olivar superior de três espécies neotropicais de morcegos (Noctilio leporinus, Phyllostomus hastatus e Carollia perspicillata) com diferentes comportamentos de forrageamento

Abstract The understanding of the echolocation by studying different auditory nuclei of echolocating bats can be an important link in elucidating questions arising in relation to their foraging behavior. The superior olivary complex (SOC) is the primary center for processing the binaural cues used in sound localization since echo locating bats rely on acoustic cues to navigate and capture prey while in flight. The present study was taken to test the hypothesis that the SOC of echolocating neotropical bats with different foraging behavior will exhibit morphological variations in relative size, degree of complexity and spatial distribution. The brains were collected from six male adult bats of each species: Noctilio leporinus (fish eating), Phyllostomus hastatus (carnivorous/omnivorous) and Carollia perspicillata (fruit eating). They were double-embedded and transverse serial sections were cut and stained with cresyl fast violet. The SOC measured as 640 ± 70 µm in the N. leporinus bat, 480 ± 50 µm in the P. hastatus and 240 ± 30 µm in the C. perspicillata bat. The principal nuclei of the SOC of in all three bats were the LSO, MSO and MNTB. The MSO and LSO were very well developed in N. leporinus bats. The MSO of N. leporinus bat subdivided into DMSO and VMSO. The main cell type of cells present in MSO and LSO are dark staining multipolar cells in all the bats studied. The well-developed MSO and LSO of N. leporinus bats indicate that these bats are highly sensitive to low frequency sounds and interaural intensity differences, which help these bats to forage over water by using various types of echolocation signals. The average size of SOC in P. hastatus and C. perspicillata bats can be attributed to the fact that these bats use vision and smell along with echolocation to forage the food.

Resumo O entendimento da ecolocalização pelo estudo de diferentes núcleos auditivos de morcegos pode ser um elo importante na elucidação das inúmeras questões que surgem em relação ao seu comportamento de forrageamento. O complexo olivar superior (SOC) é o principal centro de processamento das pistas binaurais usadas na localização do som, já que os morcegos ecolocalizadores contam com sinais acústicos para navegar e capturar as presas durante o vôo. O presente estudo foi realizado para testar a hipótese de que morcegos que usam a ecolocalização para diferentes comportamentos de forrageamento irão variar na estrutura, tamanhos relativos e grau de complexidade e distribuição espacial do grupo SOC. Os cérebros foram coletados de seis machos adultos de morcego de cada espécie: Noctilio leporinus (piscívoro), Phyllostomus hastatus (carnívoros/onívoros) e Carollia perspicillata (frugívoro). Eles foram seccionados em série e transversalmente, cortados e corados com coloração rápida cresil-violeta. tolet. O grupo SOC foi medido como 640 ± 70 µm no morcego N. leporinus, 480 ± 50 µm no P. hastatus e 240 ± 30 µm no morcego C. perspicillata. Os principais núcleos do grupo SOC dos três morcegos foram o LSO e o MSO e o MNTB. O MSO e o LSO foram muito bem desenvolvidos em morcegos N. leporinus. A MSO de N. leporinus foi subdividida em DMSO e VMSO. O principal tipo de células presentes na MSO e LSO são as células multipolares de coloração escura em todos os morcegos. Os MSO bem desenvolvidos e LSO de morcegos N. leporinus indicam que estes morcegos são altamente sensíveis a sons de baixa frequência e diferenças de intensidade interaural, que ajudaram estes morcegos a se alimentarem na superfície da água usando vários tipos de sinais de ecolocalização. O tamanho médio de SOC em morcegos de P. hastatus e C. perspicillata pode ser atribuído ao fato destes morcegos usarem visão e olfato junto com a ecolocalização para forragear.

Glycinergic Transmission in the Presence and Absence of Functional GlyT2: Lessons From the Auditory Brainstem.

Synaptic transmission is controlled by re-uptake systems that reduce transmitter concentrations in the synaptic cleft and recycle the transmitter into presynaptic terminals. The re-uptake systems are thought to ensure cytosolic concentrations in the terminals that are sufficient for reloading empty synaptic vesicles (SVs). Genetic deletion of glycine transporter 2 (GlyT2) results in severely disrupted inhibitory neurotransmission and ultimately to death. Here we investigated the role of GlyT2 at inhibitory glycinergic synapses in the mammalian auditory brainstem. These synapses are tuned for resilience, reliability, and precision, even during sustained high-frequency stimulation when endocytosis and refilling of SVs probably contribute substantially to efficient replenishment of the readily releasable pool (RRP). Such robust synapses are formed between MNTB and LSO neurons (medial nucleus of the trapezoid body, lateral superior olive). By means of patch-clamp recordings, we assessed the synaptic performance in controls, in GlyT2 knockout mice (KOs), and upon acute pharmacological GlyT2 blockade. Via computational modeling, we calculated the reoccupation rate of empty release sites and RRP replenishment kinetics during 60-s challenge and 60-s recovery periods. Control MNTB-LSO inputs maintained high fidelity neurotransmission at 50 Hz for 60 s and recovered very efficiently from synaptic depression. During 'marathon-experiments' (30,600 stimuli in 20 min), RRP replenishment accumulated to 1,260-fold. In contrast, KO inputs featured severe impairments. For example, the input number was reduced to ~1 (vs. ~4 in controls), implying massive functional degeneration of the MNTB-LSO microcircuit and a role of GlyT2 during synapse maturation. Surprisingly, neurotransmission did not collapse completely in KOs as inputs still replenished their small RRP 80-fold upon 50 Hz | 60 s challenge. However, they totally failed to do so for extended periods. Upon acute pharmacological GlyT2 inactivation, synaptic performance remained robust, in stark contrast to KOs. RRP replenishment was 865-fold in marathon-experiments, only ~1/3 lower than in controls. Collectively, our empirical and modeling results demonstrate that GlyT2 re-uptake activity is not the dominant factor in the SV recycling pathway that imparts indefatigability to MNTB-LSO synapses. We postulate that additional glycine sources, possibly the antiporter Asc-1, contribute to RRP replenishment at these high-fidelity brainstem synapses.

Intrinsic and Miniature Postsynaptic Current Changes in Rat Principal Neurons of the Lateral Superior Olive after Unilateral Auditory Deprivation at an Early Age.

Unilateral auditory deprivation results in lateralization changes in the central auditory system, interfering with the integration of binaural information and thereby leading to a decrease in binaural auditory functions such as sound localization. Principal neurons of the lateral superior olive (LSO) are responsible for computing the interaural intensity differences that are critical for sound localization in the horizontal plane. To investigate changes caused by unilateral auditory deprivation, electrophysiological activity was recorded from LSO principal neurons in control rats and rats with unilateral cochlear ablation. At one week after unilateral cochlear ablation, the excitability of LSO principal neurons on the side ipsilateral to the ablation (the ablated side) was greater than that on the side contralateral to the ablation (the intact side); however, the input resistance increased on both sides. Furthermore, by analysing the miniature inhibitory postsynaptic currents and miniature excitatory postsynaptic currents, we found that unilateral auditory deprivation weakened the inhibitory driving force on the intact side, whereas it strengthened the excitatory driving force on the ablated side. In summary, asymmetric changes in the electrophysiological activity of LSO principal neurons were found on both sides at postnatal day 19, one week after unilateral cochlear ablation.

A Vector-Based Method to Analyze the Topography of Glial Networks.

Anisotropy of tracer-coupled networks is a hallmark in many brain regions. In the past, the topography of these networks was analyzed using various approaches, which focused on different aspects, e.g., position, tracer signal, or direction of coupled cells. Here, we developed a vector-based method to analyze the extent and preferential direction of tracer spreading. As a model region, we chose the lateral superior olive-a nucleus that exhibits specialized network topography. In acute slices, sulforhodamine 101-positive astrocytes were patch-clamped and dialyzed with the GJ-permeable tracer neurobiotin, which was subsequently labeled with avidin alexa fluor 488. A predetermined threshold was used to differentiate between tracer-coupled and tracer-uncoupled cells. Tracer extent was calculated from the vector means of tracer-coupled cells in four 90° sectors. We then computed the preferential direction using a rotating coordinate system and post hoc fitting of these results with a sinusoidal function. The new method allows for an objective analysis of tracer spreading that provides information about shape and orientation of GJ networks. We expect this approach to become a vital tool for the analysis of coupling anisotropy in many brain regions.

An auditory brainstem nucleus as a model system for neuronal metabolic demands.

The correlation between neuronal activity and metabolism is essential for coding, plasticity, neurological disorders and the interpretation of functional neuroimaging data. Most likely, metabolic requirements depend upon neuron type, and macroscopic energy demands vary with brain region. However, specific needs of individual neuron types are enigmatic. Therefore, we monitored metabolic activity in the lateral superior olive (LSO), an auditory brainstem nucleus containing only one neuron type. LSO neurons exhibit extreme but well-described biophysics with firing rates of several hundred hertz and low input resistances of a few megaohms. We recorded changes in NADH and flavin adenine dinucleotide (FAD) autofluorescence and O2 concentration in acute brainstem slices of Mongolian gerbils (Meriones unguiculatus) following electrical stimulation. The LSO shows the typical biphasic NADH/FAD response up to a physiologically relevant frequency of 400 Hz. In the same animal, we compared the LSO with the hippocampal CA1 region and the cerebral cortex. The rate of NADH/FADH2 consumption and regeneration was slowest in LSO. However, frequency dependence was only similar during the consumption phase but varied during regeneration within the three brain regions. Changes in NADH, FAD and O2 levels and blocking metabolic reactions indicate a pronounced contribution of mitochondrial oxidative phosphorylation in the LSO which is known for the other brain regions as well. Lactate transport and interconversion are involved in LSO metabolism as we found in immunohistochemical and pharmacological experiments. Our findings show that the LSO represents an apt, biophysically distinct model for brain metabolism and that neuronal properties determine metabolic needs.

Mice Lacking the Alpha9 Subunit of the Nicotinic Acetylcholine Receptor Exhibit Deficits in Frequency Difference Limens and Sound Localization.

Sound processing in the cochlea is modulated by cholinergic efferent axons arising from medial olivocochlear neurons in the brainstem. These axons contact outer hair cells in the mature cochlea and inner hair cells during development and activate nicotinic acetylcholine receptors composed of α9 and α10 subunits. The α9 subunit is necessary for mediating the effects of acetylcholine on hair cells as genetic deletion of the α9 subunit results in functional cholinergic de-efferentation of the cochlea. Cholinergic modulation of spontaneous cochlear activity before hearing onset is important for the maturation of central auditory circuits. In α9KO mice, the developmental refinement of inhibitory afferents to the lateral superior olive is disturbed, resulting in decreased tonotopic organization of this sound localization nucleus. In this study, we used behavioral tests to investigate whether the circuit anomalies in α9KO mice correlate with sound localization or sound frequency processing. Using a conditioned lick suppression task to measure sound localization, we found that three out of four α9KO mice showed impaired minimum audible angles. Using a prepulse inhibition of the acoustic startle response paradigm, we found that the ability of α9KO mice to detect sound frequency changes was impaired, whereas their ability to detect sound intensity changes was not. These results demonstrate that cholinergic, nicotinic α9 subunit mediated transmission in the developing cochlear plays an important role in the maturation of hearing.

Enhanced Excitatory Connectivity and Disturbed Sound Processing in the Auditory Brainstem of Fragile X Mice.

Hypersensitivity to sounds is one of the prevalent symptoms in individuals with Fragile X syndrome (FXS). It manifests behaviorally early during development and is often used as a landmark for treatment efficacy. However, the physiological mechanisms and circuit-level alterations underlying this aberrant behavior remain poorly understood. Using the mouse model of FXS (Fmr1 KO), we demonstrate that functional maturation of auditory brainstem synapses is impaired in FXS. Fmr1 KO mice showed a greatly enhanced excitatory synaptic input strength in neurons of the lateral superior olive (LSO), a prominent auditory brainstem nucleus, which integrates ipsilateral excitation and contralateral inhibition to compute interaural level differences. Conversely, the glycinergic, inhibitory input properties remained unaffected. The enhanced excitation was the result of an increased number of cochlear nucleus fibers converging onto one LSO neuron, without changing individual synapse properties. Concomitantly, immunolabeling of excitatory ending markers revealed an increase in the immunolabeled area, supporting abnormally elevated excitatory input numbers. Intrinsic firing properties were only slightly enhanced. In line with the disturbed development of LSO circuitry, auditory processing was also affected in adult Fmr1 KO mice as shown with single-unit recordings of LSO neurons. These processing deficits manifested as an increase in firing rate, a broadening of the frequency response area, and a shift in the interaural level difference function of LSO neurons. Our results suggest that this aberrant synaptic development of auditory brainstem circuits might be a major underlying cause of the auditory processing deficits in FXS.SIGNIFICANCE STATEMENT Fragile X Syndrome (FXS) is the most common inheritable form of intellectual impairment, including autism. A core symptom of FXS is extreme sensitivity to loud sounds. This is one reason why individuals with FXS tend to avoid social interactions, contributing to their isolation. Here, a mouse model of FXS was used to investigate the auditory brainstem where basic sound information is first processed. Loss of the Fragile X mental retardation protein leads to excessive excitatory compared with inhibitory inputs in neurons extracting information about sound levels. Functionally, this elevated excitation results in increased firing rates, and abnormal coding of frequency and binaural sound localization cues. Imbalanced early-stage sound level processing could partially explain the auditory processing deficits in FXS.

Developmental Emergence of Phenotypes in the Auditory Brainstem Nuclei of Fmr1 Knockout Mice.

Fragile X syndrome (FXS), the most common monogenic cause of autism, is often associated with hypersensitivity to sound. Several studies have shown abnormalities in the auditory brainstem in FXS; however, the emergence of these auditory phenotypes during development has not been described. Here, we investigated the development of phenotypes in FXS model [Fmr1 knockout (KO)] mice in the ventral cochlear nucleus (VCN), medial nucleus of the trapezoid body (MNTB), and lateral superior olive (LSO). We studied features of the brainstem known to be altered in FXS or Fmr1 KO mice, including cell size and expression of markers for excitatory (VGLUT) and inhibitory (VGAT) synapses. We found that cell size was reduced in the nuclei with different time courses. VCN cell size is normal until after hearing onset, while MNTB and LSO show decreases earlier. VGAT expression was elevated relative to VGLUT in the Fmr1 KO mouse MNTB by P6, before hearing onset. Because glial cells influence development and are altered in FXS, we investigated their emergence in the developing Fmr1 KO brainstem. The number of microglia developed normally in all three nuclei in Fmr1 KO mice, but we found elevated numbers of astrocytes in Fmr1 KO in VCN and LSO at P14. The results indicate that some phenotypes are evident before spontaneous or auditory activity, while others emerge later, and suggest that Fmr1 acts at multiple sites and time points in auditory system development.

Cytoarchitecture of the superior olivary complex of three neotropical species of bats (Noctilio leporinus, Phyllostomus hastatus and Carollia perspicillata) with different foraging behavior

Abstract The understanding of the echolocation by studying different auditory nuclei of echolocating bats can be an important link in elucidating questions arising in relation to their foraging behavior. The superior olivary complex (SOC) is the primary center for processing the binaural cues used in sound localization since echo locating bats rely on acoustic cues to navigate and capture prey while in flight. The present study was taken to test the hypothesis that the SOC of echolocating neotropical bats with different foraging behavior will exhibit morphological variations in relative size, degree of complexity and spatial distribution. The brains were collected from six male adult bats of each species: Noctilio leporinus (fish eating), Phyllostomus hastatus (carnivorous/omnivorous) and Carollia perspicillata (fruit eating). They were double-embedded and transverse serial sections were cut and stained with cresyl fast violet. The SOC measured as 640 ± 70 µm in the N. leporinus bat, 480 ± 50 µm in the P. hastatus and 240 ± 30 µm in the C. perspicillata bat. The principal nuclei of the SOC of in all three bats were the LSO, MSO and MNTB. The MSO and LSO were very well developed in N. leporinus bats. The MSO of N. leporinus bat subdivided into DMSO and VMSO. The main cell type of cells present in MSO and LSO are dark staining multipolar cells in all the bats studied. The well-developed MSO and LSO of N. leporinus bats indicate that these bats are highly sensitive to low frequency sounds and interaural intensity differences, which help these bats to forage over water by using various types of echolocation signals. The average size of SOC in P. hastatus and C. perspicillata bats can be attributed to the fact that these bats use vision and smell along with echolocation to forage the food.

Resumo O entendimento da ecolocalização pelo estudo de diferentes núcleos auditivos de morcegos pode ser um elo importante na elucidação das inúmeras questões que surgem em relação ao seu comportamento de forrageamento. O complexo olivar superior (SOC) é o principal centro de processamento das pistas binaurais usadas na localização do som, já que os morcegos ecolocalizadores contam com sinais acústicos para navegar e capturar as presas durante o vôo. O presente estudo foi realizado para testar a hipótese de que morcegos que usam a ecolocalização para diferentes comportamentos de forrageamento irão variar na estrutura, tamanhos relativos e grau de complexidade e distribuição espacial do grupo SOC. Os cérebros foram coletados de seis machos adultos de morcego de cada espécie: Noctilio leporinus (piscívoro), Phyllostomus hastatus (carnívoros/onívoros) e Carollia perspicillata (frugívoro). Eles foram seccionados em série e transversalmente, cortados e corados com coloração rápida cresil-violeta. tolet. O grupo SOC foi medido como 640 ± 70 µm no morcego N. leporinus, 480 ± 50 µm no P. hastatus e 240 ± 30 µm no morcego C. perspicillata. Os principais núcleos do grupo SOC dos três morcegos foram o LSO e o MSO e o MNTB. O MSO e o LSO foram muito bem desenvolvidos em morcegos N. leporinus. A MSO de N. leporinus foi subdividida em DMSO e VMSO. O principal tipo de células presentes na MSO e LSO são as células multipolares de coloração escura em todos os morcegos. Os MSO bem desenvolvidos e LSO de morcegos N. leporinus indicam que estes morcegos são altamente sensíveis a sons de baixa frequência e diferenças de intensidade interaural, que ajudaram estes morcegos a se alimentarem na superfície da água usando vários tipos de sinais de ecolocalização. O tamanho médio de SOC em morcegos de P. hastatus e C. perspicillata pode ser atribuído ao fato destes morcegos usarem visão e olfato junto com a ecolocalização para forragear.

Different tonotopic regions of the lateral superior olive receive a similar combination of afferent inputs.

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.