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Drumming is a non-vocal auditory display producing airborne as well as seismic vibrations by tapping body extremities on a surface. It is mostly described as an alarm signal but is also discussed to signal dominance or mating quality. To clarify the function of drumming in Mongolian gerbils (Meriones unguiculatus), we compared the occurrence of drumming during predator, opposite-sex and same-sex encounters. We tested 48 captive Mongolian gerbils (Meriones unguiculatus) in three experiments. In predator experiments, subjects were exposed alone or with their cagemate to aerial and terrestrial predator dummies. In social encounter experiments, familiar and unfamiliar male-female dyads and same-sex dyads were confronted. For the same-sex encounters, a dominance index was calculated for each subject based on the number of won and lost conflicts. Drumming and drumming-call combinations were counted, and a multi-parametric sound analysis was performed. In all experiments drumming and drumming-call combinations occurred. In predator experiments, more subjects drummed when confronted with the predator stimulus than in the habituation phase. In social encounter experiments, more subjects drummed when facing an unfamiliar than a familiar conspecific. In addition, the accompanying call type and body posture of the sender differed between experiments. Thus, we suggest that whereas drumming signals an increased arousal state of the sender, the accompanying call type and the body posture signal context specific information., (© 2024. The Author(s).)

Based on the auditory periphery and the small head size, Etruscan shrews (Suncus etruscus) approximate ancestral mammalian conditions. The auditory brainstem in this insectivore has not been investigated. Using labelling techniques, we assessed the structures of their superior olivary complex (SOC) and the nuclei of the lateral lemniscus (NLL). There, we identified the position of the major nuclei, their input pattern, transmitter content, expression of calcium binding proteins (CaBPs) and two voltage-gated ion channels. The most prominent SOC structures were the medial nucleus of the trapezoid body (MNTB), the lateral nucleus of the trapezoid body (LNTB), the lateral superior olive (LSO) and the superior paraolivary nucleus (SPN). In the NLL, the ventral (VNLL), a specific ventrolateral VNLL (VNLLvl) cell population, the intermediate (INLL) and dorsal (DNLL) nucleus, as well as the inferior colliculus's central aspect were discerned. INLL and VNLL were clearly separated by the differential distribution of various marker proteins. Most labelled proteins showed expression patterns comparable to rodents. However, SPN neurons were glycinergic and not GABAergic and the overall CaBPs expression was low. Next to the characterisation of the Etruscan shrew's auditory brainstem, our work identifies conserved nuclei and indicates variable structures in a species that approximates ancestral conditions., (© 2024. The Author(s).)

The dorsal nucleus of the lateral lemniscus (DNLL) is a GABAergic, reciprocally connected auditory brainstem structure that continues to develop postnatally in rodents. One key feature of the DNLL is the generation of a strong, prolonged, ionotropic, GABA A receptor-mediated inhibition. Possible GABA B receptor-mediated signalling is unexplored in the DNLL. Here, we used Mongolian gerbils of either sex to describe GABA B receptor-mediated modulation of postsynaptic potassium currents and synaptic inputs in postnatal (P) animals of days 10/11 and 23-28. Throughout development, we observed the presence of a Baclofen-activated GABA B receptor-enhanced potassium outward conductance that is capable of suppressing action potential generation. In P10/11, old gerbils GABA B receptor activation enhances glutamatergic and suppresses ionotropic GABAergic synaptic transmission. During development, this differential modulation becomes less distinct, because in P22-28, old animals Baclofen-activated GABA B receptors rather enhance ionotropic GABAergic synaptic transmission, whereas glutamatergic transmission is both enhanced and suppressed. Blocking GABA B receptors causes an increase in ionotropic GABAergic transmission in P10/11 old gerbils that was independent on stimulation frequency but depended on the type of short-term plasticity. Together with the lack of Baclofen-induced changes in the synaptic paired-pulse ratio of either input type, we suggest that GABA B receptor-mediated modulation is predominantly postsynaptic and activates different signalling cascades. Thus, we argue that in DNLL neurons, the GABA B receptor is a post-synaptically located signalling hub that alters signalling cascades during development for distinct targets., (© 2024 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

The evolution of novel motor behaviors requires modifications in the central pattern generators (CPGs) controlling muscle activity. How such changes gradually lead to novel behaviors remains enigmatic due to the long time course of evolution. Rattlesnakes provide a unique opportunity to investigate how a locomotor CPG was evolutionarily modified to generate a novel behavior-in this case, acoustic signaling. We show that motoneurons (MNs) in the body and tail spinal cord of rattlesnakes possess fundamentally different physiological characteristics, which allow MNs in the tail to integrate and transmit CPG output for controlling superfast muscles with high temporal precision. Using patch-clamp electrophysiology, we demonstrate that these differences in locomotor and rattle MNs are mainly determined by KV7 2/3 potassium channels. However, although KV7 2/3 exerted a significantly different influence on locomotor and rattle MN physiology, single-cell RNA-seq unexpectedly did not reveal any differences in KV7 2/3 channels' expression. VIDEO ABSTRACT., Competing Interests: Declaration of interests The authors declare no conflict of interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Intracerebral drug delivery is an experimental approach for the treatment of drug-resistant epilepsies that allows for pharmacological intervention in targeted brain regions. Previous studies have shown that targeted pharmacological inhibition of the subthalamic nucleus (STN) via modulators of the GABAergic system produces antiseizure effects. However, with chronic treatment, antiseizure effects are lost as tolerance develops. Here, we report that chronic intrasubthalamic microinfusion of valproate (VPA), an antiseizure medication known for its wide range of mechanisms of action, can produce long-lasting antiseizure effects over three weeks in rats. In the intravenous pentylenetetrazole seizure-threshold test, seizure thresholds were determined before and during chronic VPA application (480 μg/d, 720 μg/d, 960 μg/d) to the bilateral STN. Results indicate a dose-dependent variation in VPA-induced antiseizure effects with mean increases in seizure threshold of up to 33%, and individual increases of up to 150%. The lowest VPA dose showed a complete lack of tolerance development with long-lasting antiseizure effects. Behavioral testing with all doses revealed few, acceptable adverse effects. VPA concentrations were high in STN and low in plasma and liver. In vitro electrophysiology with bath applied VPA revealed a reduction in spontaneous firing rate, increased background membrane potential, decreased input resistance and a significant reduction in peak NMDA, but not AMPA, receptor currents in STN neurons. Our results suggest an advantage of VPA over purely GABAergic modulators in preventing tolerance development with chronic intrasubthalamic drug delivery and provide first mechanistic insights in intracerebral pharmacotherapy targeting the STN., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

In the brain, microglia are involved in immune responses and synaptic maturation. During early development, these cells invade the brain, proliferate, and morphologically mature to achieve coverage of the surrounding tissue with their fine processes. Their developmental proliferation overlaps with the postnatal development of neuronal circuits. Within the superior olivary complex (SOC), an auditory brainstem structure, microglia, and their early postnatal development have been documented. A quantification over the full developmental profile of the arrangement and morphological changes in single microglia cells is missing. Here, we used immunofluorescence labeling to quantify their distribution, morphological changes, and coverage during early and late postnatal development in the SOC of Mongolian gerbils. Microglia distributed rather homogenously within each nucleus with a bias to the nucleus borders at postnatal day (P) 5 and more centrally in the nucleus in mature stages. We found a nucleus-specific transient increase in microglia cell number and density reaching its peak at P17 with a subsequent decline to P55 values. Length and branching of microglia protrusions increased especially after P12. The stronger ramification together with the increase in cell density allows coverage of the surrounding tissue from P5 to mature stages, despite the large developmental increase in nucleus size. The transient increase in density during synaptic refinement in SOC nuclei suggests that microglia are important during the pruning period, compensating for developmental increase in tissue volume, and that in mature stages their main function appears tissue surveillance., (© 2023 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

The lateral lemniscus encompasses processing stages for binaural hearing, suppressing spurious frequencies and frequency integration. Within the lemniscal fibres three nuclei can be identified, termed after their location as dorsal, intermediate and ventral nucleus of the lateral lemniscus (DNLL, INLL and VNLL). While the DNLL and VNLL have been functionally and anatomically characterized, less is known about INLL neurons. Here, we quantitatively describe the morphology, the cellular orientation and distribution of synaptic contact sites along dendrites in mature Mongolian gerbils. INLL neurons are largely non-inhibitory and morphologically heterogeneous with an overall perpendicular orientation regarding the lemniscal fibers. Dendritic ranges are heterogeneous and can extend beyond the nucleus border. INLL neurons receive VGluT1/2 containing glutamatergic and a mix of GABA- and glycinergic inputs distributed over the entire dendrite. Input counts suggest that numbers of excitatory exceed the inhibitory contact sites. Axonal projections indicate connectivity to ascending and descending auditory structures. Our data show that INLL neurons form a morphologically heterogeneous continuum and incoming auditory information is processed on thin dendrites of various length and biased to perpendicular orientation. Together with the different axonal projection patterns, this indicates that the INLL is a highly complex structure that might hold many unexplored auditory functions., (© 2023. Springer Nature Limited.)

In mammals, common rules for the encoding of arousal and physical characteristics of the sender are suggested based on a similar vocal production apparatus. In this study, we want to investigate to what extent vocalizations of developing Mongolian gerbil pups fulfill these rules. We recorded vocalizations of 28 Mongolian gerbil pups in four developmental stages using a separation paradigm, suggested to induce different arousal levels. For low arousal, a pup was placed in an arena isolated from its siblings and parents; for high arousal, the pup was additionally stressed through the simulation of a predator. An unsupervised cluster analysis revealed three call types: ultrasonic (USV), audible vocalizations (ADV), and transitions between both (USV-ADV). The USV and USV-ADV rate showed an age-dependent decrease, contrasting an age-dependent increase for ADVs. Vocal correlates for the encoding of arousal were found for USVs and of physical characteristics for USVs and ADVs. However, the pattern of encoding these cues differed between call types and only partly confirmed the common rules suggested for mammals. Our results show that divergent encoding patterns do not only differ between species but also between call types within a species, indicating that coding rules can be shaped by socio-ecological factors or call type specific production mechanisms.

Comparative analysis of evolutionarily conserved neuronal circuits between phylogenetically distant mammals highlights the relevant mechanisms and specific adaptations to information processing. The medial nucleus of the trapezoid body (MNTB) is a conserved mammalian auditory brainstem nucleus relevant for temporal processing. While MNTB neurons have been extensively investigated, a comparative analysis of phylogenetically distant mammals and the spike generation is missing. To understand the suprathreshold precision and firing rate, we examined the membrane, voltage-gated ion channel and synaptic properties in Phyllostomus discolor (bat) and in Meriones unguiculatus (rodent) of either sex. Between the two species, the membrane properties of MNTB neurons were similar at rest with only minor differences, while larger dendrotoxin (DTX)-sensitive potassium currents were found in gerbils. Calyx of Held-mediated EPSCs were smaller and frequency dependence of short-term plasticity (STP) less pronounced in bats. Simulating synaptic train stimulations in dynamic clamp revealed that MNTB neurons fired with decreasing success rate near conductance threshold and at increasing stimulation frequency. Driven by STP-dependent conductance decrease, the latency of evoked action potentials increased during train stimulations. The spike generator showed a temporal adaptation at the beginning of train stimulations that can be explained by sodium current inactivation. Compared with gerbils, the spike generator of bats sustained higher frequency input-output functions and upheld the same temporal precision. Our data mechanistically support that MNTB input-output functions in bats are suited to sustain precise high-frequency rates, while for gerbils, temporal precision appears more relevant and an adaptation to high output-rates can be spared. SIGNIFICANCE STATEMENT Neurons in the mammalian medial nucleus of the trapezoid body (MNTB) convey precise, faithful inhibition vital for binaural hearing and gap detection. The MNTB's structure and function appear evolutionarily well conserved. We compared the cellular physiology of MNTB neurons in bat and gerbil. Because of their adaptations to echolocation or low frequency hearing both species are model systems for hearing research, yet with largely overlapping hearing ranges. We find that bat neurons sustain information transfer with higher ongoing rates and precision based on synaptic and biophysical differences in comparison to gerbils. Thus, even in evolutionarily conserved circuits species-specific adaptations prevail, highlighting the importance for comparative research to differentiate general circuit functions and their specific adaptations., (Copyright © 2023 the authors.)

Bats are a natural reservoir for many viruses and are considered to play an important role in the interspecies transmission of viruses. To analyze the susceptibility of bat airway cells to infection by viruses of other mammalian species, we developed an airway organoid culture model derived from airways of Carollia perspicillata. Application of specific antibodies for fluorescent staining indicated that the cell composition of organoids resembled those of bat trachea and lungs as determined by immunohistochemistry. Infection studies indicated that Carollia perspicillata bat airway organoids (AOs) from the trachea or the lung are highly susceptible to infection by two different porcine influenza A viruses. The bat AOs were also used to develop an air-liquid interface (ALI) culture system of filter-grown epithelial cells. Infection of these cells showed the same characteristics, including lower virulence and enhanced replication and release of the H1N1/2006 virus compared to infection with H3N2/2007. These observations agreed with the results obtained by infection of porcine ALI cultures with these two virus strains. Interestingly, lectin staining indicated that bat airway cells only contain a small amount of alpha 2,6-linked sialic acid, the preferred receptor determinant for mammalian influenza A viruses. In contrast, large amounts of alpha 2,3-linked sialic acid, the preferred receptor determinant for avian influenza viruses, are present in bat airway epithelial cells. Therefore, bat airway cells may be susceptible not only to mammalian but also to avian influenza viruses. Our culture models, which can be extended to other parts of the airways and to other species, provide a promising tool to analyze virus infectivity and the transmission of viruses both from bats to other species and from other species to bats. IMPORTANCE We developed an organoid culture system derived from the airways of the bat species Carollia perspicillata . Using this cell system, we showed that the airway epithelium of these bats is highly susceptible to infection by influenza viruses of other mammalian species and thus is not a barrier for interspecies transmission. These organoids provide an almost unlimited supply of airway epithelial cells that can be used to generate well-differentiated epithelial cells and perform infection studies. The establishment of the organoid model required only three animals, and can be extended to other epithelia (nose, intestine) as well as to other species (bat and other animal species). Therefore, organoids promise to be a valuable tool for future zoonosis research on the interspecies transmission of viruses (e.g., bat → intermediate host → human).

The structure of the mammalian auditory brainstem is evolutionarily highly plastic, and distinct nuclei arrange in a species-dependent manner. Such anatomical variability is present in the superior olivary complex (SOC) and the nuclei of the lateral lemniscus (LL). Due to the structure-function relationship in the auditory brainstem, the identification of individual nuclei supports the understanding of sound processing. Here, we comparatively describe the nucleus arrangement and the expression of functional markers in the auditory brainstem of the two bat species Phyllostomus discolor and Carollia perspicillata. Using immunofluorescent labeling, we describe the arrangement and identity of the SOC and LL nuclei based on the expression of synaptic markers (vesicular glutamate transporter 1 and glycine transporter 2), calcium-binding proteins, as well as the voltage-gated ion channel subunits Kv1.1 and HCN1. The distribution of excitatory and inhibitory synaptic labeling appears similar between both species and matches with that of other mammals. The detection of calcium-binding proteins indicates species-dependent differences and deviations from other mammals. Kv1.1 and HCN1 show largely the same expression pattern in both species, which diverges from other mammals, indicating functional adaptations in the cellular physiology of bat neurons., (© 2022 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

Large glutamatergic, somatic synapses mediate temporally precise information transfer. In the ventral nucleus of the lateral lemniscus, an auditory brainstem nucleus, the signal of an excitatory large somatic synapse is sign inverted to generate rapid feedforward inhibition with high temporal acuity at sound onsets, a mechanism involved in the suppression of spurious frequency information. The mechanisms of the synaptically driven input-output functions in the ventral nucleus of the lateral lemniscus are not fully resolved. Here, we show in Mongolian gerbils of both sexes that, for stimulation frequencies up to 200 Hz, the EPSC kinetics together with short-term plasticity allow for faithful transmission with only a small increase in latency. Glutamatergic currents are exclusively mediated by AMPARs and NMDARs. Short-term plasticity is frequency-dependent and composed of an initial facilitation followed by depression. Physiologically relevant output generation is limited by the decrease in synaptic conductance through short-term plasticity (STP). At this endbulb synapse, STP acts as a low pass filter and increases the dynamic range of the conductance dependent input-output relation, while NMDAR signaling slightly increases the sensitivity of the input-output function. Our computational model shows that STP-mediated filtering limits the intensity dependence of the spike output, thus maintaining selectivity to sound transients. Our results highlight the interaction of cellular features that together give rise to the computations in the circuit. SIGNIFICANCE STATEMENT Auditory information processing in the brainstem is a prerequisite for generating our auditory representation of the environment. Thereby, many processing steps rely on temporally precise filtering. Precise feedforward inhibition is a key motif in auditory brainstem processing and produced through sign inversion at several large somatic excitatory synapses. A particular feature of the ventral nucleus of the lateral lemniscus is to produce temporally precise onset inhibition with little temporal variance independent of sound intensity. Our cell-physiology and modeling data explain how the synaptic characteristics of different current components and their short-term plasticity are tuned to establish sound intensity-invariant onset inhibition that is crucial for filtering out spurious frequency information., (Copyright © 2022 the authors.)

A high prevalence of AA-amyloidosis was identified in a breeding colony of northern tree shrews ( Tupaia belangeri ) in a retrospective analysis, with amyloid deposits in different organs being found in 26/36 individuals (72%). Amyloid deposits, confirmed by Congo red staining, were detected in kidneys, intestines, skin, and lymph nodes, characteristic of systemic amyloidosis. Immunohistochemically, the deposited amyloid was intensely positive with anti-AA-antibody (clone mc4), suggesting AA-amyloidosis. The kidneys were predominantly affected (80%), where amyloid deposits ranged from mild to severe and was predominantly located in the renal medulla. In addition, many kidneys contained numerous cysts with atrophy of the renal parenchyma. There was no significant association between concurrent neoplastic or inflammatory processes and amyloidosis. The lack of distinctive predisposing factors suggests a general susceptibility of captive T. belangeri to develop amyloidosis. Clinical and laboratory findings of a female individual with pronounced kidney alterations were indicative of renal failure. The observed tissue tropism with pronounced kidney alterations, corresponding renal dysfunction, and an overall high prevalence suggests amyloidosis as an important disease in captive tree shrews.

To counterbalance long-term environmental changes, neuronal circuits adapt the processing of sensory information. In the auditory system, ongoing background noise drives long-lasting adaptive mechanism in binaural coincidence detector neurons in the superior olive. However, the compensatory cellular mechanisms of the binaural neurons in the medial superior olive (MSO) to long-term background changes are unexplored. Here we investigated the cellular properties of MSO neurons during long-lasting adaptations induced by moderate omnidirectional noise exposure. After noise exposure, the input resistance of MSO neurons of mature Mongolian gerbils was reduced, likely due to an upregulation of hyperpolarisation-activated cation and low voltage-activated potassium currents. Functionally, the long-lasting adaptations increased the action potential current threshold and facilitated high frequency output generation. Noise exposure accelerated the occurrence of spontaneous postsynaptic currents. Together, our data suggest that cellular adaptations in coincidence detector neurons of the MSO to continuous noise exposure likely increase the sensitivity to differences in sound pressure levels.

Neurons in the medial superior olive (MSO) detect 10 µs differences in the arrival times of a sound at the two ears. Such acuity requires exquisitely precise integration of binaural synaptic inputs. There is substantial understanding of how neuronal phase locking of afferent MSO structures, and MSO membrane biophysics subserve such high precision. However, we still lack insight into how the entirety of excitatory inputs is integrated along the MSO dendrite under sound stimulation. To understand how the dendrite integrates excitatory inputs as a whole, we combined anatomic quantifications of the afferent innervation in gerbils of both sexes with computational modeling of a single cell. We present anatomic data from confocal and transmission electron microscopy showing that single afferent fibers follow a single dendrite mostly up to the soma and contact it at multiple (median 4) synaptic sites, each containing multiple independent active zones (the overall density of active zones is estimated as 1.375 per μm 2 ). Thus, any presynaptic action potential may elicit temporally highly coordinated synaptic vesicle release at tens of active zones, thereby achieving secure transmission. Computer simulations suggest that such an anatomic arrangement boosts the amplitude and sharpens the time course of excitatory postsynaptic potentials by reducing current sinks and more efficiently recruiting subthreshold potassium channels. Both effects improve binaural coincidence detection compared with single large synapses at the soma. Our anatomic data further allow for estimation of a lower bound of 7 and an upper bound of 70 excitatory fibers per dendrite. SIGNIFICANCE STATEMENT Passive dendritic propagation attenuates the amplitude of postsynaptic potentials and widens their temporal spread. Neurons in the medial superior olive, with their large bilateral dendrites, however, can detect coincidence of binaural auditory inputs with submillisecond precision, a computation that is in stark contrast to passive dendritic processing. Here, we show that dendrites can counteract amplitude attenuation and even decrease the temporal spread of postsynaptic potentials, if active subthreshold potassium conductances are triggered in temporal coordination along the whole dendrite. Our anatomic finding that axons run in parallel to the dendrites and make multiple synaptic contacts support such coordination since incoming action potentials would depolarize the dendrite at multiple sites within a brief time interval., (Copyright © 2021 the authors.)

The auditory system relies on temporal precise information transfer, requiring an interplay of synchronously activated inputs and rapid postsynaptic integration. During late postnatal development synaptic, biophysical, and morphological features change to enable mature auditory neurons to perform their appropriate function. How the number of minimal required input fibers and the relevant EPSC time course integrated for action potential generation changes during late postnatal development is unclear. To answer these questions, we used in vitro electrophysiology in auditory brainstem structures from pre-hearing onset and mature Mongolian gerbils of either sex. Synaptic and biophysical parameters changed distinctively during development in the medial nucleus of the trapezoid body (MNTB), the medial superior olive (MSO), and the ventral and dorsal nucleus of the lateral lemniscus (VNLL and DNLL). Despite a reduction in input resistance in most cell types, all required fewer inputs in the mature stage to drive action potentials. Moreover, the EPSC decay time constant is a good predictor of the EPSC time used for action potential generation in all nuclei but the VNLL. Only in MSO neurons, the full EPSC time course is integrated by the neuron's resistive element, while otherwise, the relevant EPSC time matches only 5-10% of the membrane time constant, indicating membrane charging as a dominant role for output generation. We conclude, that distinct developmental programs lead to a general increase in temporal precision and integration accuracy matched to the information relaying properties of the investigated nuclei., (Copyright © 2020 Kladisios, Fischer and Felmy.)

G M1 -gangliosidosis is caused by a reduced activity of β-galactosidase ( Glb1 ), resulting in intralysosomal accumulations of G M1 . The aim of this study was to reveal the pathogenic mechanisms of G M1 -gangliosidosis in a new Glb1 knockout mouse model. Glb1 -/- mice were analyzed clinically, histologically, immunohistochemically, electrophysiologically and biochemically. Morphological lesions in the central nervous system were already observed in two-month-old mice, whereas functional deficits, including ataxia and tremor, did not start before 3.5-months of age. This was most likely due to a reduced membrane resistance as a compensatory mechanism. Swollen neurons exhibited intralysosomal storage of lipids extending into axons and amyloid precursor protein positive spheroids. Additionally, axons showed a higher kinesin and lower dynein immunoreactivity compared to wildtype controls. Glb1 -/- mice also demonstrated loss of phosphorylated neurofilament positive axons and a mild increase in non-phosphorylated neurofilament positive axons. Moreover, marked astrogliosis and microgliosis were found, but no demyelination. In addition to the main storage material G M1 , G A1 , sphingomyelin, phosphatidylcholine and phosphatidylserine were elevated in the brain. In summary, the current Glb1 -/- mice exhibit a so far undescribed axonopathy and a reduced membrane resistance to compensate the functional effects of structural changes. They can be used for detailed examinations of axon-glial interactions and therapy trials of lysosomal storage diseases.

The development of sensory circuits is partially guided by sensory experience. In the medial superior olive (MSO), these refinements generate precise coincidence detection to localize sounds in the azimuthal plane. Glycinergic inhibitory inputs to the MSO, which tune the sensitivity to interaural time differences, undergo substantial structural and functional refinements after hearing onset. Whether excitation and calcium signaling in the MSO are similarly affected by the onset of acoustic experience is unresolved. To assess the time window and mechanism of excitatory and calcium-dependent refinements during late postnatal development, we quantified EPSCs and calcium entry in MSO neurons of Mongolian gerbils of either sex raised in a normal and in an activity altered, omnidirectional white noise environment. Global dendritic calcium transients elicited by action potentials disappeared rapidly after hearing onset. Local synaptic calcium transients decreased, leaving a GluR2 lacking AMPAR-mediated influx as the only activity-dependent source in adulthood. Exposure to omnidirectional white noise accelerated the decrease in calcium entry, leaving membrane properties unaffected. Thus, sound-driven activity accelerates the excitatory refinement and shortens the period of activity-dependent calcium signaling around hearing onset. Together with earlier reports, our findings highlight that excitation, inhibition, and biophysical properties are differentially sensitive to distinct features of sensory experience. SIGNIFICANCE STATEMENT Neurons in the medial superior olive, an ultra-fast coincidence detector for sound source localization, acquire their specialized function through refinements during late postnatal development. The refinement of inhibitory inputs that convey sensitivity to relevant interaural time differences is instructed by the experience of sound localization cues. Which cues instruct the refinement of excitatory inputs, calcium signaling, and biophysical properties is unknown. Here we demonstrate a time window for activity- and calcium-dependent refinements limited to shortly after hearing onset. Exposure to omnidirectional white noise, which suppresses sound localization cues but increases overall activity, accelerates the refinement of calcium signaling and excitatory inputs without affecting biophysical membrane properties. Thus, the refinement of excitation, inhibition, and intrinsic properties is instructed by distinct cues., (Copyright © 2020 the authors.)

Coincidence detector neurons of the medial superior olive (MSO) are sensitive to interaural time differences in the range of a few tens of microseconds. The biophysical basis for this remarkable acuity is a short integration time constant of the membrane, which is achieved by large low voltage-activated potassium and hyperpolarization-activated inward cation conductances. Additional temporal precision is thought to be achieved through a sub-cellular distribution of low voltage-activated potassium channel expression biased to the soma. To evaluate the contribution of potassium channels, we investigated the presence and sub-cellular distribution profile of seven potassium channel sub-units in adult MSO neurons of gerbils. We find that low- and high voltage-activated potassium channels are present with distinct sub-cellular distributions. Overall, low voltage-activated potassium channels appear to be biased to the soma while high voltage-activated potassium channels are more evenly distributed and show a clear expression at distal dendrites. Additionally, low voltage-activated potassium channel sub-units co-localize with glycinergic inputs while HCN1 channels co-localize more with high voltage-activated potassium channels. Functionally, high voltage-activated potassium currents are already active at low voltages near the resting potential. We describe a possible role of high voltage-activated potassium channels in modulating EPSPs in a computational model and contributing to setting the integration time window of coincidental inputs. Our data shows that MSO neurons express a large set of different potassium channels with distinct functional relevance.

In natural environments our auditory system is exposed to multiple and diverse signals of fluctuating amplitudes. Therefore, to detect, localize, and single out individual sounds the auditory system has to process and filter spectral and temporal information from both ears. It is known that the overall sound pressure level affects sensory signal transduction and therefore the temporal response pattern of auditory neurons. We hypothesize that the mammalian binaural system utilizes a dynamic mechanism to adjust the temporal filters in neuronal circuits to different overall sound pressure levels. Previous studies proposed an inhibitory mechanism generated by the reciprocally coupled dorsal nuclei of the lateral lemniscus (DNLL) as a temporal neuronal-network filter that suppresses rapid binaural fluctuations. Here we investigated the consequence of different sound levels on this filter during binaural processing. Our in vivo and in vitro electrophysiology in Mongolian gerbils shows that the integration of ascending excitation and contralateral inhibition defines the temporal properties of this inhibitory filter. The time course of this filter depends on the synaptic drive, which is modulated by the overall sound pressure level and N-methyl-D-aspartate receptor (NMDAR) signaling. In psychophysical experiments we tested the temporal perception of humans and show that detection and localization of two subsequent tones changes with the sound pressure level consistent with our physiological results. Together our data support the hypothesis that mammals dynamically adjust their time window for sound detection and localization within the binaural system in a sound level dependent manner.

In sensory systems, the neuronal representation of external stimuli is enhanced along the sensory pathway. In the auditory system, strong enhancement of binaural information takes place between the brainstem and the midbrain; however, the underlying cellular mechanisms are unknown. Here we investigated the transformation of binaural information in the dorsal nucleus of the lateral lemniscus (DNLL), a nucleus that connects the binaural nuclei in the brainstem and the inferior colliculus in the midbrain. We used in vitro and in vivo electrophysiology in adult Mongolian gerbils to show that N-methyl-D-aspartate receptor (NMDARs) play a critical role in neuronal encoding of stimulus properties in the DNLL. While NMDARs increase firing rates, the timing and the accuracy of the neuronal responses remain unchanged. NMDAR-mediated excitation increases the information about the acoustic stimulus. Taken together, our results show that NMDARs in the DNLL enhance the auditory information content in adult mammal brainstem., (© 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

The peripheral deafness gene Mir96 is expressed in both the cochlea and central auditory circuits. To investigate whether it plays a role in the auditory system beyond the cochlea, we characterized homozygous Dmdo/Dmdo mice with a point mutation in miR-96. Anatomical analysis demonstrated a significant decrease in volume of auditory nuclei in Dmdo/Dmdo mice. This decrease resulted from decreased cell size. Non-auditory structures in the brainstem of Dmdo/Dmdo mice or auditory nuclei of the congenital deaf Cldn14-/- mice revealed no such differences. Electrophysiological analysis in the medial nucleus of the trapezoid body (MNTB) showed that principal neurons fired preferentially multiple action potentials upon depolarization, in contrast to the single firing pattern prevalent in controls and Cldn14-/- mice. Immunohistochemistry identified significantly reduced expression of two predicted targets of the mutated miR-96, Kv1.6 and BK channel proteins, possibly contributing to the electrophysiological phenotype. Microscopic analysis of the Dmdo/Dmdo calyx of Held revealed a largely absent compartmentalized morphology, as judged by SV2-labeling. Furthermore, MNTB neurons from Dmdo/Dmdo mice displayed larger synaptic short-term depression, slower AMPA-receptor decay kinetics and a larger NMDA-receptor component, reflecting a less matured stage. Again, these synaptic differences were not present between controls and Cldn14-/- mice. Thus, deafness genes differentially affect the auditory brainstem. Furthermore, our study identifies miR-96 as an essential gene regulatory network element of the auditory system which is required for functional maturation in the peripheral and central auditory system alike.

Auditory signals carry relevant information on a large range of time scales from below milliseconds to several seconds. Different stages in the auditory brainstem are specialized to extract information in specific frequency domains. One biophysical mechanism to facilitate frequency specific processing are membrane potential resonances. Here, we provide data from three different brainstem nuclei that all exhibit high-frequency subthreshold membrane resonances that are all most likely based on low-threshold potassium currents. Fitting a linear model, we argue that, as long as neurons possess active subthreshold channels, the main determinant for their resonance behavior is the steady state membrane time constant. Tuning this leak conductance can shift membrane resonance frequencies over more than a magnitude and therefore provide a flexible mechanism to tune frequency-specific auditory processing.

Pseudorabies virus (PRV), a neurovirulent α-herpesvirus, spreads between neurons at synaptic connections. PRV-infected neurons have been shown to exhibit functional deficits with the attenuated PRV152 Bartha strain negatively influencing neuronal functioning in in vitro model systems. However, the impact of this attenuated PRV152 Bartha strain on the native central nervous system has not been fully explored. Using a combination of in vivo stereotactic injections and post-hoc in vitro whole-cell recordings, we investigated the functional impact of PRV152 Bartha in the auditory system of juvenile Mongolian gerbils. The specificity of this virus strain to spread exclusively trans-synaptically in a retrograde fashion and the well-defined structure of the ascending auditory brainstem pathways allowed us to determine the physiological alterations in primary and secondary infected neurons. We find at primary and secondary infections sites, the inferior colliculus (IC) and dorsal nucleus of the lateral lemniscus respectively, a reduced excitability of infected cells. The loss of excitability is manifested by an increase in current threshold and a loss of action potential generation. The minor changes in the approximated passive membrane parameters induced by the infection cannot explain the full loss in excitability, indicating that channel densities and properties have changed. This impact on neuronal functioning might contribute to the lethal neurovirulent effects of PRV viruses as vital neuronal circuits might cease activity. Since the detrimental effects of the attenuated PRV152 Bartha strain are reduced compared to wild-type strains, it comprises an excellent tool to study the neuropathological mechanisms of viral infections., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Animals employ mechano-sensory systems to detect and explore their environment. Mechano-sensation encompasses stimuli such as constant pressure, surface movement or vibrations at various intensities that need to be segregated in the central nervous system. Besides different receptor structures, sensory filtering via intrinsic response properties could provide a convenient way to solve this problem. In leech, three major mechano-sensory cell types can be distinguished, according to their stimulus sensitivity, as nociceptive, pressure and touch cells. Using intracellular recordings, we show that the different mechano-sensory neuron classes in Hirudo medicinalis differentially respond supra-threshold to distinct frequencies of sinusoidal current injections between 0.2 and 20 Hz. Nociceptive cells responded with a low-pass filter characteristic, pressure cells as high-pass filters and touch cells as an intermediate band-pass filter. Each class of mechano-sensory neurons is thus intrinsically tuned to a specific frequency range of voltage oscillation that could help segregate mechano-sensory information centrally., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

The identification and characterization of organization principals is essential for the understanding of neural function of brain areas. The inferior colliculus (IC) represents a midbrain nexus involved in numerous aspects of auditory processing. Likewise, neurons throughout the IC are tuned to a diverse range of specific stimulus features. Yet beyond a topographic arrangement of the cochlea-inherited frequency tuning, the functional organization of the IC is not well understood. Particularly, a common principle that links the diverse tuning characteristics is unknown. Here we used in vitro patch clamp recordings combined with laser-uncaging, and in vivo single cell recordings to study the spatial and functional organization principles of the central IC. We identified a topographic bias of ascending synaptic input timing that is balanced between inhibition and excitation and co-varies with in vivo first-spike latency. This bias was paralleled post-synaptically by differences in biophysical membrane properties and firing patterns, with integrating neurons predominantly found in the dorso-medial part, and coincidence-detector neurons biased to the ventro-lateral IC. Importantly, these cellular and network features translated into distinct temporal processing capabilities irrespectively of the neurons' characteristic frequency. Our data therefore imply that heterogeneity of synaptic inputs, intrinsic properties and temporal processing are functional principles that underlie the spatial organization of the central IC., (Copyright © 2016 Elsevier B.V. All rights reserved.)

To capture the context of sensory information, neural networks must process input signals across multiple timescales. In the auditory system, a prominent change in temporal processing takes place at an inhibitory GABAergic synapse in the dorsal nucleus of the lateral lemniscus (DNLL). At this synapse, inhibition outlasts the stimulus by tens of milliseconds, such that it suppresses responses to lagging sounds, and is therefore implicated in echo suppression. Here, we untangle the cellular basis of this inhibition. We demonstrate with in vivo whole-cell patch-clamp recordings in Mongolian gerbils that the duration of inhibition increases with sound intensity. Activity-dependent spillover and asynchronous release translate the high presynaptic firing rates found in vivo into a prolonged synaptic output in acute slice recordings. A key mechanism controlling the inhibitory time course is the passive integration of the hyperpolarizing inhibitory conductance. This prolongation depends on the synaptic conductance amplitude. Computational modeling shows that this prolongation is a general mechanism and relies on a non-linear effect caused by synaptic conductance saturation when approaching the GABA reversal potential. The resulting hyperpolarization generates an efficient activity-dependent suppression of action potentials without affecting the threshold or gain of the input-output function. Taken together, the GABAergic inhibition in the DNLL is adjusted to the physiologically relevant duration by passive integration of inhibition with activity-dependent synaptic kinetics. This change in processing timescale combined with the reciprocal connectivity between the DNLLs implements a mechanism to suppress the distracting localization cues of echoes and helps to localize the initial sound source reliably., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Optical visualization of neural network activity is limited by imaging system-dependent technical tradeoffs. To overcome these constraints, we have developed a powerful low-cost and flexible imaging system with high spectral variability and unique spatio-temporal precision for simultaneous optical recording and manipulation of neural activity of large cell groups. The system comprises eight high-power light-emitting diodes, a camera with a large metal-oxide-semiconductor sensor and a high numerical aperture water-dipping objective. It allows fast and precise control of excitation and simultaneous low noise imaging at high resolution. Adjustable apertures generated two independent areas of variable size and position for simultaneous optical activation and image capture. The experimental applicability of this system was explored in semi-isolated preparations of larval axolotl (Ambystoma mexicanum) with intact inner ear organs and central nervous circuits. Cyclic galvanic stimulation of semicircular canals together with glutamate- and γ-aminobutyric acid (GABA)-uncaging caused a corresponding modulation of Ca(2+) transients in central vestibular neurons. These experiments revealed specific cellular properties as well as synaptic interactions between excitatory and inhibitory inputs, responsible for spatio-temporal-specific sensory signal processing. Location-specific GABA-uncaging revealed a potent inhibitory shunt of vestibular nerve afferent input in the predominating population of tonic vestibular neurons, indicating a considerable impact of local and commissural inhibitory circuits on the processing of head/body motion-related signals. The discovery of these previously unknown properties of vestibular computations demonstrates the merits of our novel microscope system for experimental applications in the field of neurobiology., (© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Passive and active membrane properties determine the voltage responses of neurons. Within the auditory brain stem, refinements in these intrinsic properties during late postnatal development usually generate short integration times and precise action-potential generation. This developmentally acquired temporal precision is crucial for auditory signal processing. How the interactions of these intrinsic properties develop in concert to enable auditory neurons to transfer information with high temporal precision has not yet been elucidated in detail. Here, we show how the developmental interaction of intrinsic membrane parameters generates high firing precision. We performed in vitro recordings from neurons of postnatal days 9-28 in the ventral nucleus of the lateral lemniscus of Mongolian gerbils, an auditory brain stem structure that converts excitatory to inhibitory information with high temporal precision. During this developmental period, the input resistance and capacitance decrease, and action potentials acquire faster kinetics and enhanced precision. Depending on the stimulation time course, the input resistance and capacitance contribute differentially to action-potential thresholds. The decrease in input resistance, however, is sufficient to explain the enhanced action-potential precision. Alterations in passive membrane properties also interact with a developmental change in potassium currents to generate the emergence of the mature firing pattern, characteristic of coincidence-detector neurons. Cholinergic receptor-mediated depolarizations further modulate this intrinsic excitability profile by eliciting changes in the threshold and firing pattern, irrespective of the developmental stage. Thus our findings reveal how intrinsic membrane properties interact developmentally to promote temporally precise information processing., (Copyright © 2015 the American Physiological Society.)

Neurons in the medial superior olive (MSO) detect microsecond differences in the arrival time of sounds between the ears (interaural time differences or ITDs), a crucial binaural cue for sound localization. Synaptic inhibition has been implicated in tuning ITD sensitivity, but the cellular mechanisms underlying its influence on coincidence detection are debated. Here we determine the impact of inhibition on coincidence detection in adult Mongolian gerbil MSO brain slices by testing precise temporal integration of measured synaptic responses using conductance-clamp. We find that inhibition dynamically shifts the peak timing of excitation, depending on its relative arrival time, which in turn modulates the timing of best coincidence detection. Inhibitory control of coincidence detection timing is consistent with the diversity of ITD functions observed in vivo and is robust under physiologically relevant conditions. Our results provide strong evidence that temporal interactions between excitation and inhibition on microsecond timescales are critical for binaural processing.

Across all sensory modalities, the effect of context-dependent neural adaptation can be observed at every level, from receptors to perception. Nonetheless, it has long been assumed that the processing of interaural time differences, which is the primary cue for sound localization, is nonadaptive, as its outputs are mapped directly onto a hard-wired representation of space. Here we present evidence derived from in vitro and in vivo experiments in gerbils indicating that the coincidence-detector neurons in the medial superior olive modulate their sensitivity to interaural time differences through a rapid, GABA(B) receptor-mediated feedback mechanism. We show that this mechanism provides a gain control in the form of output normalization, which influences the neuronal population code of auditory space. Furthermore, psychophysical tests showed that the paradigm used to evoke neuronal GABA(B) receptor-mediated adaptation causes the perceptual shift in sound localization in humans that was expected on the basis of our physiological results in gerbils.

Neurons of the medial superior olive (MSO) code for the azimuthal location of low-frequency sound sources via a binaural coincidence detection system operating on microsecond time scales. These neurons are morphologically simple and stereotyped, and anatomical studies have indicated a functional segregation of excitatory and inhibitory inputs between cellular compartments. It is thought that this morphological arrangement holds important implications for the computational task of these cells. To date, however, there has been no functional investigation into synaptic input sites or functional receptor distributions on mature neurons of the MSO. Here, functional neurotransmitter receptor maps for amino-3-hydroxyl-5-methyl-4-isoxazole propionate (AMPA), N-methyl-D-aspartate (NMDA), glycine (Gly), and ionotropic γ-aminobutyric acid (GABA(A)) receptors (Rs) were compared and complemented by their corresponding synaptic input map. We find in MSO neurons from postnatal day 20-35 gerbils that AMPARs and their excitatory inputs target the soma and dendrites. Functional GlyRs and their inhibitory inputs are predominantly refined to the somata, although a pool of functional GlyRs is present extrasynaptically on MSO dendrites. GABA(A)R responses are present throughout the cell but lack direct synaptic contact indicating an involvement in volume transmission. NMDARs are present both synaptically and extrasynaptically with an overall distribution similar to GlyRs. Interestingly, even at physiological temperatures these functional NMDARs can be potentiated by synaptically released Gly. The functional receptor and synaptic input maps produced here led to the identification of a cross talk between transmitter systems and raises the possibility that extrasynaptic receptors could be modulating leak conductances as a homeostatic mechanism.

Neurons in the medial superior olive (MSO) process microsecond interaural time differences, the major cue for localizing low-frequency sounds, by comparing the relative arrival time of binaural, glutamatergic excitatory inputs. This coincidence detection mechanism is additionally shaped by highly specialized glycinergic inhibition. Traditionally, it is assumed that the binaural inputs are conveyed by many independent fibers, but such an anatomical arrangement may decrease temporal precision. Short-term depression on the other hand might enhance temporal fidelity during ongoing activity. For the first time we show that binaural coincidence detection in MSO neurons may require surprisingly few but strong inputs, challenging long-held assumptions about mammalian coincidence detection. This study exclusively uses adult gerbils for in vitro electrophysiology, single-cell electroporation and immunohistochemistry to characterize the size and short-term plasticity of inputs to the MSO. We find that the excitatory and inhibitory inputs to the MSO are well balanced both in strength and short-term dynamics, redefining this fastest of all mammalian coincidence detector circuits.

In the mammalian medial superior olive (MSO), neurons compute the azimuthal location of sound sources by temporally precise coincidence detection. It is assumed that the dendritic morphology of MSO neurons plays a crucial role in this computational process. However, few quantitative data about the morphology of these neuronal coincidence detectors are available, limiting theoretical approaches. Such a quantitative morphological description of neurons of the mammalian MSO would also allow a comparative analysis with its avian analog, the nucleus laminaris. We used single-cell electroporation, microscopic reconstruction, and compartmentalization to extract anatomical parameters of MSO neurons and quantitatively describe their morphology and development between postnatal day 9 and 36. We found that developmental refinement occurs until P27, generating morphologically compact, cylinder-like cells with axons originating from the soma. The complexity of higher order dendrites decreases between postnatal days 9 and 21. This decrease in dendritic complexity is judged from counting and analyzing the location of dendritic branches and determining the distribution of the surface area and total length of neurons. During this developmental period, the average length of terminal branches increases about twofold, indicating an elimination of predominantly small branches. The cell volume increases more than 1.5-fold between P9 and P27, a change that can be attributed to an increase in dendritic diameter during this developmental period. The developmental profile of the morphology of MSO neurons obtained indicates that maturation is reached 2 weeks after hearing onset.

Protein recruitment during endocytosis is well characterized in fibroblasts. Since fibroblasts do not engage in regulated exocytosis, only information about protein recruitment during constitutive endocytosis is provided. Furthermore, the cortical actin of fibroblasts is characterized by stress fibers rather than a thick cortical meshwork. A cell model, which differs in these features, could provide insight into the heterogeneity of protein recruitment to constitutive and exocytosis coupled endocytotic areas. Therefore, this study investigates the sequence of protein recruitment in PC12 cells, a well documented exocytotic cell model with thick actin cortex. Using real time total-internal-reflection fluorescence microscopy it was found that at the plasma membrane steady, but not transient, dynamin-1-EGFP or -mCherry fluorescence spots that rapidly dimmed coincided with markers for constitutive endocytotic such as clathrin-LC-dsRed and transferrin-receptor-pHluorin. Clathrin-LC-dsRed and dynamin-1-EGFP were further used to determine the temporal sequence of protein recruitment to areas of constitutive endocytosis. mCherry- and EGFP-beta-actin, Arp-3-EGFP and EGFP-mAbp1 were slowly recruited before the dynamin-1-mCherry fluorescence dimmed, but their fluorescence peaked after the loss of clathrin-LC-dsRed commenced. Furthermore, mCherry-beta-actin fluorescence increased before exocytosis, indicating redistribution prior to release. Also, no average dynamin-1-mCherry recruitment was observed within 50 s to regions of exocytosis marked by NPY-mGFP. This indicates that the temporal-spatial coupling between regulated exo-and endocytosis is rather limited in PC12 cells. Furthermore, the time course of the protein recruitment to constitutive endocytotic sites might depend on the subcellular morphology such as the size of the actin cortex.

During regulated fusion of secretory granules with the plasma membrane, a fusion pore first opens and then dilates. The dilating pore allows cargo proteins from the dense core to be released into the extracellular space. Using real-time evanescent field fluorescence microscopy of live PC12 cells, it was determined how rapidly proteins of different sizes escape from single granules after fusion. Tissue plasminogen activator (tPA)-Venus is released 40-fold slower than the three times smaller neuropeptide Y [NPY-monomeric GFP (mGFP)]. An NPY bearing two mGFPs in tandem [NPY-(mGFP)(2)] as an intermediate-sized fusion probe is released most slowly. Although, the time-course of release varies substantially for a given probe. Coexpression of beta-actin, actin-related protein 3 or mAbp1 slowed the release of the two larger cargo molecules but did not affect release of NPY-mGFP or of the granule-membrane-bound probe Vamp-pHluorin. Additionally, high concentrations of cytochalasin D slowed release of the tPA-Venus. Together these results suggest that fusion pore dilation is not the only determinate of release time-course and that actin rearrangements similar to those mediating actin-mediated motility influences the time-course of release without directly interfering with the granule membrane to cell membrane connection.