Exact Distribution of the Quantal Content in Synaptic Transmission.

During electrochemical signal transmission through synapses, triggered by an action potential (AP), a stochastic number of synaptic vesicles (SVs), called the "quantal content," release neurotransmitters in the synaptic cleft. It is widely accepted that the quantal content probability distribution is a binomial based on the number of ready-release SVs in the presynaptic terminal. But the latter number itself fluctuates due to its stochastic replenishment, hence the actual distribution of quantal content is unknown. We show that exact distribution of quantal content can be derived for general stochastic AP inputs in the steady state. For fixed interval AP train, we prove that the distribution is a binomial, and corroborate our predictions by comparison with electrophysiological recordings from MNTB-LSO synapses of juvenile mice. For a Poisson train, we show that the distribution is nonbinomial. Moreover, we find exact moments of the quantal content in the Poisson and other general cases, which may be used to obtain the model parameters from experiments.

Development of synaptic fidelity and action potential robustness at an inhibitory sound localization circuit: effects of otoferlin-related deafness.

Sound localization involves information analysis in the lateral superior olive (LSO), a conspicuous nucleus in the mammalian auditory brainstem. LSO neurons weigh interaural level differences (ILDs) through precise integration of glutamatergic excitation from the cochlear nucleus (CN) and glycinergic inhibition from the medial nucleus of the trapezoid body (MNTB). Sound sources can be localized even during sustained perception, an accomplishment that requires robust neurotransmission. Virtually nothing is known about the sustained performance and the temporal precision of MNTB-LSO inputs after postnatal day (P)12 (time of hearing onset) and whether acoustic experience guides development. Here we performed whole-cell patch-clamp recordings to investigate neurotransmission of single MNTB-LSO fibres upon sustained electrical stimulation (1-200 Hz/60 s) at P11 and P38 in wild-type (WT) and deaf otoferlin (Otof) knock-out (KO) mice. At P11, WT and KO inputs performed remarkably similarly. In WTs, the performance increased drastically between P11 and P38, e.g. manifested by an 8 to 11-fold higher replenishment rate (RR) of synaptic vesicles and action potential robustness. Together, these changes resulted in reliable and highly precise neurotransmission at frequencies ≤100 Hz. In contrast, KO inputs performed similarly at both ages, implying impaired synaptic maturation. Computational modelling confirmed the empirical observations and established a reduced RR per release site for P38 KOs. In conclusion, acoustic experience appears to contribute massively to the development of reliable neurotransmission, thereby forming the basis for effective ILD detection. Collectively, our results provide novel insights into experience-dependent maturation of inhibitory neurotransmission and auditory circuits at the synaptic level. KEY POINTS: Inhibitory glycinergic inputs from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO) are involved in sound localization. This brainstem circuit performs reliably throughout life. How such reliability develops is unknown. Here we investigated the role of acoustic experience on the functional maturation of MNTB-LSO inputs at juvenile (postnatal day P11) and young adult ages (P38) employing deaf mice lacking otoferlin (KO). We analysed neurotransmission at single MNTB-LSO fibres in acute brainstem slices employing prolonged high-frequency stimulation (1-200 Hz/60 s). At P11, KO inputs still performed normally, as manifested by normal synaptic attenuation, fidelity, replenishment rate, temporal precision and action potential robustness. Between P11 and P38, several synaptic parameters increased substantially in wild-type mice, collectively resulting in high-fidelity and temporally precise neurotransmission. In contrast, maturation of synaptic fidelity was largely absent in KOs after P11. Collectively, reliable neurotransmission at inhibitory MNTB-LSO inputs develops under the guidance of acoustic experience.

Topographic map refinement and synaptic strengthening of a sound localization circuit require spontaneous peripheral activity.

KEY POINTS: Loss of the calcium sensor otoferlin disrupts neurotransmission from inner hair cells. Central auditory nuclei are functionally denervated in otoferlin knockout mice (Otof KOs) via gene ablation confined to the periphery. We employed juvenile and young adult Otof KO mice (postnatal days (P)10-12 and P27-49) as a model for lacking spontaneous activity and deafness, respectively. We studied the impact of peripheral activity on synaptic refinement in the sound localization circuit from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO). MNTB in vivo recordings demonstrated drastically reduced spontaneous spiking and deafness in Otof KOs. Juvenile KOs showed impaired synapse elimination and strengthening, manifested by broader MNTB-LSO inputs, imprecise MNTB-LSO topography and weaker MNTB-LSO fibres. The impairments persisted into young adulthood. Further functional refinement after hearing onset was undetected in young adult wild-types. Collectively, activity deprivation confined to peripheral protein loss impairs functional MNTB-LSO refinement during a critical prehearing period. ABSTRACT: Circuit refinement is critical for the developing sound localization pathways in the auditory brainstem. In prehearing mice (hearing onset around postnatal day (P)12), spontaneous activity propagates from the periphery to central auditory nuclei. At the glycinergic projection from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO) of neonatal mice, super-numerous MNTB fibres innervate a given LSO neuron. Between P4 and P9, MNTB fibres are functionally eliminated, whereas the remaining fibres are strengthened. Little is known about MNTB-LSO circuit refinement after P20. Moreover, MNTB-LSO refinement upon activity deprivation confined to the periphery is largely unexplored. This leaves a considerable knowledge gap, as deprivation often occurs in patients with congenital deafness, e.g. upon mutations in the otoferlin gene (OTOF). Here, we analysed juvenile (P10-12) and young adult (P27-49) otoferlin knockout (Otof KO) mice with respect to MNTB-LSO refinement. MNTB in vivo recordings revealed drastically reduced spontaneous activity and deafness in knockouts (KOs), confirming deprivation. As RNA sequencing revealed Otof absence in the MNTB and LSO of wild-types, Otof loss in KOs is specific to the periphery. Functional denervation impaired MNTB-LSO synapse elimination and strengthening, which was assessed by glutamate uncaging and electrical stimulation. Impaired elimination led to imprecise MNTB-LSO topography. Impaired strengthening was associated with lower quantal content per MNTB fibre. In young adult KOs, the MNTB-LSO circuit remained unrefined. Further functional refinement after P12 appeared absent in wild-types. Collectively, we provide novel insights into functional MNTB-LSO circuit maturation governed by a cochlea-specific protein. The central malfunctions in Otof KOs may have implications for patients with sensorineuronal hearing loss.

GABA is a modulator, rather than a classical transmitter, in the medial nucleus of the trapezoid body-lateral superior olive sound localization circuit.

KEY POINTS: The lateral superior olive (LSO), a brainstem hub involved in sound localization, integrates excitatory and inhibitory inputs from the ipsilateral and the contralateral ear, respectively. In gerbils and rats, inhibition to the LSO reportedly shifts from GABAergic to glycinergic within the first three postnatal weeks. Surprisingly, we found no evidence for synaptic GABA signalling during this time window in mouse LSO principal neurons. However, we found that presynaptic GABAB Rs modulate Ca2+ influx into medial nucleus of the trapezoid body axon terminals, resulting in reduced synaptic strength. Moreover, GABA elicited strong responses in LSO neurons that were mediated by extrasynaptic GABAA Rs. RNA sequencing revealed highly abundant δ subunits, which are characteristic of extrasynaptic receptors. Whereas GABA increased the excitability of neonatal LSO neurons, it reduced the excitability around hearing onset. Collectively, GABA appears to control the excitability of mouse LSO neurons via extrasynaptic and presynaptic signalling. Thus, GABA acts as a modulator, rather than as a classical transmitter. ABSTRACT: GABA and glycine mediate fast inhibitory neurotransmission and are coreleased at several synapse types. Here we assessed the contribution of GABA and glycine in synaptic transmission between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO), two nuclei involved in sound localization. Whole-cell patch-clamp experiments in acute mouse brainstem slices at postnatal days (P) 4 and 11 during pharmacological blockade of GABAA receptors (GABAA Rs) and/or glycine receptors demonstrated no GABAergic synaptic component on LSO principal neurons. A GABAergic component was absent in evoked inhibitory postsynaptic currents and miniature events. Coimmunofluorescence experiments revealed no codistribution of the presynaptic GABAergic marker GAD65/67 with gephyrin, a postsynaptic marker for GABAA Rs, corroborating the conclusion that GABA does not act synaptically in the mouse LSO. Imaging experiments revealed reduced Ca2+ influx into MNTB axon terminals following activation of presynaptic GABAB Rs. GABAB R activation reduced the synaptic strength at P4 and P11. GABA appears to act on extrasynaptic GABAA Rs as demonstrated by application of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, a δ-subunit-specific GABAA R agonist. RNA sequencing showed high mRNA levels for the δ-subunit in the LSO. Moreover, GABA transporters GAT-1 and GAT-3 appear to control extracellular GABA. Finally, we show an age-dependent effect of GABA on the excitability of LSO neurons. Whereas tonic GABA increased the excitability at P4, leading to spike facilitation, it decreased the excitability at P11 via shunting inhibition through extrasynaptic GABAA Rs. Taken together, we demonstrate a modulatory role of GABA in the murine LSO, rather than a function as a classical synaptic transmitter.

Poor transcript-protein correlation in the brain: negatively correlating gene products reveal neuronal polarity as a potential cause.

Transcription, translation, and turnover of transcripts and proteins are essential for cellular function. The contribution of those factors to protein levels is under debate, as transcript levels and cognate protein levels do not necessarily correlate due to regulation of translation and protein turnover. Here we propose neuronal polarity as a third factor that is particularly evident in the CNS, leading to considerable distances between somata and axon terminals. Consequently, transcript levels may negatively correlate with cognate protein levels in CNS regions, i.e., transcript and protein levels behave reciprocally. To test this hypothesis, we performed an integrative inter-omics study and analyzed three interconnected rat auditory brainstem regions (cochlear nuclear complex, CN; superior olivary complex, SOC; inferior colliculus, IC) and the rest of the brain as a reference. We obtained transcript and protein sets in these regions of interest (ROIs) by DNA microarrays and label-free mass spectrometry, and performed principal component and correlation analyses. We found 508 transcript|protein pairs and detected poor to moderate transcript|protein correlation in all ROIs, as evidenced by coefficients of determination from 0.34 to 0.54. We identified 57-80 negatively correlating gene products in the ROIs and intensively analyzed four of them for which the correlation was poorest. Three cognate proteins (Slc6a11, Syngr1, Tppp) were synaptic and hence candidates for a negative correlation because of protein transport into axon terminals. Thus, we systematically analyzed the negatively correlating gene products. Gene ontology analyses revealed overrepresented transport/synapse-related proteins, supporting our hypothesis. We present 30 synapse/transport-related proteins with poor transcript|protein correlation. In conclusion, our analyses support that protein transport in polar cells is a third factor that influences the protein level and, thereby, the transcript|protein correlation. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* and *Open Data* because it provided all relevant information to reproduce the study in the manuscript and because it made the data publicly available. The data can be accessed at https://osf.io/ha28n/. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Synaptic reliability and temporal precision are achieved via high quantal content and effective replenishment: auditory brainstem versus hippocampus.

KEY POINTS: Auditory brainstem neurons involved in sound source localization are equipped with several morphological and molecular features that enable them to compute interaural level and time differences. As sound source localization works continually, synaptic transmission between these neurons should be reliable and temporally precise, even during sustained periods of high-frequency activity. Using patch-clamp recordings in acute brain slices, we compared synaptic reliability and temporal precision in the seconds-minute range between auditory and two types of hippocampal synapses; the latter are less confronted with temporally precise high-frequency transmission than the auditory ones. We found striking differences in synaptic properties (e.g. continually high quantal content) that allow auditory synapses to reliably release vesicles at much higher rate than their hippocampal counterparts. Thus, they are indefatigable and also in a position to transfer information with exquisite temporal precision and their performance appears to be supported by very efficient replenishment mechanisms. ABSTRACT: At early stations of the auditory pathway, information is encoded by precise signal timing and rate. Auditory synapses must maintain the relative timing of events with submillisecond precision even during sustained and high-frequency stimulation. In non-auditory brain regions, e.g. telencephalic ones, synapses are activated at considerably lower frequencies. Central to understanding the heterogeneity of synaptic systems is the elucidation of the physical, chemical and biological factors that determine synapse performance. In this study, we used slice recordings from three synapse types in the mouse auditory brainstem and hippocampus. Whereas the auditory brainstem nuclei experience high-frequency activity in vivo, the hippocampal circuits are activated at much lower frequencies. We challenged the synapses with sustained high-frequency stimulation (up to 200 Hz for 60 s) and found significant performance differences. Our results show that auditory brainstem synapses differ considerably from their hippocampal counterparts in several aspects, namely resistance to synaptic fatigue, low failure rate and exquisite temporal precision. Their high-fidelity performance supports the functional demands and appears to be due to the large size of the readily releasable pool and a high release probability, which together result in a high quantal content. In conjunction with very efficient vesicle replenishment mechanisms, these properties provide extremely rapid and temporally precise signalling required for neuronal communication at early stations of the auditory system, even during sustained activation in the minute range.

L-type Calcium Channel Cav1.2 Is Required for Maintenance of Auditory Brainstem Nuclei.

Cav1.2 and Cav1.3 are the major L-type voltage-gated Ca(2+) channels in the CNS. Yet, their individual in vivo functions are largely unknown. Both channel subunits are expressed in the auditory brainstem, where Cav1.3 is essential for proper maturation. Here, we investigated the role of Cav1.2 by targeted deletion in the mouse embryonic auditory brainstem. Similar to Cav1.3, loss of Cav1.2 resulted in a significant decrease in the volume and cell number of auditory nuclei. Contrary to the deletion of Cav1.3, the action potentials of lateral superior olive (LSO) neurons were narrower compared with controls, whereas the firing behavior and neurotransmission appeared unchanged. Furthermore, auditory brainstem responses were nearly normal in mice lacking Cav1.2. Perineuronal nets were also unaffected. The medial nucleus of the trapezoid body underwent a rapid cell loss between postnatal days P0 and P4, shortly after circuit formation. Phosphorylated cAMP response element-binding protein (CREB), nuclear NFATc4, and the expression levels of p75NTR, Fas, and FasL did not correlate with cell death. These data demonstrate for the first time that both Cav1.2 and Cav1.3 are necessary for neuronal survival but are differentially required for the biophysical properties of neurons. Thus, they perform common as well as distinct functions in the same tissue.

ADF/Cofilin Controls Synaptic Actin Dynamics and Regulates Synaptic Vesicle Mobilization and Exocytosis.

Actin is a regulator of synaptic vesicle mobilization and exocytosis, but little is known about the mechanisms that regulate actin at presynaptic terminals. Genetic data on LIMK1, a negative regulator of actin-depolymerizing proteins of the ADF/cofilin family, suggest a role for ADF/cofilin in presynaptic function. However, synapse physiology is fully preserved upon genetic ablation of ADF in mice, and n-cofilin mutant mice display defects in postsynaptic plasticity, but not in presynaptic function. One explanation for this phenomenon is overlapping functions of ADF and n-cofilin in presynaptic physiology. Here, we tested this hypothesis and genetically removed ADF together with n-cofilin from synapses. In double mutants for ADF and n-cofilin, synaptic actin dynamics was impaired and more severely affected than in single mutants. The resulting cytoskeletal defects heavily affected the organization, mobilization, and exocytosis of synaptic vesicles in hippocampal CA3-CA1 synapses. Our data for the first time identify overlapping functions for ADF and n-cofilin in presynaptic physiology and vesicle trafficking. We conclude that n-cofilin is a limiting factor in postsynaptic plasticity, a function which cannot be substituted by ADF. On the presynaptic side, the presence of either ADF or n-cofilin is sufficient to control actin remodeling during vesicle release.

Neuroproteomics in the auditory brainstem: candidate proteins for ultrafast and precise information processing.

In the mammalian auditory brainstem, the cochlear nuclear complex (CN) and the superior olivary complex (SOC) feature structural and functional specializations for ultrafast (<1 ms) and precise information processing. Their proteome, the basis for structure and function, has been rarely analyzed so far. Here we identified and quantified the protein profiles of three major auditory brainstem regions of adult rats, the CN, the SOC, and the inferior colliculus (IC). The rest of the brain served as a reference. Via label-free quantitative mass spectrometry and 2-D DIGE/MALDI-MS, we identified 584 and 297 proteins in the plasma membrane/synaptic vesicle proteome and the cytosolic proteome, respectively. 'Region-typical' proteins, i.e., those with higher abundance in one region than in the other three, were considered candidates for functional specializations. Key proteins were validated via Western blots and immunohistochemistry. Functional annotation clustering revealed an overrepresentation of neurofilament proteins among the CN+SOC-typical proteins. These are related to regulation of axon diameter and, thereby, conduction velocity. Interestingly, the sets of synapse-associated proteins differed between regions. For example, synaptotagmin-2 (Syt2), a Ca2+ sensor for fast exocytosis, was CN+SOC+IC-typical, whereas Syt1 was CN+SOC+IC-atypical. Together, our quantitative comparison of protein profiles has revealed several interesting candidate proteins for ultrafast and precise information processing.

α2δ3 is essential for normal structure and function of auditory nerve synapses and is a novel candidate for auditory processing disorders.

The auxiliary subunit α2δ3 modulates the expression and function of voltage-gated calcium channels. Here we show that α2δ3 mRNA is expressed in spiral ganglion neurons and auditory brainstem nuclei and that the protein is required for normal acoustic responses. Genetic deletion of α2δ3 led to impaired auditory processing, with reduced acoustic startle and distorted auditory brainstem responses. α2δ3(-/-) mice learned to discriminate pure tones, but they failed to discriminate temporally structured amplitude-modulated tones. Light and electron microscopy analyses revealed reduced levels of presynaptic Ca(2+) channels and smaller auditory nerve fiber terminals contacting cochlear nucleus bushy cells. Juxtacellular in vivo recordings of sound-evoked activity in α2δ3(-/-) mice demonstrated impaired transmission at these synapses. Together, our results identify a novel role for the α2δ3 auxiliary subunit in the structure and function of specific synapses in the mammalian auditory pathway and in auditory processing disorders.

Synaptic plasticity in the auditory system: a review.

Synaptic transmission via chemical synapses is dynamic, i.e., the strength of postsynaptic responses may change considerably in response to repeated synaptic activation. Synaptic strength is increased during facilitation, augmentation and potentiation, whereas a decrease in synaptic strength is characteristic for depression and attenuation. This review attempts to discuss the literature on short-term and long-term synaptic plasticity in the auditory brainstem of mammals and birds. One hallmark of the auditory system, particularly the inner ear and lower brainstem stations, is information transfer through neurons that fire action potentials at very high frequency, thereby activating synapses >500 times per second. Some auditory synapses display morphological specializations of the presynaptic terminals, e.g., calyceal extensions, whereas other auditory synapses do not. The review focuses on short-term depression and short-term facilitation, i.e., plastic changes with durations in the millisecond range. Other types of short-term synaptic plasticity, e.g., posttetanic potentiation and depolarization-induced suppression of excitation, will be discussed much more briefly. The same holds true for subtypes of long-term plasticity, like prolonged depolarizations and spike-time-dependent plasticity. We also address forms of plasticity in the auditory brainstem that do not comprise synaptic plasticity in a strict sense, namely short-term suppression, paired tone facilitation, short-term adaptation, synaptic adaptation and neural adaptation. Finally, we perform a meta-analysis of 61 studies in which short-term depression (STD) in the auditory system is opposed to short-term depression at non-auditory synapses in order to compare high-frequency neurons with those that fire action potentials at a lower rate. This meta-analysis reveals considerably less STD in most auditory synapses than in non-auditory ones, enabling reliable, failure-free synaptic transmission even at frequencies >100 Hz. Surprisingly, the calyx of Held, arguably the best-investigated synapse in the central nervous system, depresses most robustly. It will be exciting to reveal the molecular mechanisms that set high-fidelity synapses apart from other synapses that function much less reliably.

Epicocconone staining: a powerful loading control for Western blots.

Western blot analysis is routinely employed for quantifying differences in protein levels between samples. To control equal loading and to arithmetically compensate loading differences, immunodetection of housekeeping proteins is commonly used. Due to potential biases, this approach has been criticized. Here, we evaluate epicocconone-based total protein staining (E-ToPS) as an alternative. We compared it with two other total protein stainings (Coomassie and Sypro Ruby) and with immunodetection of housekeeping proteins (ß-tubulin and glyceraldehyde 3-phosphate dehydrogenase). Evaluation comprised both the natural and the synthetic epicocconone compound. Both compounds produced highly congruent results and showed more sensitive (≤ 1 µg) and less variable staining properties than the other variants. The high sensitivity of E-ToPS, covering minute protein amounts, makes it a powerful loading control, especially for precious samples. Regarding biological and technical variances, E-ToPS outperformed immunostaining against ß-tubulin and glyceraldehyde 3-phosphate dehydrogenase. Furthermore, E-ToPS had no impact on subsequent immunodetection, allowing for an early control of proper loading prior to immunodetection. In contrast to earlier studies, we found logarithmic staining properties for E-ToPS, which should be considered when using it for arithmetic normalization. In conclusion, we demonstrate the superior power of E-ToPS as a loading control for Western blots.

Time-dependent gene expression analysis of the developing superior olivary complex.

The superior olivary complex (SOC) is an essential auditory brainstem relay involved in sound localization. To identify the genetic program underlying its maturation, we profiled the rat SOC transcriptome at postnatal days 0, 4, 16, and 25 (P0, P4, P16, and P25, respectively), using genome-wide microarrays (41,012 oligonucleotides (oligos)). Differences in gene expression between two consecutive stages were highest between P4 and P16 (3.6%) and dropped to 0.06% between P16 and P25. To identify SOC-related genetic programs, we also profiled the entire brain at P4 and P25. The number of differentially expressed oligonucleotides between SOC and brain almost doubled from P4 to P25 (4.4% versus 7.6%). These data demonstrate considerable molecular specification around hearing onset, which is rapidly finalized. Prior to hearing onset, several transcription factors associated with the peripheral auditory system were up-regulated, probably coordinating the development of the auditory system. Additionally, crystallin-γ subunits and serotonin-related genes were highly expressed. The molecular repertoire of mature neurons was sculpted by SOC-related up- and down-regulation of voltage-gated channels and G-proteins. Comparison with the brain revealed a significant enrichment of hearing impairment-related oligos in the SOC (26 in the SOC, only 11 in the brain). Furthermore, 29 of 453 SOC-related oligos mapped within 19 genetic intervals associated with hearing impairment. Together, we identified sequential genetic programs in the SOC, thereby pinpointing candidates that may guide its development and ensure proper function. The enrichment of hearing impairment-related genes in the SOC may have implications for restoring hearing because central auditory structures might be more severely affected than previously appreciated.

Functional analysis of the inhibitory neurotransmitter transporters GlyT1, GAT-1, and GAT-3 in astrocytes of the lateral superior olive.

Neurotransmitter clearance from the synaptic cleft is a major function of astrocytes and requires neurotransmitter transporters. In the rodent lateral superior olive (LSO), a conspicuous auditory brainstem center, both glycine and GABA mediate synaptic inhibition. However, the main inhibitory input from the medial nucleus of the trapezoid body (MNTB) appears to be glycinergic by postnatal day (P) 14, when circuit maturation is almost accomplished. Using whole-cell patch-clamp recordings at P3-20, we analyzed glycine transporters (GlyT1) and GABA transporters (GAT-1, GAT-3) in mouse LSO astrocytes, emphasizing on their developmental regulation. Application of glycine or GABA induced a dose- and age-dependent inward current and a respective depolarization. The GlyT1-specific inhibitor sarcosine reduced the maximal glycine-induced current (IGly (max) ) by about 60%. The GAT-1 and GAT-3 antagonists NO711 and SNAP5114, respectively, reduced the maximal GABA-induced current (IGABA (max) ) by about 35%. Furthermore, [Cl(-) ]o reduction decreased IGly (max) and IGABA (max) by about 85 to 95%, showing the Cl(-) dependence of GlyT and GAT. IGABA (max) was stronger than IGly (max) , and the ratio increased developmentally from 1.6-fold to 3.7-fold. Together, our results demonstrate the functional presence of the three inhibitory neurotransmitter transporters GlyT1, GAT-1, and GAT-3 in LSO astrocytes. Furthermore, the uptake capability for GABA was higher than for glycine, pointing toward eminent GABAergic signaling in the LSO. GABA may originate from another source than the MNTB-LSO synapses, namely from another projection or from reversal of astrocytic GATs. Thus, neuronal signaling in the LSO appears to be more versatile than previously thought. GLIA 2014;62:1992-2003.

Retrocochlear function of the peripheral deafness gene Cacna1d.

Hearing impairment represents the most common sensory deficit in humans. Genetic mutations contribute significantly to this disorder. Mostly, only malfunction of the ear is considered. Here, we assessed the role of the peripheral deafness gene Cacna1d, encoding the L-type channel Ca(v)1.3, in downstream processing of acoustic information. To this end, we generated a mouse conditional Cacna1d-eGFP(flex) allele. Upon pairing with Egr2::Cre mice, Ca(v)1.3 was ablated in the auditory brainstem, leaving the inner ear intact. Structural assessment of the superior olivary complex (SOC), an essential auditory brainstem center, revealed a dramatic volume reduction (43-47%) of major nuclei in young adult Egr2::Cre;Cacna1d-eGFP(flex) mice. This volume decline was mainly caused by a reduced cell number (decline by 46-56%). Abnormal formation of the lateral superior olive was already present at P4, demonstrating an essential perinatal role of Ca(v)1.3 in the SOC. Measurements of auditory brainstem responses demonstrated a decreased amplitude in the auditory nerve between 50 and 75 dB stimulation in Egr2::Cre;Cacna1d-eGFP(flex) knockout mice and increased amplitudes in central auditory processing centers. Immunohistochemical studies linked the amplitude changes in the central auditory system to reduced expression of K(v)1.2. No changes were observed for K(v)1.1, KCC2, a determinant of inhibitory neurotransmission, and choline acetyltransferase, a marker of efferent olivocochlear neurons. Together, these analyses identify a crucial retrocochlear role of Ca(v)1.3 and demonstrate that mutations in deafness genes can affect sensory cells and neurons alike. As a corollary, hearing aids have to address central auditory processing deficits as well.

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.

Synaptic refinement of an inhibitory topographic map in the auditory brainstem requires functional Cav1.3 calcium channels.

Synaptic refinement via the elimination of inappropriate synapses and strengthening of appropriate ones is crucially important for the establishment of specific, topographic neural circuits. The mechanisms driving these processes are poorly understood, particularly concerning inhibitory projections. Here, we address the refinement of an inhibitory topographic projection in the auditory brainstem in functional and anatomical mapping studies involving patch-clamp recordings in combination with minimal and maximal stimulation, caged glutamate photolysis, and single axon tracing. We demonstrate a crucial dependency of the refinement on Ca(V)1.3 calcium channels: Ca(V)1.3(-/-) mice displayed virtually no elimination of projections up to hearing onset. Furthermore, strengthening was strongly impaired, in line with a reduced number of axonal boutons. The mediolateral topography was less precise and the shift from a mixed GABA/glycinergic to a purely glycinergic transmission before hearing onset did not occur. Together, our findings provide evidence for a Ca(V)1.3-dependent mechanism through which both inhibitory circuit formation and determination of the neurotransmitter phenotype are achieved.

Profilin1 is required for glial cell adhesion and radial migration of cerebellar granule neurons.

Cerebellar granule neurons (CGNs) exploit Bergmann glia (BG) fibres for radial migration, and cell-cell contacts have a pivotal role in this process. Nevertheless, little is known about the mechanisms that control CGN-BG interaction. Here we demonstrate that the actin-binding protein profilin1 is essential for CGN-glial cell adhesion and radial migration. Profilin1 ablation from mouse brains leads to a cerebellar hypoplasia, aberrant organization of cerebellar cortex layers and ectopic CGNs. Conversely, neuronal progenitor proliferation, tangential migration of neurons and BG morphology appear to be independent of profilin1. Our mouse data and the mapping of developmental neuropathies to the chromosomal region of PFN1 suggest a similar function for profilin1 in humans.

An inhibitory glycinergic projection from the cochlear nucleus to the lateral superior olive.

Auditory brainstem neurons in the lateral superior olive (LSO) receive excitatory input from the ipsilateral cochlear nucleus (CN) and inhibitory transmission from the contralateral CN via the medial nucleus of the trapezoid body (MNTB). This circuit enables sound localization using interaural level differences. Early studies have observed an additional inhibitory input originating from the ipsilateral side. However, many of its details, such as its origin, remained elusive. Employing electrical and optical stimulation of afferents in acute mouse brainstem slices and anatomical tracing, we here describe a glycinergic projection to LSO principal neurons that originates from the ipsilateral CN. This inhibitory synaptic input likely mediates inhibitory sidebands of LSO neurons in response to acoustic stimulation.

Cav1.3 calcium channels are required for normal development of the auditory brainstem.

Within the Ca(v)1 family of voltage-gated calcium channels, Ca(v)1.2 and Ca(v)1.3 channels are the predominant subtypes in the brain. Whereas specific functions for each subtype were described in the adult brain, their role in brain development is poorly understood. Here we assess the role of Ca(v)1.3 subunits in the activity-dependent development of the auditory brainstem. We used Ca(v)1.3-deficient (Ca(v)1.3(-/-)) mice because these mice lack cochlea-driven activity that deprives the auditory centers from peripheral input. We found a drastically reduced volume in all auditory brainstem centers (range 25-59%, total 35%), which was manifest before hearing onset. A reduction was not obvious outside the auditory system. The lateral superior olive (LSO) was strikingly malformed in Ca(v)1.3(-/-) mice and had fewer neurons (1/3 less). The remaining LSO neurons displayed normal dendritic trees and received functional glutamatergic input, yet they fired action potentials predominantly with a multiple pattern upon depolarization, in contrast to the single firing pattern prevalent in controls. The latter finding appears to be due to a reduction of dendrototoxin-sensitive potassium conductances, presumably mediated through the K(v)1.2 subtype. Fura2 imaging provided evidence for functional Ca(v)1.3 channels in the LSO of wild-type mice. Our results imply that Ca(v)1.3 channels are indispensable for the development of the central auditory system. We propose that the unique LSO phenotype in Ca(v)1.3(-/-) mice, which hitherto was not described in other hereditary deafness models, is caused by the synergistic contribution of two factors: on-site loss of Ca(v)1.3 channels in the neurons plus lack of peripheral input.