The principal neurons of the medial nucleus of the trapezoid body and NG2+ glial cells receive coordinated excitatory synaptic input

Glial cell processes are part of the synaptic structure and sense spillover of transmitter, while some glial cells can even receive direct synaptic input. Here, we report that a defined type of glial cell in the medial nucleus of the trapezoid body (MNTB) receives excitatory glutamatergic synaptic input from the calyx of Held (CoH). This giant glutamatergic terminal forms an axosomatic synapse with a single principal neuron located in the MNTB. The NG2 glia, as postsynaptic principal neurons, establish synapse-like structures with the CoH terminal. In contrast to the principal neurons, which are known to receive excitatory as well as inhibitory inputs, the NG2 glia receive mostly, if not exclusively, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor–mediated evoked and spontaneous synaptic input. Simultaneous recordings from neurons and NG2 glia indicate that they partially receive synchronized spontaneous input. This shows that an NG2⁺ glial cell and a postsynaptic neuron share presynaptic terminals.     

Developmental changes in intrinsic excitability of principal neurons in the rat medial nucleus of the trapezoid body

The calyx of Held synapse is a giant axosomatic synapse that has a fast relay function within the sound localization circuit of the brainstem. In the adult, each principal neuron of the medial nucleus of the trapezoid body (MNTB) is contacted by a single calyx terminal. In rodents, the calyx of Held synapse forms around the third postnatal day (P3). Here, we studied the developmental changes in the intrinsic excitability of the principal neurons during the first postnatal week by making whole-cell recordings from brainstem slices. In slices from P0-1 rats, about 20% of the principal neurons were spontaneously active, whereas after P3, no spontaneously active cells were observed. Already at P0, principal neurons received both glutamatergic and GABAergic/glycinergic inputs. The occurrence of spontaneous action potentials depended upon the presence of spontaneous glutamatergic inputs; summation of only a few quanta was enough to reach action potential threshold. The main cause for this high excitability was a high resting membrane resistance, which decreased at least four-fold during the first postnatal week. A relatively slow decay of synaptic currents and a relatively depolarized membrane potential may have contributed as well. We conclude that the decrease in the excitability of principal neurons in the MNTB matches the increase of the strength of the synaptic inputs resulting from the formation and maturation of the calyx of Held synapse during the first postnatal week. This decrease in excitability will make it progressively more difficult for non-calyceal inputs to trigger action potentials.     

Role of BMP Signaling for the Formation of Auditory Brainstem Nuclei and Large Auditory Relay Synapses

Large excitatory synapses are found at specific points in the neuronal circuits of the auditory brainstem, to enable fast information transfer and the preservation of acoustic timing information. The extracellular cues and signaling mechanisms that lead to the development of these specialized synaptic connections, exemplified by the calyx of Held in the medial nucleus of the trapezoid body (MNTB), are still largely unknown. Here, we investigate the role of BMP signaling for the early development of the ventral cochlear nucleus (VCN) and MNTB, and for the initial formation of the calyx of Held synaptic connection. We used conditional alleles of two BMP type‐1 receptors in the background of a constitutive BMPR1b knock‐out (KO), or else a conditional allele of SMAD4. The conditional alleles were recombined by the Krox20^Cre mouse line that is active around mid‐gestation in rhombomeres (r) 3 and 5 from which the VCN and MNTB are derived; alternatively, virus‐mediated Cre‐expression was performed early postnatally in the VCN. The data show that embryonic SMAD‐dependent BMP‐signaling in r3 and r5 contributes to the histogenesis of auditory brainstem nuclei. On the other hand, BMP‐receptor signaling early postnatally in presynaptic neurons of the calyx of Held projection is necessary for correct axon branch retraction, which suggests a cell‐autonomous role of presynaptic BMP‐receptors in synapse elimination at the developing calyx of Held. Thus, our work dissects developmentally early and late roles of BMP‐signaling for the formation of auditory brainstem nuclei, and the highly specialized synaptic connectivity in these structures.     

Neuron-derived FGF9 is essential for scaffold formation of Bergmann radial fibers and migration of granule neurons in the cerebellum

Although fibroblast growth factor 9 (FGF9) is widely expressed in the central nervous system (CNS), the function of FGF9 in neural development remains undefined. To address this question, we deleted the Fgf9 gene specifically in the neural tube and demonstrated that FGF9 plays a key role in the postnatal migration of cerebellar granule neurons. Fgf9-null mice showed severe ataxia associated with disrupted Bergmann fiber scaffold formation, impaired granule neuron migration, and upset Purkinje cell maturation. Ex vivo cultured wildtype or Fgf9-null glia displayed a stellate morphology. Coculture with wildtype neurons, but not Fgf9-deficient neurons, or treating with FGF1 or FGF9 induced the cells to adopt a radial glial morphology. In situ hybridization showed that Fgf9 was expressed in neurons and immunostaining revealed that FGF9 was broadly distributed in both neurons and Bergmann glial radial fibers. Genetic analyses revealed that the FGF9 activities in cerebellar development are primarily transduced by FGF receptors 1 and 2. Furthermore, inhibition of the MAP kinase pathway, but not the PI3K/AKT pathway, abrogated the FGF activity to induce glial morphological changes, suggesting that the activity is mediated by the MAP kinase pathway. This work demonstrates that granule neurons secrete FGF9 to control formation of the Bergmann fiber scaffold, which in turn, guides their own inward migration and maturation of Purkinje cells.     

EphB signaling regulates target innervation in the developing and deafferented auditory brainstem

Precision in auditory brainstem connectivity underlies sound localization. Cochlear activity is transmitted to the ventral cochlear nucleus (VCN) in the mammalian brainstem via the auditory nerve. VCN globular bushy cells project to the contralateral medial nucleus of the trapezoid body (MNTB), where specialized axons terminals, the calyces of Held, encapsulate MNTB principal neurons. The VCN-MNTB pathway is an essential component of the circuitry used to compute interaural intensity differences that are used for localizing sounds. When input from one ear is removed during early postnatal development, auditory brainstem circuitry displays robust anatomical plasticity. The molecular mechanisms that control the development of auditory brainstem circuitry and the developmental plasticity of these pathways are poorly understood. In this study we examined the role of EphB signaling in the development of the VCN-MNTB projection and in the reorganization of this pathway after unilateral deafferentation. We found that EphB2 and EphB3 reverse signaling are critical for the normal development of the projection from VCN to MNTB, but that successful circuit assembly most likely relies upon the coordinated function of many EphB proteins. We have also found that ephrin-B reverse signaling repels induced projections to the ipsilateral MNTB after unilateral deafferentation, suggesting that similar mechanisms regulate these two processes.     

Deficiency of neural recognition molecule NB-2 affects the development of glutamatergic auditory pathways from the ventral cochlear nucleus to the superior olivary complex in mouse

Neural recognition molecule NB-2/contactin 5 is expressed transiently during the first postnatal week in glutamatergic neurons of the central auditory system. Here, we investigated the effect of NB-2 deficiency on the auditory brainstem in mouse. While almost all principal neurons are wrapped with the calyces of Held in the medial nucleus of the trapezoid body (MNTB) in wild type, 8% of principal neurons in NB-2 knockout (KO) mice lack the calyces of Held at postnatal day (P) 6. At P10 and P15, apoptotic principal neurons were detected in NB-2 KO mice, but not in wild type. Apoptotic cells were also increased in the ventral cochlear nucleus (VCN) of NB-2 KO mice, which contains bushy neurons projecting to the MNTB and the lateral superior olive (LSO). At the age of 1 month, the number of principal neurons in the MNTB and of glutamatergic synapses in the LSO was reduced in NB-2 KO mice. Finally, interpeak latencies for auditory brainstem response waves II-III and III-IV were significantly increased in NB-2 KO mice. Together, these findings suggest that NB-2 deficiency causes a deficit in synapse formation and then induces apoptosis in MNTB and VCN neurons, affecting auditory brainstem function.     

Neuron–astrocyte interactions in the medial nucleus of the trapezoid body

The calyx of Held (CoH) synapse serves as a model system to analyze basic mechanisms of synaptic transmission. Astrocyte processes are part of the synaptic structure and contact both pre- and postsynaptic membranes. In the medial nucleus of the trapezoid body (MNTB), midline stimulation evoked a current response that was not mediated by glutamate receptors or glutamate uptake, despite the fact that astrocytes express functional receptors and transporters. However, astrocytes showed spontaneous Ca²⁺ responses and neuronal slow inward currents (nSICs) were recorded in the postsynaptic principal neurons (PPNs) of the MNTB. These currents were correlated with astrocytic Ca²⁺ activity because dialysis of astrocytes with BAPTA abolished nSICs. Moreover, the frequency of these currents was increased when Ca²⁺ responses in astrocytes were elicited. NMDA antagonists selectively blocked nSICs while D-serine degradation significantly reduced NMDA-mediated currents. In contrast to previous studies in the hippocampus, these NMDA-mediated currents were rarely synchronized.     

Methods for patch clamp capacitance recordings from the calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.     

Reliability of Synaptic Transmission at the Synapses of Held In Vivo under Acoustic Stimulation

Background

The giant synapses of Held play an important role in high-fidelity auditory processing and provide a model system for synaptic transmission at central synapses. Whether transmission of action potentials can fail at these synapses has been investigated in recent studies. At the endbulbs of Held in the anteroventral cochlear nucleus (AVCN) a consistent picture emerged, whereas at the calyx of Held in the medial nucleus of the trapezoid body (MNTB) results on the reliability of transmission remain inconsistent. In vivo this discrepancy could be due to the difficulty in identifying failures of transmission.

Methods/Findings

We introduce a novel method for detecting unreliable transmission in vivo. Based on the temporal relationship between a cells'' waveform and other potentials in the recordings, a statistical test is developed that provides a balanced decision between the presence and the absence of failures. Its performance is quantified using simulated voltage recordings and found to exhibit a high level of accuracy. The method was applied to extracellular recordings from the synapses of Held in vivo. At the calyces of Held failures of transmission were found only rarely. By contrast, at the endbulbs of Held in the AVCN failures were found under spontaneous, excited, and suppressed conditions. In accordance with previous studies, failures occurred most abundantly in the suppressed condition, suggesting a role for inhibition.

Conclusions/Significance

Under the investigated activity conditions/anesthesia, transmission seems to remain largely unimpeded in the MNTB, whereas in the AVCN the occurrence of failures is related to inhibition and could be the basis/result of computational mechanisms for temporal processing. More generally, our approach provides a formal tool for studying the reliability of transmission with high statistical accuracy under typical in vivo recording conditions.     

TGF-? Regulates Enamel Mineralization and Maturation through KLK4 Expression

Transforming growth factor-ß (TGF-ß) signaling plays an important role in regulating crucial biological processes such as cell proliferation, differentiation, apoptosis, and extracellular matrix remodeling. Many of these processes are also an integral part of amelogenesis. In order to delineate a precise role of TGF-ß signaling during amelogenesis, we developed a transgenic mouse line that harbors bovine amelogenin promoter-driven Cre recombinase, and bred this line with TGF-ß receptor II floxed mice to generate ameloblast-specific TGF-ß receptor II conditional knockout (cKO) mice. Histological analysis of the teeth at postnatal day 7 (P7) showed altered enamel matrix composition in the cKO mice as compared to the floxed mice that had enamel similar to the wild-type mice. The µCT and SEM analyses revealed decreased mineral content in the cKO enamel concomitant with increased attrition and thinner enamel crystallites. Although the mRNA levels remained unaltered, immunostaining revealed increased amelogenin, ameloblastin, and enamelin localization in the cKO enamel at the maturation stage. Interestingly, KLK4 mRNA levels were significantly reduced in the cKO teeth along with a slight increase in MMP-20 levels, suggesting that normal enamel maturation is regulated by TGF-ß signaling through the expression of KLK4. Thus, our study indicates that TGF-ß signaling plays an important role in ameloblast functions and enamel maturation.     

Short-term plasticity at the calyx of held

Synapses show widely varying degrees of short-term facilitation and depression. Several mechanisms have been proposed to underlie short-term plasticity, but the contributions of presynaptic mechanisms have been particularly difficult to study because of the small size of synaptic boutons in the mammalian brain. Here we review the functional properties of the calyx of Held, a giant nerve terminal that has shed new light on the general mechanisms that control short-term plasticity. The calyx of Held has also provided fresh insights into the strategies used by synapses to extend their dynamic range of operation and preserve the timing of sensory stimuli.     

Expression of members of the Fgf family and their receptors during midfacial development

Members of the FGF family play diverse roles in patterning, cell proliferation and differentiation during embryogenesis. To begin to address their function during craniofacial development we have analyzed the expression of 18 members of the Fgf family (Fgf1-15, -17, -18 and -20) and the four members of the FGF-receptor family in the prospective midfacial region between E9.5 and E11.5 by whole-mount in situ hybridization. We show that at E9.5, Fgf3, -8, -9, -10 and -17 are broadly expressed in midfacial ectoderm. Concomitant with the outgrowth of the nasal processes at E10.5, expression of Fgf3, -8, -9, -10, -15, -17 and -18 was detected in spatially restricted regions of ectoderm at the edge of the nasal pit and at the oral edge of the medial nasal process. Expression of Fgf8, Fgf9, Fgf10 and Fgf17 was still observed in these domains at E11.5. In contrast to the restricted expression patterns of the ligands, FgfR1 and FgfR2 were broadly expressed in facial mesenchyme and ectoderm, respectively, indicating a wide competence of midfacial tissue to respond to FGF signaling.     

Runx1 is involved in the fusion of the primary and the secondary palatal shelves

Runx1 is expressed in medial edge epithelial (MEE) cells of the palatal shelf. Conditionally rescued Runx1^−/− mice showed limited clefting in the anterior junction between the primary and the secondary palatal shelves, but not in the junction between the secondary palates. In wild type mice, the fusing epithelial surface exhibited a rounded cobblestone-like appearance, while such cellular prominence was less evident in the Runx1 mutants. We also found that Fgf18 was expressed in the mesenchyme underlying the MEE and that locally applied FGF18 induced ectopic Runx1 expression in the epithelium of the palatal explants, indicating that Runx1 was induced by mesenchymal Fgf18 signaling. On the other hand, unpaired palatal explant cultures revealed the presence of anterior-posterior (A-P) differences in the MEE fates and fusion mechanism. Interestingly, the location of anterior clefting in Runx1 mutants corresponded to the region with different MEE behavior. These data showed a novel function of Runx1 in morphological changes in the MEE cells in palatal fusion, which is, at least in part, regulated by the mesenchymal Fgf signaling via an epithelial-mesenchymal interaction.     

Hopf bifurcation in the presomitic mesoderm during the mouse segmentation

Vertebrae and ribs arise from embryonic tissues called somites. Somites arise sequentially from the unsegmented embryo tail, called presomitic mesoderm (PSM). The pace of somite formation is controlled by gene products such as hairy and enhancer of split 7 (Hes7) whose expression oscillates in the PSM. In addition to the cyclic genes, there is a gradient of fibroblast growth factor 8 (Fgf8) mRNA from posterior to anterior PSM. Recent experiments have shown that in the absence of Fgf signaling, Hes7 oscillations in the anterior and posterior PSM are lost. On the other hand, Notch mutants reduce the amplitude of posterior Hes7 oscillations and abolish anterior Hes7 oscillations. To understand these phenotypes, we delineated and simulated a logical and a delay differential equation (DDE) model with similar network topology in wild-type and mutant situations. Both models reproduced most wild-type and mutant phenotypes suggesting that the chosen topology is robust to explain these phenotypes. Numerical continuation of the model showed that even in the wild-type situation, the system changed from sustained to damped, i.e. a Hopf bifurcation occurred, when the Fgf concentration decreased in the PSM. This numerical continuation analysis further indicated that the most sensitive parameters for the oscillations are the parameters of Hes7 followed by those of Lunatic fringe (Lfng) and Notch1. In the wild-type, the damping of Hes7 oscillations was not so strong so that cells reached the new somites before they lose Hes7 oscillations. By contrast, in the fibroblast growth factor receptor 1 (Fgfr1) conditional knock-out (cKO) mutant simulation, Notch signaling was not able to maintain sustained Hes7 oscillations. Our analysis suggests that Fgf signaling makes cells enter an oscillatory state of Hes7 expression. After moving to the anterior PSM, where Fgf signaling is missing, Notch signaling compensates the damping of Hes7 oscillations in the anterior PSM.     

In vivo and in vitro comparison of the effects of FGF-2 null and haplo-insufficiency on bone formation in mice

We previously reported that deletion of the Fgf2 gene (Fgf2-/-) resulted in decreased bone mass in adult mice. This study examines the effect of haplo-insuffiency (Fgf2+/-) on bone loss in vertebrae from these mutant mice. Fgf2+/+ mice attained peak bone mass at 8-9 months of age. In contrast BMD was significantly reduced in vertebrae from adult (8-9) Fgf2+/- mice. Exogenous FGF-2 rescued reduced bone nodule formation in Fgf2+/- and Fgf2-/- cultures. Runx2 mRNA was reduced in cultures from Fgf2+/- and Fgf2-/- mice. FGF receptor2 mRNA and protein were markedly reduced in Fgf2+/- and Fgf2-/- mice. Decreased bone formation in Fgf2 mutant mice may correlate with impaired FGFR signaling, decreased Runx2 gene expression.