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Across many parts of the nervous system, initial neural circuit assembly involves stereotyped stages of early axonal proliferation within target territories, selective reinforcement of a subset of inputs and the removal of exuberant or unneeded connections via either activity or experience-dependent mechanisms, in a process referred to as synaptic competition. The mechanisms and associated structural transformations that underlie and define these stages of neurodevelopment have been examined in a variety of brain regions including the peripheral neuromuscular junction, the central climbing fiber innervation of cerebellar Purkinje cells and retinal ganglion cell innervation of the thalamic lateral geniculate nucleus. Each of these connections are considered representative examples of sites of synaptic competition. We and others have established the globular bushy cell (GBC) innervation of principal cells (PC) of the medial nucleus of the trapezoid body (MNTB) in the auditory brainstem via the large calyx of Held (CH) as an additional representative, or model, system for the study of terminal growth and competition. We utilized dense reconstruction of pre and postsynaptic elements from serial block-face scanning electron microscopy (SBEM) image volumes collected during key developmental timepoints from both prehearing (postnatal days (P)2, 3, 4, 6, & 9) and adult (P30) mice. In this document, I report our most recent studies of MNTB ultrastructure, which revealed additional structural elements that define the progressive growth, competition and subsequent maturation of the CH and its innervation of PCs. We found evidence that all developing CHs extend 3 types of collateral arbors including short filopodia (5.85 µm), and growth cone tipped collaterals of sufficient length to innervate adjacent cells or cells within 60 µm, but in some cases up to 100 µm, away from the innervation territory of the CH. Moreover, as the proportion of polyinnervated PCs with competing CH inputs declines from 23% at P6 to 11% at P9, we found a significant reduction in the length of CH collaterals until few if any were found by maturity (P30). Using the first live imaging data collected from a living brain slice with the lattice light sheet microscope, we revealed with high spatial and temporal resolution that CH collaterals exhibited some of the fastest motility recorded in developing neural systems (average growth cone rate of motility: 2.1 µm per min). We also observed interactions among long filopodial collaterals that resulted in retraction and altered growth trajectories suggesting possible repulsive contact-mediated guidance of CH growth and innervation. These may be the predominant locations where competing CHs physically interact, since they typically expand over non-overlapping territories across the cell surface of the PC soma. Postsynaptically, we provided the first systematic description of two types of invaginating spine (type 1 and 2) that reside specifically within recesses underneath and penetrating the developing CH. Similar to the emergence of CH collaterals, we found a strong association of type 1 spine emergence with the initial expansion of the terminal. Moreover, for the first time, somatic spine mats, composed of so-called type 2 spines that intertwine to assemble a macrostructure called the somatic spine mat, were described in the vertebrate CNS. From inspection of EM images, we suggest three modes of intercellular communication between the CH and its synaptic partner, the PC: chemical synaptic transmission, signaling via cell adhesion proteins, trans-endocytosis of spine mats into the CH. We hypothesize that the unifying event to synchronize CH growth and emergence of collaterals and postsynaptic somatic processes is a transformation of spontaneous activity to a bursting pattern. Preliminary experiments reveal that interference with synaptic transmission using viral vector induced expression of tetanus toxin leads to morphological changes in the CH and slower growth of the postsynaptic cell body. Further experiments are proposed that combine electrophysiology and dynamic imaging with molecular and physiological perturbation to further explore these linkages.

Neural circuit formation is a complex process involving coordinated communication between neurons, glia and vascular-associated cells (VACs). Each cell type is responsible for a unique transcriptional and translational contribution to tissue maturation. Deciphering the intercellular signaling patterns which guide neural circuit formation during normal development is thus an essential step in understanding which components of neural circuit formation go awry in neurodevelopmental disorders. The medial nucleus of the trapezoid body (MNTB), located in the auditory brainstem, was used as a model system to study the dynamics of neural circuit formation because it contains a mostly homogeneous population of postsynaptic neurons and the largest nerve terminal in the central nervous system, the calyx of Held (CH). We conducted an extensive cell counting study using light microscopy to characterize changes in the neuronal:nonneuronal cell ratio across development of the MNTB. Using cell type-specific antibodies we obtained the relative percentages of each cell type at P3 and P6, key timepoints for CH growth and refinement to mono-innervation. Significant increases in the glial cell percentage are due mostly to an increase in percentage of oligodendrocytes. Using proliferation and mitotic markers, we demonstrated that oligodendrocytes locally divide within the boundaries of the MNTB during this timeframe. Changes in the neuronal:nonneuronal cell ratio aided in interpretation of transcriptional data obtained from a previously conducted developmental microarray study performed in the MNTB. In the microarray study, many up-regulated transcripts were known to be more highly expressed in glial cells in other brain regions. By connecting cell numbers to transcript level, we were able to determine whether a transcript highly expressed in glial cells was detected as up-regulated due to increases in cell number or due to true transcriptional up-regulation on a per cell basis. In a second study, single cell RNA-Sequencing (scRNA-Seq) allowed us to generate cell type-specific transcriptional profiles for each major cell type in the MNTB. These data added cell type-specific expression information to the developmentally regulated transcripts identified in the microarray study, many of which were previously unassigned to a specific cell type. We identified transcripts related to several major intercellular signaling pathways at P3, including FGF, Delta-Notch, TGFβ and VEGF pathways. In many cases the direction of signaling between cell types could be determined based on expression patterns of transcripts encoding for ligands and their cognate receptors. Most interestingly, we were able to tie transcriptional data with structural changes that occur during MNTB tissue maturation, such as perineuronal net formation and angiogenesis. The combination of cell counting data, temporal transcriptional data at the tissue level and cell type-specific transcriptional data at the single cell level offers a broad picture of the process of neural circuit formation in the MNTB and provides a solid foundation to develop and test new mechanistic hypotheses.

In the auditory brainstem, the connection between globular bushy cells of the anteroventral cochlear nucleus and principal cells (PCs) of the medial nucleus of the trapezoid body (MNTB) is created by one of the largest nerve terminals in the central nervous system, the calyx of Held (CH). The characteristics of the CH:MNTB connection—a short developmental period (48-72 hours), accessibility for recording from pre- and postsynaptic components, and clear monoinnervated end point—make this system an ideal model system for studying nervous system development. Model systems undergo stereotyped stages of development, including exuberant overinnervation, competition between terminals, and a refinement of innervation through removal of weak inputs. However, unlike other similar model systems (climbing fiber:Purkinje cell, retinal ganglion cells:dorsolateral geniculate nucleus), it has been a long-standing question whether the CH:MNTB system undergoes competition. We investigated the innervation state of PCs using the novel technique of segmentation and 3D reconstruction of PCs and their associated inputs across important developmental timepoints (postnatal days (P)2,3,4,6,9,30). This was accomplished by application of serial block-face scanning electron microscopy (SBEM), a method of serial section electron microscopy providing high spatial resolution (~4-10nm) and a high degree of alignment between images with very little section loss. Applying this technique, we show early exuberant innervation of PCs (P2), establish that competition is a common process, and pinpoint the 24-hour period from P3-P4 as a uniquely active day in CH growth during which terminal contact with PCs increases at a rate exceeding 200 µm2/day. Common morphological characteristics of the CH:MNTB connection also became qualitatively evident based on 3D reconstructions, particularly an eccentric PC nucleus and preference for polarized terminal growth. Based on these observations, we undertook a quantitative study of polarity in CH:MNTB development using our 3D reconstructions. The results of this investigation demonstrate a novel polarity in development of both the CH and PC; developing PCs are characterized by eccentrically placed nuclei that establish an “intrasomatic polarity” that persists through young adulthood (P30). This polarity appears to define a unique territory opposite of the nuclear location that is amenable to growth of the calyx, is enriched in dendrites, and is selectively enlarged as the principal cell matures to create glia-free surface area for innervation. To our knowledge, this is the first report of a polarity program in the coordinated pre- and postsynaptic development of a non-laminar brain region. Additionally, our findings have codified a progression of dendritic pruning in the maturation of principal cells that may influence and be influenced by the developmental state of the cell. Taken in totality, these results indicate a highly polarized, systemic competitive process in the MNTB during the development of the calyx of Held and suggests potential mediators of competition that deserve further study.

The auditory brainstem has been the area of much focus in recent years due to its advantages in circuitry and synaptic connections. The endbulbs and calyces of Held are large, glutamatergic synapses within the AVCN and MNTB, respectively. Because of their large size and accessibility these synapses have offered the ability to study pre- and post-synaptic mechanisms of neurotransmission within the central nervous system. The calyx of Held synapse is the main excitatory input into the MNTB principal cells, containing hundreds of individual synaptic specializations, or release sites. Previous studies have identified physiological and morphological changes at the endbulb of Held synapse but not the calyx of Held synapse. However, changes in inhibitory innervation at the MNTB were observed as a result of altered input. In this study we have utilized the calyx of Held synapse, apposing inhibitory terminals within the MNTB of CBA/J and congenitally deaf mice (dn/dn) to examine the role of altered input on synaptic strength. The dn/dn strain is of particular importance because they exhibit no spontaneous activity within the VIIIth nerve fiber, lending itself as a valuable model in the altered input studies, particularly an absence of afferent input. Serial electron microscopic analysis of the fine structure of the calyx of Held and inhibitory contacts onto the MNTB principal cells were compared between normal and deaf. Strikingly, the calyceal active zones within the adult deaf animals were significantly larger compared with normal hearing animals despite the fact there were no changes in excitatory currents. Examination of the inhibitory terminals confirmed the increase in mIPSC frequency manifested in an increase in inhibitory synapses expressed on the MNTB principal cells.

This experiment analyzes synaptic differences in the central auditory pathway between normal hearing and congenitally deaf (dn/dn) mice, and provides valuable insight into central changes that correspond with human congenital deafness. Specifically, this experiment analyzes developmental expression of the Calcium (Ca2+)-binding proteins Calretinin (CR), Calbindin D-28k (CB) and Parvalbumin (PV) in large excitatory synapses in the anteroventral cochlear nucleus (AVCN) and the medial nucleus of the trapezoid body (MNTB) of normal and dn/dn mice. Immunofluorescence imaging with primary antibodies detecting CR, CB or PV was used to analyze the expression of each at 9 days, 13 days, 20 days, 30 days and 49 days postnatal in normal and dn/dn mice. Results indicated that Ca2+-binding expression was similar at each location in normal and dn/dn mice at 9 days postnatal, prior to opening of the ear canal and the onset of hearing (which occurs around 11 days postnatal) . In normal mice, patterns of Ca2+-binding protein expression changed progressively after the onset of hearing. In dn/dn mice (which completely lack auditory nerve activity), however, patterns of expression did not change after the onset of hearing, suggesting that patterns of Ca2+-binding protein expression change during development in normal mice in response to evoked auditory nerve activity, and that patterns of Ca2+-binding protein expression do not change during development in dn/dn mice due to lack of evoked auditory nerve activity. As a result, Ca2+ buffering is impaired in synapses located in the AVCN and MNTB of dn/dn mice.