Identification of Signaling Mechanisms Determining the Development of Large Excitatory Synapses in the Lower Auditory System

In the brainstem auditory circuit of mammals and birds, excitatory synapses with extraordinarily large size have evolved, which ensure fast membrane potential signaling mediated by large, multiquantal excitatory postsynaptic currents (EPSCs). The so-called "calyx of Held" synapses are formed by the globular bushy cells (GBCs) in the ventral cochlear nucleus (VCN) onto the contralateral medial nucleus of the trapezoid body (MNTB) neurons. One the other hand, the spherical bushy cells (SBCs) in the VCN project to the lateral superior olive (LSO), where they form small bouton-like synapses. Therefore, the lower auditory brainstem circuits constitute an example of synapse-specificity, in which presynaptic neuron pools are connected through highly specific synapses to their postsynaptic partner neurons. Calyx of Held synapses are formed at postnatal day 2 - 4 in rodents in a target-cell specific manner and prior to the onset of hearing. The molecular signaling pathways which drive the formation of these synapses are unknown. Here we identify bone morphogenetic protein (BMP) signaling as an essential signaling pathway in the development of calyces of Held. Through unbiased genome-wide transcriptome analyses in rats and mice, BMPs were identified as candidates for diffusible signaling molecules which are expressed at a higher level in the MNTB (the target area of calyces of Held), as compared to the LSO. The microarray analysis was validated by quantitative PCR (qPCR) and in-situ hybridisation. A conditional knock-out (KO) of BMP-receptor 1a (BMPR1a) in the lower auditory system, in the genetic background of a conventional KO of BMP-receptor 1b (BMPR1b), was then used to address the role of BMP-receptor signaling for calyx of Held formation in vivo. Genetically knocking out BMPR1a/1b activity in the auditory brainstem led to a drastic deficit at the calyx of Held synapses both morphologically and functionally, as well as to the persistence of multiple innervation of MNTB neurons. This study therefore shows that BMP signaling drives the development of the large calyx of Held synapses. The study offers a possible mechanism for the specificity of a large excitatory synapse formation in the central nervous systems (CNS). In addition, the study shows a novel function of BMP signaling in synaptogenesis in the mammalian CNS.

BMP signaling specifies the development of a large and fast CNS synapse

Enida Gjoni, Graham Knott, Elin Kristina Kronander, Ralf Schneggenburger, Le Xiao

excitatory synapses with multiple active zones ensure reliable and fast information transfer at specific points in neuronal circuits. However, the mechanisms that determine synapse size in CNS circuits are largely unknown. Here we use the calyx of Held synapse, a major relay in the auditory system, to identify and study signaling pathways that specify large nerve terminal size and fast synaptic transmission. Using genome-wide screening, we identified bone morphogenetic proteins (BMPs) as candidate signaling molecules in the area of calyx synapses. Conditional deletion of BMP receptors in the auditory system of mice led to aberrations of synapse morphology and function specifically at the calyx of Held, with impaired nerve terminal growth, loss of monoinnervation and less mature transmitter release properties. Thus, BMP signaling specifies large and fast-transmitting synapses in the auditory system in a process that shares homologies with, but also extends beyond, retrograde BMP signaling at Drosophila neuromuscular synapses.

Nature Publishing Group2013

Genetic dissection of the role of BMP signaling for the development of the auditory brainstem nuclei and the large calyx of Held synapse

Elin Kristina Kronander

Synapses are key elements for communication within the complex network of neurons that make up our brain. Interestingly, synaptic connections can differ greatly in form and function, depending on the specific circuit into which they are embedded. In this PhD thesis, I use a large excitatory synaptic connection in the auditory brainstem, the calyx of Held, as a model to study the molecular mechanisms of synapse development. We are especially interested in the role of bone morphogenetic protein (BMP) signaling in this process. BMPs are extracellular morphogens with signaling roles early in the development of multicellular animals, and in brain development. In the first part of this PhD thesis, I have established an organotypic culture model of the auditory brainstem, in which large calyx-like synapses form in vitro. I could demonstrate the usefulness of this culture system for studying molecular mechanisms of nerve terminal growth, by showing that the BMP-receptor inhibitor, LDN-193189, leads to a reduction in the size of nerve terminals forming in vitro. In the second part, I have used mouse genetics, especially floxed alleles of BMP-receptors and the SMAD4 protein, coupled with morphological analysis and electrophysiological measurements, to investigate the role of BMP signaling in the development of large calyx of Held nerve terminals. Genetic inactivation of BMP type I receptors early-on (~ embryonic day 9 (E9) led to a significantly smaller ventral cochlear nucleus (VCN) and medial nucleus of the trapezoid body (MNTB), indicating that BMP signaling is involved in the histogenesis of these auditory brainstem nuclei. These deficits were in part caused by the intracellular SMAD - signaling pathway, as revealed by conditional inactivation of the SMAD4 gene. To circumvent morphological changes observed upon the early inactivation of BMP receptors, I investigated the onset of Cre expression in a Parvalbumin (PV) Cre mouse line where recombination happens in presynaptic VCN neurons at ~ E15, and in postsynaptic MNTB neurons at postnatal day (P) 6. Conditional inactivation of BMP-receptors using the PVCre mouse line did not lead to morphological alterations. However, in these conditional KO mice, the functional development of the calyx of Held synapse was not significantly altered when measured at P6 (only the presynaptic receptors should be removed), nor at P14 or at P21, at which both pre- and postsynaptic receptors should be removed. Thus, presynaptic BMP-receptor activation alone seems unlikely to be necessary for calyx of Held growth. Also, ongoing BMP-signaling in both pre- and postsynaptic compartments seems dispensable for later maturation processes and the maintenance of the calyx of Held synapse. Finally, I removed BMP receptors specifically in the presynaptic neuron pool via virus-mediated expression of Cre-recombinase in newborn mice. I then observed subtle changes in the branching pattern of presynaptic axons and a slight increase in double-innervations. Taken together, this study shows that BMP signaling plays a role in the histogenesis of auditory brainstem structures and partially in the growth of calyx of Held synapses and elimination of competing synaptic connections. This work also offers a deeper understanding into the use of mouse genetic tools for studying the development of specific synaptic connections and establishes a novel culture model for studying such connections in vitro.

EPFL2016

Structural architecture of the Neuronal-Glial-Vascular system

Eleftherios Zisis

Brain functionality relies on the neuronal-glial-vascular (NGV) ensemble for energy support. However, the details of the complex biological mechanisms involved in these processes and the microscopic interactions between these three components are not yet fully understood. Astrocytes, an essential component of the NGV ensemble, extend ramified processes to the vasculature and to neuronal synapses to provide neuronal energy support, homeostatic control, and multi-directional signaling with the vasculature, neurons, and neighboring astrocytes. As key links between the components of the NGV circuitry, the astrocytes are essential for drug delivery in the brain and are involved in the progression of neurodegenerative diseases. Therefore, comprehensive models of the neuronal-glial-vascular (NGV) ensemble are essential for understanding the role of astrocytes in the formation and function of the complex networks within the brain and its pathological conditions. A complete computational model of this ensemble has not yet been developed. This thesis presents the first model of a data-driven digital reconstruction of the neuronal-glial-vascular (NGV) ensemble at a micrometer anatomical resolution. Combining the sparse literature data and the few available detailed biological reconstructions, I have computationally generated the structural architecture of a neocortical NGV circuit that forms a functional column of the juvenile rat neocortex and consists of neurons, protoplasmic astrocytes, and the microvasculature, including their pairwise connectivities. This data-driven approach allows for incremental refinement as more experimental data become available, new biophysical models get published, and new questions arise. The NGV circuit is validated against a plethora of literature sources to ensure its biological fidelity: it successfully reproduces the spatial organization of the astrocytes, their morphological characteristics as well as their volume occupancy, and the overlap with their neighbors. The power of the computational model of NGV lies in its ability to serve as a framework for addressing long-standing questions that cannot be experimentally investigated due to the complexity of the microscopic systems and the limitations of current techniques to observe all components simultaneously. In this thesis, I have performed experiments to investigate why astrocytes acquire their particular shapes, the organizational principles of NGV that lead to the observed biological network architectures, and the effect of astrocytic density on endfoot organization. The circuit's structural analysis showed that astrocytes optimize their positions and spacing from each other to provide the vasculature with a uniform coverage for trophic support and signaling. By increasing the density of astrocytes in NGV circuits, I discovered a limit in astrocyte's ability to make perivascular projections because of the vascular spatial occupancy, which constrains the extent of astrocyte morphology. Thus, their role in linking vasculature to neurons constrains their organization via the proportional relationship of density and microdomain shrinkage due to contact spacing. By addressing these questions, I demonstrated how this model can serve as an exploratory tool that provides a window into the complexity of the NGV architecture.