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

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.


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