Abstract

The calyx of Held, a large axo-somatic relay synapse containing hundreds of presynaptic active zones, is possibly the largest nerve terminal in the mammalian CNS. Studying its initial growth in-vitro might provide insights into the specification of synaptic connection size in the developing brain. However, attempts to maintain calyces of Held in organotypic cultures have not been fruitful in past studies. Here, we describe an organotypic slice culture method in which calyces of Held form in-vitro. We made coronal brainstem slices with an optimized slice angle using newborn mice in which calyces have not yet formed; the presynaptic bushy cells were genetically labeled using the Math5 promoter. After six to nine days of culturing, we readily observed large Math5—positive nerve terminals in the medial nucleus of the trapezoid body (MNTB), but not in the neighboring lateral superior olive nucleus (LSO). These calyx—like synapses expressed the Ca2+- sensor Synaptotagmin-2 (Syt-2) and the Ca2+ binding protein Parvalbumin (PV), two markers of developing calyces of Held in vivo. Application of the BMP inhibitor LDN-193189 significantly inhibited the growth of calyx synapses, demonstrating the feasibility of long-term pharmacological manipulation using this organotypic culture method. These experiments provide a method for organotypic culturing of calyces of Held, and show that the formation of calyx—like synapses onto MNTB neurons can be preserved in-vitro. Furthermore, our study adds pharmacological evidence for a role of BMP-signaling in the formation of large calyx of Held synapses.

Highlights

  • The calyx of Held synapse of the auditory brainstem has become a model system to study the biophysical mechanisms of presynaptic function, because of its large presynaptic nerve terminal accessible to direct patch-clamp recordings [1,2,3,4]

  • A newborn mouse at a time was removed from the cage, and killed by decapitation without prior anesthesia

  • We first verified whether the Math5Cre line used here drives reporter gene expression sufficiently early and strong to allow for labelling of bushy cell axons at around birth

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Summary

Introduction

The calyx of Held synapse of the auditory brainstem has become a model system to study the biophysical mechanisms of presynaptic function, because of its large presynaptic nerve terminal accessible to direct patch-clamp recordings [1,2,3,4]. There is considerable recent interest in understanding how this large, and highly specialized synapse is formed initially [5,6,7,8,9], because understanding the mechanisms of synapse size specification promises to yield insights into how synaptic circuits are wired during brain development. A slice culture of the growing calyx of Held and by a post-doctoral fellowship to NM (MarieCurie program of the European Union, IEF-235223Calyx-MMFF) http://ec.europa.eu/research/ mariecurieactions/index_en.htm. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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