An organotypic slice culture to study the formation of calyx of Held synapses in-vitro

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.

Specific synaptic input strengths determine the computational properties of excitation–inhibition integration in a sound localization circuit

“…; Kronander et al . ) revealed small EPSCs of ∼50 pA amplitudes at the threshold for successful stimulation (Fig. ); similar observations were made with optogenetic stimulation of excitatory inputs under the PV‐promoter (Fig.…”

Kinetics of transmitter release at the calyx of Held synapse

“…The next step is to examine the molecular mechanisms of transmitter release and vesicle replenishment. Recent developments in slice and dissociated culture, 63 , 64 ) together with imaging 51 ) and possible molecular perturbations 65 – 67 ) offer unique possibilities for gaining detailed knowledge on the molecular mechanisms of synaptic transmission. Recent developments in optical microscopy have also revealed physical correlates of the transmitter release processes, as postulated by electrophysiology.…”

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

“…Brain sections of 30–40 µm thickness were obtained using a sliding Microtome (HYRAX S30; Zeiss). Processing of the sections for immunohistochemistry was done as described before (Kronander et al ., ). Primary antibodies were: goat anti‐MafB (Santa Cruz sc10022; RRID AB_648633, dilution 1/500); rabbit anti‐Calbindin D‐28k (Swant CB‐38a, RRID AB_10000340, dilution 1/500); chicken anti‐GFP (AbCam 13970 RRID AB_300798 dilution 1/1000); rabbit anti‐Parvalbumin 25 (PV‐25 RRID AB_10000344, dilution 1/2000); mouse anti‐Synaptotagmin 2 (znp‐1, ZIRC; RRID AB_10013783, dilution 1/500).…”

“…The maturation of functional parameters of synaptic transmission which takes places at the calyx of Held around hearing onset has been studied intensely (Joshi and Wang, ; Taschenberger et al ., ; Takahashi, ); hearing onset occurs in rodents at ~ postnatal days (P) 10–14. Nevertheless, the molecular mechanisms which drive the initial formation of the characteristic 1:1 connection of the calyx of Held synapse are only beginning to be understood (Toyoshima et al ., ; Xiao et al ., ; Dimitrov et al ., ; Kronander et al ., ).…”