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

Détails

Ressource 1Télécharger: pone.0175964 (1).pdf (4316.50 [Ko])
Etat: Public
Version: Final published version
ID Serval
serval:BIB_B02731B862FB
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
An organotypic slice culture to study the formation of calyx of Held synapses in-vitro.
Périodique
PloS one
Auteur⸱e⸱s
Kronander E., Michalski N., Lebrand C., Hornung J.P., Schneggenburger R.
ISSN
1932-6203 (Electronic)
ISSN-L
1932-6203
Statut éditorial
Publié
Date de publication
2017
Peer-reviewed
Oui
Volume
12
Numéro
4
Pages
e0175964
Langue
anglais
Notes
Publication types: Journal Article
Publication Status: epublish
Résumé
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.

Mots-clé
Animals, Auditory Pathways, Axons/drug effects, Axons/physiology, Axons/ultrastructure, Basic Helix-Loop-Helix Transcription Factors/analysis, Basic Helix-Loop-Helix Transcription Factors/genetics, Bone Morphogenetic Proteins/antagonists & inhibitors, Brain Stem/cytology, Brain Stem/drug effects, Brain Stem/physiology, Brain Stem/ultrastructure, Mice, Nerve Tissue Proteins/analysis, Nerve Tissue Proteins/genetics, Organ Culture Techniques/methods, Parvalbumins/analysis, Promoter Regions, Genetic, Pyrazoles/pharmacology, Pyrimidines/pharmacology, Synapses/drug effects, Synapses/physiology, Synapses/ultrastructure, Synaptotagmin II/analysis
Pubmed
Open Access
Oui
Création de la notice
25/04/2017 18:28
Dernière modification de la notice
20/08/2019 15:19
Données d'usage