Organotypic coculture preparation for the study of developmental synapse elimination in mammalian brain

• Published in: The Journal of neuroscience 2012-08, Vol.32 (34), p.11657-11670
• Main Authors: Uesaka, Naofumi, Mikuni, Takayasu, Hashimoto, Kouichi, Hirai, Hirokazu, Sakimura, Kenji, Kano, Masanobu
• Format: Article
• Language: English
• Subjects: Age Factors; Animals; Animals, Newborn; Biophysics; Cell Adhesion Molecules, Neuronal - genetics; Cell Adhesion Molecules, Neuronal - metabolism; Cell Line, Transformed; Cerebellum - cytology; Cerebellum - embryology; Cerebellum - growth & development; Coculture Techniques; Electric Stimulation; Electroporation; Embryo, Mammalian; Excitatory Amino Acid Agents - pharmacology; Excitatory Postsynaptic Potentials - drug effects; Excitatory Postsynaptic Potentials - genetics; Green Fluorescent Proteins - genetics; Humans; Medulla Oblongata - cytology; Membrane Proteins - metabolism; Mice; Mice, Inbred C57BL; Mice, Knockout; MicroRNAs - metabolism; Mutation - genetics; Nerve Fibers - physiology; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Neurons - drug effects; Neurons - physiology; Organ Culture Techniques; Patch-Clamp Techniques; Receptors, Glutamate - deficiency; Receptors, N-Methyl-D-Aspartate - genetics; Receptors, N-Methyl-D-Aspartate - metabolism; RNA Interference - physiology; Statistics, Nonparametric; Synapses - drug effects; Synapses - physiology; Transfection
• ISSN: 0270-6474; 1529-2401; 1529-2401
• DOI: 10.1523/jneurosci.1097-12.2012

We developed an organotypic coculture preparation allowing fast and efficient identification of molecules that regulate developmental synapse elimination in the mammalian brain. This coculture consists of a cerebellar slice obtained from rat or mouse at postnatal day 9 (P9) or P10 and a medullary explant containing the inferior olive dissected from rat at embryonic day 15. We verified that climbing fibers (CFs), the axons of inferior olivary neurons, formed functional synapses onto Purkinje cells (PCs) in the cerebellum of cocultures. PCs were initially reinnervated by multiple CFs with similar strengths. Surplus CFs were eliminated subsequently, and the remaining CFs became stronger. These changes are similar to those occurring in developing cerebellum in vivo. Importantly, the changes in CF innervations in cocultures involved the same molecules required for CF synapse elimination in vivo, including NMDA receptor, type 1 metabotropic glutamate receptor and glutamate receptor δ2 (GluRδ2). We demonstrate that gain- and loss-of-function analyses can be efficiently performed by lentiviral-mediated overexpression and RNAi-induced knockdown of GluRδ2. Using this approach, we identified neuroligin-2 as a novel molecule that promotes CF synapse elimination in postsynaptic PCs. Thus, our coculture preparation will greatly facilitate the elucidation of molecular mechanisms of synapse elimination.