MOLEDULARBIOLOGICAL ANALYSIS OF SYNAPTSE FORMATION IN CEREBELLAR PURKINJE CELL

2001 Fiscal Year Final Research Report Summary

• Project/Area Number: 09680804
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Neuroscience in general
• Research Institution: AKITA UNIVERSITY
• Principal Investigator: MEGURO Hiroyuki Akita University School of Medicine, Research Associate, 医学部, 助手 (60291093)
• Project Period (FY): 1997 – 2000
• Keywords: SAP97 / NMDA receptor / PSD protein / Synapse / Hippocumpal primary culture / memory formation / Subtraction cloning / in situ

Research Abstract

Rat was treated with 3AP which selectively destroy the climbing fiber of Purkinje cell. After synapses were reformed between climbing and parallel fiber, RNAs were purified from either treated or mock-treated cerebellum. Subtraction cloning was done between them. Unfortunately northern plot revealed that no clone was differently expressed pre- and post-treated rat cerebellum.
To use less complicated system and material seemed important. Then rat hippocumpal neuronal primary culture, which contains more than 80 % neuronal contents, was used. Selective glutamate receptor stimulator NMDA was applied in the medium. The same subtraction cloning was done between treated or mock-treated neuronal RNA. Determined by RT-PCR, one from several clones was differentially expressed. Tissue expression was determined by in situ hybridization using riboprobe. The transcript of the clone was increased in NMDA stimulated neuron and it was expressed almost every neuron. Subcloned plasmid was sequenced and database analysis confirmed the clone as SAP97, a PSD protein. SAP97 is known to locate in synapse but the exact pre or post synapse location is controversial. SAP97 is associated with several important synaptic proteins such as E-cadherin, Kv 1.3, Kir2.2, GKAP, protein 4.1 and GluR1. This research reveals a new insight that NMDA receptor stimulation induces the up-regulation of a synaptic protein (SAP97) which anchors the functional protein. This is very important because NMDA is a key receptor in memory formation and its function might be expressed through this SAP97 up-regulation. Such proteins that constitute synapse as SAP97 are also key molecules in memory formation in the light of new discoveries that electronic synaptic modulation might be brought by the modification of the structural modification of synapse, which change its shape in a second after electronic stimulation.