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Received October 24, 2002
Accepted after revision January 15, 2003
1 Department Physiology and Biophysics, Georgetown University, Washington DC, USA
2 Department Physiology and Biophysics, Georgetown University, Washington DC and Laboratory of Neurochemistry, NIDCD, NIH, Bethesda, MD, USA
3 Department Physiology and Biophysics, Georgetown University, Washington DC USA and Laboratory of Neurobiology, Zhejiang University, Hangzhou, China
4 Department Physiology and Biophysics, Georgetown University, Washington DC, USA and Laboratory of Neurobiology, Zhejiang University, Hangzhou, China
5 Laboratory of Neurochemistry, NIDCD, NIH, Bethesda, MD, USA
6 Department of Physiology and Biophysics, Georgetown University, Box 571460, Basic Science Building Room 225, Washington DC 20057-1460, USA
* To whom correspondence should be addressed. E-mail: svicin01{at}georgetown.edu.
We transfected a green fluorescent protein-tagged PSD-95 (PSD-95gfp) into cultured rat cerebellar granule cells (CGC) to investigate the role of PSD-95 in excitatory synapse maturation. Cells were grown in low potassium to favour functional synapse formation in vitro. Transfected cells displayed clear clusters of PSD-95gfp, often at the extremities of the short dendritic trees. We recorded NMDA and AMPA miniature excitatory postsynaptic currents (NMDA-, AMPA-mESPCs) in the presence of TTX and bicuculline. At DIV 7-8 PSD-95gfp-transfected cells had NMDA-mEPSCs with faster decay and smaller amplitudes than matching controls. In contrast, AMPA-mEPSC frequencies and amplitudes were increased. Whole-cell current density and ifenprodil sensitivity were reduced in PSD-95gfp cells, indicating a reduction of NR2B subunits containing NMDA receptors. No changes were observed compared to control when cells were transfected with cDNA for PSD-95gfp with palmitoylation site mutations that prevent targeting to the synapse. Overexpression of the NMDA receptor NR2A subunit, but not the NR2B subunit, prevented NMDA-mEPSC amplitude reduction when cotransfected with PSD-95gfp. PSD-95gfp overexpression produced faster NMDA-mEPSCs decay when transfected alone or with either NR2 subunit. Surface staining of the epitope-tagged NR2 subunits revealed that colocalization with PSD-95gfp was higher for NR2Aflag subunit clusters than for NR2Bflag subunit clusters. These data suggest that PSD-95 overexpression in CGCs favours synaptic maturation by allowing synaptic insertion of NR2A and depressing expression of NR2B subunits.
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