PSD-95 regulates NMDA receptors in developing cerebellar granule neurons of the rat.
We transfected a green fluorescent protein-tagged PSD-95 (PSD-95gfp) into cultured rat cerebellar granule cells (CGCs) 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- and AMPA-mESPCs) in the presence of TTX and bicuculline. At days in vitro (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-mEPSC 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 flag-tagged NR2A subunit clusters than for flag-tagged NR2B 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.
Losi
Gabriele
G
Department of Physiology and Biophysics, Georgetown University, Washington, DC 20057, USA.
Prybylowski
Kate
K
Fu
Zhanyan
Z
Luo
Jianhong
J
Wenthold
Robert J
RJ
Vicini
Stefano
S
D000465:Algorithms; D000818:Animals; D002460:Cell Line; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D000076122:Disks Large Homolog 4 Protein; D004594:Electrophysiology; D000939:Epitopes / Q000235:genetics; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010880:Piperidines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013569:Synapses / Q000502:physiology; D014162:Transfection
D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D000939:Epitopes; D018691:Excitatory Amino Acid Antagonists; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010880:Piperidines; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins; C033652:postsynaptic density proteins; D049452:Green Fluorescent Proteins; D004247:DNA; C010739:ifenprodil