Functional NMDA receptor channels generated by NMDAR2B gene transfer in rat cerebellar Purkinje cells.
The adult cerebellar Purkinje cell is an exceptional neuron in the central nervous system in that it expresses high levels of NMDAR1 (NR1) mRNA without expressing any NMDAR2 (NR2) mRNAs. It has no functional NMDA receptor (NMDAR) channels, although it receives enormous numbers of excitatory inputs. Despite the high level of NR1 mRNA expression, the presence and localization of NR1 protein in mature Purkinje cells are controversial. To examine the presence of NR1 protein and its ability to form functional NMDARs, we expressed the NR2B subunit in rat mature Purkinje neurons by Sindbis viral-mediated gene transfer. The recombinant virus encoding both the NR2B and enhanced green fluorescent protein (GFP) genes (designated as SIN-EG-NR2B) infected Purkinje cells without infecting glial cells. GFP fluorescence was detected in the soma and throughout dendrites of Purkinje cells 18-24 h postinfection. In most of GFP-positive cells, the expression of NR2B protein was detected by immunostaining with NR2B-specific antibodies. In Purkinje cells infected with SIN-EG-NR2B, the iontophoretic application of NMDA induced prominent NMDAR-mediated current responses, indicating that the exogenous NR2B was assembled with endogenous NR1 to form functional NMDARs. Furthermore, NMDAR-mediated synaptic currents were detected at both the climbing fibre and parallel fibre synapses in infected Purkinje cells. Thus, the mature Purkinje cell produces NR1 protein that is ready to combine with NR2 to form functional NMDARs in excitatory synapses.
Kakegawa
Wataru
W
Department of Physiology, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan. wkake@med.gunma-u.ac.jp
Tsuzuki
Keisuke
K
Iino
Masae
M
Ozawa
Seiji
S
D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry / Q000379:methods; D007239:Infections; D008164:Luminescent Proteins / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D016202:N-Methylaspartate / Q000494:pharmacology*; D018408:Patch-Clamp Techniques / Q000379:methods; D011689:Purkinje Cells / Q000187:drug effects / Q000502:physiology* / Q000821:virology; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000502:physiology*; D012845:Sindbis Virus / Q000502:physiology; D014633:Valine / Q000031:analogs & derivatives* / Q000494:pharmacology
D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D008164:Luminescent Proteins; C121001:NR2B NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate; D049452:Green Fluorescent Proteins; D016202:N-Methylaspartate; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; C056639:2-amino-5-phosphopentanoic acid; D014633:Valine; D008274:Magnesium