Sindbis viral-mediated expression of Ca2+-permeable AMPA receptors at hippocampal CA1 synapses and induction of NMDA receptor-independent long-term potentiation.
Gene manipulation in order to artificially express a particular gene in neurons in the central nervous system is a powerful tool for the analysis of brain function. Sindbis viral vectors have been developed to express high levels of foreign genes in postmitotic brain neurons with little transfection of glial cells. In this study, we expressed the gene encoding the unedited GluR2 (GluR-B) subunit of the AMPA-type glutamate receptor that forms inwardly rectifying and Ca2+-permeable channels, in rat CA1 hippocampal neurons in slice cultures using Sindbis viral vectors. The pyramidal cell layer of the CA1 region was injected with recombinant Sindbis viruses encoding both enhanced green fluorescent protein (GFP) and unedited GluR2. The GFP fluorescence from CA1 neurons could be detected as early as 6 h and reached a maximal level about 48 h postinfection. The inwardly rectifying and Ca2+-permeable AMPA receptors were expressed in most CA1 pyramidal cells expressing GFP. These AMPA receptors expressed by gene transfer were involved in fast excitatory neurotransmission elicited by electrical stimulation of the Schaffer collaterals in the stratum radiatum. Tetanic stimulation of Schaffer collaterals induced NMDA receptor-independent, long-term potentiation due to Ca2+ influx through the newly expressed AMPA receptors in the area densely stained with GFP. Thus, the combined use of Sindbis viral vectors with the GFP reporter allowed physiological examination of the roles of a specific gene product in synaptic function in well-characterized brain neurons.
Okada
T
T
Department of Physiology, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan.
Yamada
N
N
Kakegawa
W
W
Tsuzuki
K
K
Kawamura
M
M
Nawa
H
H
Iino
M
M
Ozawa
S
S
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D066298:In Vitro Techniques; D007202:Indicators and Reagents / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D007763:Lac Operon / Q000502:physiology; D017774:Long-Term Potentiation / Q000502:physiology*; D008164:Luminescent Proteins / Q000378:metabolism; D009474:Neurons / Q000502:physiology; D010539:Permeability; D017966:Pyramidal Cells / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018091:Receptors, AMPA / Q000378:metabolism* / Q000502:physiology; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology*; D012845:Sindbis Virus / Q000235:genetics; D013569:Synapses / Q000378:metabolism*; D014162:Transfection
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D018091:Receptors, AMPA; D016194:Receptors, N-Methyl-D-Aspartate; D049452:Green Fluorescent Proteins; C104722:glutamate receptor ionotropic, AMPA 2; D002118:Calcium