Cell type-selective expression of green fluorescent protein and the calcium indicating protein, yellow cameleon, in rat cortical primary cultures.
A cell type-specific green fluorescent protein (GFP) expression system in rat cortical primary cultures has been developed for the fluorescence labeling of brain cells. Lipid-mediated transfection (lipofection) was employed, allowing the establishment of a convenient efficient system for the analysis of individual cells. To achieve cell type-specific labeling, GFP expression vectors containing the rat neuron-specific enolase (NSE) gene promoter, human glial fibril acidic protein (GFAP) gene promoter, human elongation factor (EF-1alpha) gene promoter, or human cytomegalovirus (CMV) immediate early promoter were constructed, and their specificities examined. Vectors containing the CMV or GFAP promoter resulted primarily in GFP expression in astrocytes, while those containing the EF1-alpha or NSE promoter resulted primarily in GFP expression in neurons. This labeling system was applied to the morphological analysis of living neurons and to cell type-selective calcium imaging. Confocal microscopy revealed that individual GFP-expressing neurons had processes, which were longer than 500 microm and bore spine-like protrusions. A calcium-indicating GFP variant, yellow cameleon (YC2.1), was expressed in the same system, and cell type-selective calcium imaging performed. On pharmacological stimulation, YC2.1-expressing neurons responded to depolarizing stimuli, but not to the metabotropic glutamate receptor agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (tACPD), while astrocytes responded only to tACPD.
Tsuchiya
Remi
R
Laboratory of Cellular Neurobiology, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Yoshiki
Fumito
F
Kudo
Yoshihisa
Y
Morita
Mitsuhiro
M
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics; D001253:Astrocytes / Q000737:chemistry; D002118:Calcium / Q000032:analysis*; D018929:Cell Culture Techniques; D002540:Cerebral Cortex / Q000737:chemistry* / Q000166:cytology; D003515:Cycloleucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis / Q000235:genetics; D049452:Green Fluorescent Proteins; D017874:Immediate-Early Proteins / Q000235:genetics; D007150:Immunohistochemistry; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal; D009474:Neurons / Q000737:chemistry; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D010751:Phosphopyruvate Hydratase / Q000235:genetics; D011401:Promoter Regions, Genetic; D051381:Rats; D017208:Rats, Wistar; D018094:Receptors, Metabotropic Glutamate / Q000819:agonists; D014162:Transfection
D000956:Antigens, Viral; D005904:Glial Fibrillary Acidic Protein; D017874:Immediate-Early Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D020648:Peptide Elongation Factor 1; D018094:Receptors, Metabotropic Glutamate; C044582:immediate-early proteins, cytomegalovirus; D003515:Cycloleucine; C036361:1-amino-1,3-dicarboxycyclopentane; D049452:Green Fluorescent Proteins; D010751:Phosphopyruvate Hydratase; D002118:Calcium