Sindbis viral-mediated expression of eGFP-dopamine D1 receptors in situ with real-time two-photon microscopic detection.
Dopamine D1 receptors (D1DRs) mediate a major component of dopaminergic neurotransmission, and alterations in their synaptic and subcellular distribution may underlie a variety of neurological diseases. In order to monitor D1DR localization in real time, we subcloned a sindbis virus containing an enhanced-GFP coding region inserted at the C-terminal region of a dopamine D1 receptor (eGFP-D1DR). Two-photon excitation of expressed eGFP-D1DRs was monitored in a variety of viable neural preparations. Infection of primary cultured rat ventral striatal neurons, verified for neuronal phenotype using patch clamp electrophysiology, was induced by the simple addition of the virus to media. Parasagittal slice cultures, including the ventral tegmental area (VTA) and nucleus accumbens (NAc), were infected by manual injection below the glia surface. NAc-containing parasagittal slices prepared from mice in which the virus was administered via stereotaxic injection in vivo also displayed robust eGFP-D1DR expression. Expression of functional D1DRs following infection in baby hamster kidney (BHK) cells was monitored by DA-stimulated cAMP production that was also blocked by a selective D1 antagonist. Taken together, these findings provide the first demonstration of the functional expression and real-time imaging of eGFP-D1DRs, and indicate that sindbis virus is an effective method for D1 receptor expression in a variety of native neuronal preparations.
Diaz
Laurea M
LM
The College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, USA.
Maiya
Rajani
R
Sullivan
Matthew A
MA
Han
Yunqing
Y
Walton
Heather A
HA
Boehm
Stephen L
SL
Bergeson
Susan E
SE
Mayfield
R Dayne
RD
Morrisett
Richard A
RA
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D003199:Computer Systems; D006224:Cricetinae; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D066298:In Vitro Techniques; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D009474:Neurons / Q000378:metabolism / Q000821:virology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017447:Receptors, Dopamine D1 / Q000096:biosynthesis* / Q000235:genetics; D012845:Sindbis Virus / Q000235:genetics
D017447:Receptors, Dopamine D1; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins