Longitudinal noninvasive monitoring of transcription factor activation in cardiovascular regulatory nuclei using bioluminescence imaging.
The ability to monitor transcription factor (TF) activation in the central nervous system (CNS) has the potential to provide novel information regarding the molecular mechanisms underlying a wide range of neurobiological processes. However, traditional biochemical assays limit the mapping of TF activity to select time points. In vivo bioluminescence imaging (BLI) has emerged as an attractive technology for visualizing internal molecular events in the same animal over time. Here, we evaluated the utility of BLI, in combination with virally mediated delivery of reporter constructs to cardiovascular nuclei, for monitoring of TF activity in these discrete brain regions. Following viral gene transfer of NF-kappaB-driven luciferase reporter to the subfornical organ (SFO), BLI enabled daily measurements of baseline TF activity in the same animal for 1 mo. Importantly, systemic endotoxin, a stimulator of NF-kappaB activity, induced dramatic and dose-dependent increases in NF-kappaB-dependent bioluminescence in the SFO up to 30 days after gene transfer. Cotreatment with a dominant-negative IkappaBalpha mutant significantly prevented endotoxin-dependent NF-kappaB activation, confirming the specificity of the bioluminescence signal. NF-kappaB-dependent luminescence signals were also stable and inducible 1 mo following delivery of luciferase reporter construct to the paraventricular nucleus or rostral ventrolateral medulla. Lastly, using targeted adenoviral delivery of an AP-1 responsive luciferase reporter, we showed similar baseline and endotoxin-induced AP-1 activity in these same brain regions as with NF-kappaB reporters. These results demonstrate that BLI, in combination with virally mediated gene transfer, is a powerful method for longitudinal monitoring and quantification of TF activity in targeted CNS nuclei in vivo.
Peterson
Jeffrey R
JR
Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401, USA.
Infanger
David W
DW
Braga
Valdir A
VA
Zhang
Yulong
Y
Sharma
Ram V
RV
Engelhardt
John F
JF
Davisson
Robin L
RL
D000256:Adenoviridae; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002319:Cardiovascular System / Q000378:metabolism*; D003587:Cytomegalovirus; D017930:Genes, Reporter; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D007700:Kinetics; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014161:Transduction, Genetic
D016328:NF-kappa B; D018808:Transcription Factor AP-1; D014157:Transcription Factors