Use of an electrochemical nitric oxide sensor to detect neuronal nitric oxide production in conscious, unrestrained rats.
BACKGROUND
Amperometric sensors that directly measure nitric oxide (NO) are readily employed in pharmacologic research. While several of these sensors have been developed, none has been investigated for use in conscious, freely moving animals. An approach was developed and validated for real-time quantitation of neuronal NO production in rats without restricting locomotor activity or other potentially useful behavioral endpoints.


METHODS
Male Sprague-Dawley rats were equipped with a femoral vein or intraperitoneal cannula. A guide cannula and an amperometric NO sensor were placed in the left and right hippocampus, respectively. Following recovery, rats received a 6-h intravenous infusion of saline, L-arginine (an NO precursor; 250 or 500 mg/kg/h), or incremental intraperitoneal 7-nitroindazole (an NO synthase inhibitor; 200-mg/kg loading dose and 100 mg/kg every 2 h). The sensor recorded NO production continuously and microdialysis samples were collected incrementally throughout the experiment. Griess assay analysis of microdialysate samples was compared to sensor readings in vivo. In vitro degradation of an NO donor also was used to validate sensor performance.


RESULTS
Exogenous administration of L-arginine resulted in incremental increases in the neuronal NO signal. A reduction in NO production was observed during administration of 7-nitroindazole, a selective neuronal NO synthase inhibitor. A significant correlation was observed in vitro between the Griess assay analysis, an indirect analytical approach, and the NO sensor readings. The lack of a strong correlation between these measures in vivo is consistent with the indirect nature of the Griess assay.


CONCLUSIONS
The current approach allows real-time determination of neuronal NO production in unrestrained rats. This model will be invaluable in evaluating pharmacologic issues regarding brain tissue NO synthesis, assessing brain NO synthase as a molecular target, and establishing the effects of pharmacologic agents on neuronal NO production.
Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina, CB# 7360 Kerr Hall, Chapel Hill, NC 27599-7360, USA.
Erin L
EL
Heinzen
Gary M
GM
Pollack
D000818:Animals; D001120:Arginine / Q000008:administration & dosage* / Q000097:blood / Q000378:metabolism; D003243:Consciousness; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006624:Hippocampus / Q000187:drug effects; D007191:Indazoles / Q000493:pharmacokinetics / Q000494:pharmacology; D007262:Infusions, Intravenous; D007700:Kinetics; D008297:Male; D009474:Neurons / Q000201:enzymology; D009569:Nitric Oxide / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley
D004791:Enzyme Inhibitors; D007191:Indazoles; D009569:Nitric Oxide; D001120:Arginine; C080122:7-nitroindazole