High-sensitive liquid chromatographic method for determination of neuronal release of serotonin, noradrenaline and dopamine monitored by microdialysis in the rat prefrontal cortex.
A high-sensitive liquid chromatographic method based on precolumn derivatization and fluorescence detection allowing simultaneous determination of serotonin (5-HT), noradrenaline (NA) and dopamine (DA) in brain microdialysis samples is described. 5-HT, NA and DA were derivatized with benzylamine and 1,2-diphenylethylenediamine in the presence of potassium hexacyanoferrate(III) and glycine, which yielded to highly fluorescent and stable benzoxazoles. The derivatized samples were separated on a microbore column (150 mm x 1.0mm i.d., packed with C18 silica, 5 microm) within 60 min. The mobile phase consisted of acetonitrile-Briton-Robinson buffer (pH 7.2) (32:68, v/v) containing 5 mM Na2EDTA and 5 mM octanesulfonic acid sodium salt. The detection limits (signal-to-noise ratio of 3) for 5-HT, NA and DA were 76, 42 and 95 amol/10 microl injected on-column, respectively. Microdialysis samples were collected at 10-min intervals from the probes implanted in the prefrontal cortex of awake rats. The basal levels of 5-HT, NA and DA were 7.3 +/- 0.7, 5.3 +/- 0.31 and 8.1 +/- 0.47 fmol/5 microl (mean +/- S.E.M., n = 5). Following 90-min perfusion with tetrodotoxin (1 microM) or calcium-free Ringer solution, the DA and NA levels were reduced to about 15 and 20%, respectively and the 5-HT levels to 45 and 60% of the basal levels, respectively. Reserpine, 12h after a dose of 5mg/kg i.p., reduced the extracellular 5-HT, NA and DA concentrations to about 34, 39 and 32% of the basal levels, respectively. In conclusion, the preset microdialysis/analytical method enables simultaneous monitoring of basal and pharmacologically reduced neuronal release of 5-HT, NA and DA in the rat brain.
Yoshitake
Takashi
T
Department of Neuroscience, Karolinska Institutet, Retzius väg 8, S-171 77 Stockholm, Sweden.
Yoshitake
Shimako
S
Fujino
Kaoru
K
Nohta
Hitoshi
H
Yamaguchi
Masatoshi
M
Kehr
Jan
J
D018759:Adrenergic Uptake Inhibitors / Q000494:pharmacology; D000818:Animals; D001681:Biological Assay / Q000295:instrumentation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D004298:Dopamine / Q000032:analysis / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D008297:Male; D017551:Microdialysis / Q000295:instrumentation / Q000379:methods*; D009448:Neurochemistry / Q000295:instrumentation / Q000379:methods*; D009474:Neurons / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000032:analysis* / Q000378:metabolism; D009638:Norepinephrine / Q000032:analysis / Q000378:metabolism; D017397:Prefrontal Cortex / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012110:Reserpine / Q000494:pharmacology; D012701:Serotonin / Q000032:analysis / Q000378:metabolism; D013779:Tetrodotoxin / Q000494:pharmacology
D018759:Adrenergic Uptake Inhibitors; D018377:Neurotransmitter Agents; D012701:Serotonin; D013779:Tetrodotoxin; D012110:Reserpine; D004298:Dopamine; D009638:Norepinephrine