Assessment of nicotinic acetylcholine receptor-mediated release of [H-3]-norepinephrine from rat brain slices using a new 96-well format assay

The study of the modulatory effects of nicotinic acetylcholine receptor (nA ChR) agonists on neurotransmitter release from tissue slices has been hampe red by laborious and limiting superfusion techniques. A new methodology was developed utilizing 96-well filter plates. This new method produced compar able results to previously published data, yet expanded throughput to permi t more complete pharmacological characterization. Rat brain slices, preload ed with [H-3]-norepinephrine ([H-3]-NE), were distributed onto 96-well filt er plates. Following a 5 min preincubation, the slices were incubated for 5 min with nicotinic agonists or antagonists. (-)-Nicotine (NIC) and 1,1-dim ethyl-4-phenylpiperazine (DMPP) evoked release of [H-3]-NE from a number of brain regions and spinal cord, with the highest response seen in the hippo campus. Concentration-response curves revealed a rank order of potency of ( +/-)-epibatidine >> anatoxin-a > A-85380 > DMPP = NIC = (-)-cytisine in the hippocampus, thalamus, and frontal cortex. EC50 values were approximately 0.005, 0.2, 1, 5, 5 and 5 muM, respectively. Concentration-inhibition curve s of nicotine evoked [H-3]-NE release from hippocampal and thalamic slices resulted in a rank order of potency of mecamylamine > hexamethonium > d-tub ocurare > DH betaE. Schild analysis revealed apparent noncompetitive antago nism of [H-3]-NE release from hippocampus by mecamylamine, hexamethonium, a nd DH betaE. In contrast, DH betaE antagonism of [H-3]-dopamine release fro m striatal slices using a similar methodology was competitive. (C) 2000 Els evier Science Ltd. All rights reserved.