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

Abstract The study of the modulatory effects of nicotinic acetylcholine receptor (nAChR) agonists on neurotransmitter release from tissue slices has been hampered by laborious and limiting superfusion techniques. A new methodology was developed utilizing 96-well filter plates. This new method produced comparable results to previously published data, yet expanded throughput to permit more complete pharmacological characterization. Rat brain slices, preloaded with [ 3 H]-norepinephrine ([ 3 H]-NE), were distributed onto 96-well filter plates. Following a 5 min preincubation, the slices were incubated for 5 min with nicotinic agonists or antagonists. (−)-Nicotine (NIC) and 1,1-dimethyl-4-phenylpiperazine (DMPP) evoked release of [ 3 H]-NE from a number of brain regions and spinal cord, with the highest response seen in the hippocampus. 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. EC 50 values were approximately 0.005, 0.2, 1, 5, 5 and 5 μM, respectively. Concentration–inhibition curves of nicotine evoked [ 3 H]-NE release from hippocampal and thalamic slices resulted in a rank order of potency of mecamylamine>hexamethonium>d-tubocurare>DHβE. Schild analysis revealed apparent noncompetitive antagonism of [ 3 H]-NE release from hippocampus by mecamylamine, hexamethonium, and DHβE. In contrast, DHβE antagonism of [ 3 H]-dopamine release from striatal slices using a similar methodology was competitive.