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

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 microM, 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>DHbetaE. Schild analysis revealed apparent noncompetitive antagonism of [(3)H]-NE release from hippocampus by mecamylamine, hexamethonium, and DHbetaE. In contrast, DHbetaE antagonism of [(3)H]-dopamine release from striatal slices using a similar methodology was competitive.