Nicotinic cholinergic receptors labeled by [³H]acetylcholine in rat brain

On the basis of its ability to bind to nicotinic cholinergic receptors in electric tissue and at the neuromuscular junction, α-bungarotoxin has been widely used to identify central nicotinic receptors. However, the suitability of this ligand as a probe for nicotinic cholinergic receptors in the central nervous system has been questioned. The use of [³H]acetylcholine of high specific activity to measure cholinergic binding sites in rat is reported here. In the presence of a cholinesterase inhibitor to prevent hydrolysis and atropine to block muscarinic cholinergic receptors, [³H]acetylcholine bound rapidly, reversibly, and with high affinity to rat brain membranes (K(D) = 12.3 ± 0.8 nM, B(max) = 4.6 ± 0.1 pmoles/g of tissue). Kinetic analyses revealed a half-time for association of 3.2 min and a half-time for dissociation of 2.5 min. The K(D) of 12.0 nM calculated from the kinetic experiments was in excellent agreement with that calculated from equilibrium experiments. Subcellular distribution studies indicated that these binding sites were located primarily in the synaptosomal fraction. In competition studies, nicotinic agonists were more than 1000 times more potent than ganglionic and neuromuscular blocking drugs in displacing [³H]acetylcholine binding. Cytisine and (-)-nicotine had the highest affinity (K(I) = 1-6 nM) for the [³H]acetylcholine binding site. The highest levels of binding were found in the thalamus, cortex, superior colliculus, and striatum, whereas the lowest were found in the pyriform cortex and hippocampus. The K(D) values in several of these areas were found to be similar, whereas the density of binding sites varied. A lack of correlation between the regional distribution of sites labeled by [³H]acetylcholine and those labeled by ¹²⁵I-labeled α-bungarotoxin suggests that these two binding sites are not located on the same molecule.