High-resolution localization of acetylcholinesterase at the rat neuromuscular junction

To overcome the limited ultrastructural resolution of conventional acetylcholinesterase (AChE) ultrahistochemistry, acetylcholine (ATCh) was used to reduce the rate of enzymic thiocholine liberation. The conventionally limited resolution is mainly due to the high focal activity of the enzyme in neural structures, because cleavage of substrate is faster than histochemical trapping reactions. Therefore, using the copper-thiocholine method, we investigated the reduction of thiocholine liberation by acetylcholine (ACh). As examined biochemically, the apparent Ki for ACh was close to the Km for ATCh. The ACh/ATCh ratio, therefore, determined the reduction of thiocholine production in histochemical experiments. In addition, the morphological appearance of the precipitated reaction product after its changes during the histochemical procedure was monitored using electric eel AChE immobilized on Sepharose 4B. The improved fine structural resolution at 40- to 100-fold excess of ACh over ATCh is demonstrated at the neuromuscular junction of rat lumbricalis muscle. The highest focal enzyme activity is found at the presynaptic membrane and in the secondary cleft, but not on top of the junctional folds, indicating the separation of esterase and nicotinic receptors. The physiological events during neuromuscular transmission are discussed on the basis of the new "gradient switch hypothesis" suggested in this report.