Cytochemical evidence that acetylcholine is a neurotransmitter of neurons that make excitatory and inhibitory outputs in the locust ocellar visual system

Three different cytochemical methods were used to detect acetylcholine in l arge, second-order neurons of locust ocelli (L-neurons). The first method u sed polyclonal antibodies raised against choline cleaved from acetylcholine and then conjugated with native protein, and this revealed strong staining for acetylcholine in axons whose number, size, and location indicated that they were of L-neurons. A corresponding staining pattern was found using t he second method with a polyclonal antiserum against choline acetyltransfer ase (ChAT). The third method was the histochemical detection at the electro n microscope level of acetylcholinesterase, the enzyme responsible for the breakdown of acetylcholine. We found that this enzyme is located in synapti c clefts of L-neurons in both of the brain regions where L-neurons are know n to make excitatory and inhibitory output synapses. Acetylcholinesterase w as confined to synaptic sites, which is consistent with a role in synaptic transmission at these synapses. Taken together, the findings suggest that L -neurons use acetylcholine as a neurotransmitter. J. Comp. Neurol. 416:345- 355, 2000. (C) 2000 Wiley-Liss, Inc.