There are three criteria used to determine whether a chemical serves as a neurotransmitter: (1) is the molecule synthesized by and localized in the presynaptic neuron? (2) Is the molecule released upon stimulation? (3) When experimentally repeated, does the molecule mimic the effects in vitro versus when the presynaptic neuron releases it naturally? In regards to illustrating that ACh fulfills the criteria, the first criteria would have to be satisfied by either performing in situ hybridization or immunocytochemistry. A person would then have to satisfy criteria 2 by taking a sample of the fluid surrounding the presynaptic neuron and then test it for the molecule of interest. The molecule would then have to be isolated, ionized, and pipetted
