Neurotransmitters are chemical substances that are involved in the nerve impulse generation and propagation. There are many neurotransmitter molecules that either have an excitatory effect on an inhibitory effect on the action potentials. For example, acetylcholine, glutamate, γ-amino butyric acid (GABA).
There is a certain set of criteria a chemical must fulfill to be categorized as a neurotransmitter. Broadly, there are three steps in an experiment that aims to determine if a given chemical is a neurotransmitter or not.
The first step in the experimental setup is to confirm that the molecule, acetylcholine (ACh), is in fact being synthesized and localized in a particular neuron. This can be proved by:
The second step involves experiments that prove that ACh is actually released by the neurons upon stimulation. A way to confirm the hypothesis can be to work with slices of brain tissue which are alive in-vitro. These slices are bathed in a solution that is hyperkalemic, or contains high concentration of potassium. As a consequence the membrane is depolarized and stimulates the release of ACh from the presynaptic terminal. Additionally, it can be proved that the neurotransmitter release is calcium dependent by removing the concentration of calcium ions from the bathing solution. Optogenetic allows activation of one specific synapse at a time. It involves genetic methods that cause the neurons to express light-sensitive proteins. Stimulating these neurons with the flashes of light can specifically activate the synapse.
Lastly, ACh must produce a response in the postsynaptic membrane in-vitro similar to its functioning when released from the presynaptic neuron in the nervous system. Microiontophoresis is commonly used to prove this. In this technique, a glass pipette with a fine tip is filled with ionized solution. The tip is then situated next to the postsynaptic membrane, and ACh is injected into the cell slowly by maintaining electrical current through the pipette. Therefore, a microelectrode can be used to measure the membrane potential changes caused by the desired neurotransmitter.
Following are the characteristic, a chemical must fulfill to be categorized as a neurotransmitter:
The first characteristic can be experimentally confirmed by procedures like immunohistochemistry and in-situ hybridization. These procedures uniquely mark the chemicals and identify the area where they are localized.
The second characteristic can be confirmed by incubating the brain tissue in vitro, in a solution that depolarizes the neurons, thereby providing a stimulus for the release of neurotransmitters.
The third characteristics can be experimentally proved by the technique of microiontophoresis which allows application of chemicals into the postsynaptic cell. The changes in the membrane potential of postsynaptic cells developed due to this can be measured using microelectrode.