Chapter 6, End of Chapter, Review Questions, Exercise 1

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:

1. Immunochemistry- Acetylcholine molecules can be chemically purified and injected under the skin, or the bloodstream of an organism, where it elicits an immune response. During an immune response, plasma B-cells synthesize antibodies. These antibodies bind tightly to antigens on the foreign molecules which are acetylcholine neurotransmitters. These antibodies bind to ACh with high specificity and affinity, whereas it shows little or no affinity for other chemicals in the brain. These antibodies can be recovered from the blood sample of the organism, and can be tagged by fluorophores which are colorful markers that enable to trace the antigen-antibody binding by emitting fluorescent light. When these fluorescent-anti-ACh antibodies are applied to sections of the brain, they stain the neurons that contain this neurotransmitter. Demonstrating that ACh is synthesized, and stored in the same neuron can satisfy the above criterion. 
2. In-situ hybridisation- Another way to establish that the ACh is synthesized and stored in particular neurons is in-situ hybridization. Each protein, or neurotransmitter molecule is synthesized from mRNA. If the sequence of the mRNA is known, a complementary sequence can be created, known as probes. These probes can be synthesized using radiolabeled isotopes. When the probes are incubated with the cell, they will bind to the complementary sequence of mRNA producing ACh which is known as the process of hybridization. This can be observed by autoradiography, much like an X-ray film which results in the viewing of the distributed radioactivity. A modification of this method utilizes fluorescent labelled probes and is called fluorescent in-situ hybridization (FISH).

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.