Chapter 6 Neurotransmitter Systems

Introduction There are three classes of neurotransmitters (as discussed in Chapter 5) Amino acids, amines, and peptides There are a variety of different ways of defining particular transmitter systems: By the molecule synthetic machinery packaging reuptake and degradation as well as others It is important to note that all neurotransmitter systems can be described by the above 5 ways.

Neurotransmitter systems have five different ways they can be described.

Henry Dale – received the Nobel Prize for Medicine in 1926 for his work on synaptic transmission. He is also well known for his coining of the term “cholinergic.”

System #1: Cholinergic System acetylcholine (Ach) System #2 : Noadrenergic System (adrenergic derived from “adrenalin” the origin of NE norepinephrine (NE) System #3: Glutamatergic System glutamate System #4: GABAergic System gamma-aminobutryic acid (GABA) System #5: Peptidergic System various peptide neurotransmitters

Experimental Design Associated With With the myriad of different chemicals produced in neurons, how do we define a “neurotransmitter”? the chemical must elicit change in another neuron or cell Experimental Design Associated With Identifying Neurotransmitters However, because the chemicals are in minutely small quantities and because it is challenging to discern which chemical is actually affecting a receptor, the following criteria are used to assess experimentally if something is a neurotransmitter: the chemical must be synthesized and stored in a presynaptic neuron (Criteria A) the chemical must be released by the presynaptic neuron upon stimulation (Criteria B) exogenous application of the chemical will elicit a response that is similar to endogenous release (Criteria C) In the next several slides, we will look at a variety of ways to establish the above criteria.

Studying Transmitter Localization I. Immunocytochemistry (ICC)– a technique used to localize production of chemicals (molecules) to particular cells Steps in this technique: Purified molecule of interest is injected into an animal to provoke an immune response (keep in mind these are protein-based molecules, or if smaller, they are coupled to larger proteins). The antibodies that the host animal produces can be collected, purified and tagged with a marker (radioactive, fluorescent. or another type). These tagged antibodies (designed by the host to attack the “invader” injection, will now bind to any sample of nervous system tissue with the same molecule when they are applied. This will then allow for identification of brain, spinal cord, and other nervous system tissues that have the same suspected neurotransmitter compounds. This technique helps to show localization of production and storage (Criteria A)

Please note that in this slide of the rat brain, ICC has been used to identify vasopressin containing neurons and therefore identify vasopressin producing regions of the brain.

II. In situ hybridization – a technique where the identification and localization of a synthesized protein or peptide occurs (at the cell level). This technique is essentially detecting mRNA. A complementary strand to a known mRNA is made in the lab (this is called a probe). When the probe binds to mRNA, this is the process of hybridization. The probe is labeled in some fashion (like with ICC, this can be radioactivity, fluorescent dye, or something else) The probe will “stick” to the mRNA of particular neurons containing that mRNA sequence and therefore will identify it. Radioactive isotopes are still most commonly used in this process and are detected via autoradiography (basically with X-ray film). I. and II. in the preceding detect potential neurotransmitters via TWO different routes. Can you identify these routes?

A variant of traditional In-Situ Hybridization that uses fluorescent labeled probes and allows the use of a light microscope to view.

Studying Transmitter Release Studying transmitter release is fairly effective in the PNS, but it is much more difficult in the CNS because the range of different chemicals in the numerous different regions of the brain and spinal cord make isolation very difficult. One technique that can be used to approximate the study of CNS transmitter release to a REGION (not an individual neuron) of the brain or spinal cord involves in vitro techniques where slices of brain or spinal cord are kept alive in physiological saline (specifically neurophysiological saline). With this technique, and then by bathing the tissue with a different solution very high in potassium, membrane depolarization can be stimulated. A challenge remains however… just because a neurotransmitter candidate is RELEASED, does not mean it is a neurotransmitter… it could be simply a secondary chemical that is released or some other form of artifact. This ambiguity makes it difficult to establish Criteria B with certainty in the CNS.

Studying Synaptic Mimicry Microionophoresis – a technique that helps to establish Criteria C, that responses are able to be ellicited by exogenous application of the neurotransmitter candidate. The neurotransmitter candidate is dissolved in solution and acquires an electrical charge. A pulled micropipette is used to insert into a neuron the solution of interest. If the response of the exogenous injection is the same as the endogenous effect, it has been shown that the molecule of interest is a neurotransmitter according to Criteria C. A typical pipette puller like this can make a glass tube with openings only a few micons in diameter.

Three Crucial Terms Used for Studying Neurotransmitters: Agonist - a chemical that binds to a receptor of a cell and triggers a response by that cell. Agonists often mimic the action of a naturally occurring substance. Antagonist - whereas an agonist causes an action, an antagonist blocks the action of the agonist. Inverse Agonist – compound that causes an action opposite to that of the agonist.

Studying Receptors Neuropharmacological Analaysis – the discovery of TWO different cholinergic receptor types for acetylcholine (Ach) that act differently was made through this technique. The first type has been called the MUSCARINIC receptor. Muscarine is an agonist and its effect is to stop the heart. It works in a much similar way to Ach on cardiac muscle. Ach Receptor Subtype 2 is the receptor type found on cardiac cells. Muscarine – a type of poison found in some mushrooms.

Moto-Taxi Driver (1999) at a coffee shop in Bangalore, India He is stimulating his nicotinic receptors.

The second type has been called the NICOTINIC receptor. Nicotine is an agonist and its effect is to affect the Ach receptors on skelletal muscle. Ach Receptor Subtype 1 is the receptor type found on skelletal muscle cells.