Neurotransmitter Systems

Question 1

What are the three major classes of neurotransmitters?

Answer 1

Amino acids, amines, and peptides.

Question 2

What is included in a neurotransmitter system?

Answer 2

In addition to the molecule itself, a neurotransmitter system includes all the molecular machinery responsible for transmitter synthesis, vesicular packaging, reuptake and degradation, and transmitter action

Question 3

What was the first molecule positively identified as a neurotransmitter in the 1920s?

Answer 3

acetylcholine, or ACh

Question 4

What word is used to describe the cells that produce and release ACh?

Answer 4

Cholinergic

Question 5

What term was given to the neurons that use the amine neurotransmitter norepinephrine?

Answer 5

noradrenergic

Question 6

What other name is there for norepinephrine and what is the initials given?

Answer 6

(NE is known as noradrenaline in United Kingdom.)

Question 7

Name the elements of neurotransmitter systems in order from the presynaptic axon terminal to the postsynaptic dendrite (9)

Answer 7

• Neurotransmitter synthesising enzymes
• Synaptic vesicle transporters
• Reuptake transporters
• Degradative enzymes

-Transmitter-gated ion channels
-G-protein-coupled receptors
-G-proteins
G-protein gated ion channels
-Second messenger cascades

Question 8

What criteria must a chemical in the brain meet to be considered a neurotransmitter? (3)

Answer 8

1. The molecule must be synthesized and stored in the presynaptic neuron.
2. The molecule must be released by the presynaptic axon terminal upon
   stimulation.
3. The molecule, when experimentally applied, must produce a response
   in the postsynaptic cell that mimics the response produced by the release of neurotransmitter from the presynaptic neuron.

Question 9

Name two of the most important techniques used today to show that the molecule is, in fact, localised in, and synthesised by, particular neurons.

Answer 9

immunocytochemistry and in situ hybridization.

Question 10

What is immunocytochemistry used for?

Answer 10

The method of immunocytochemistry is used to anatomically localize particular molecules to particular cells.

Question 11

What other name is given to immunocytochemistry and when?

Answer 11

When the same technique is applied to thin sections of tissue, including brain, it is often referred to as immunohistochemistry.

Question 12

Describe the principle behind immunocytochemistry

Answer 12

Once the neurotransmitter candidate has been chemically purified, it is injected under the skin or into the blood- stream of an animal where it stimulates an immune response. (Often, to evoke or enhance the immune response, the molecule is chemically coupled to a larger molecule.) One feature of the immune response is the generation of large proteins called antibodies. Antibodies can bind tightly to specific sites on the foreign molecule, also known as the antigen—in this case, the transmitter candidate. The best antibodies for immunocy- tochemistry bind very tightly to the transmitter of interest and bind very little or not at all to other chemicals in the brain. These specific antibody molecules can be recovered from a blood sample of the immunized ani- mal and chemically tagged with a colorful marker that can be seen with a microscope. When these labeled antibodies are applied to a section of brain tissue, they will color just those cells that contain the transmitter candidate. By using several different antibodies, each labeled with a different marker color, it is possible to distinguish several types of cells in the same region of the brain

Question 13

What can satisfy the criterion that the molecule be localized in, and synthesized by, a particular neuron?

Answer 13

Immunocytochemistry can be used to localize any molecule for which a specific antibody can be generated, including the synthesizing enzymes for transmitter candidates. Demonstration that the transmitter candidate and its synthesizing enzyme are contained in the same neuron—or better yet, in the same axon terminal—can help satisfy the criterion that the molecule be localized in, and synthesized by, a particular neuron.

Question 14

Describe the principle of Hybridization

Answer 14

proteins are assembled by the ribosomes according to instructions from specific mRNA molecules. There is a unique mRNA molecule for every polypeptide synthesized by a neuron. The mRNA transcript consists of the four different nucleic acids linked together in various sequences to form a long strand. Each nucleic acid has the unusual property that it will bind most tightly to one other complementary nucleic acid. Thus, if the sequence of nucleic acids in a strand of mRNA is known, it is possible to construct in the lab a com- plementary strand that will stick, like a strip of Velcro, to the mRNA molecule.

Question 15

What is the complementary strand called?

Answer 15

a probe

Question 16

How can hybridisation be used to determine whether a neuron is synthesising a molecule?

Answer 16

In order to see if the mRNA for a particular peptide is localised in a neuron, we chemically label the appropriate probe so it can be detected, apply it to a section of brain tissue, allow time for the probes to stick to any complementary mRNA strands, then wash away all the extra probes that have not stuck. Finally, we search for neurons that contain the label.

Question 17

How can labelled cells be visualised in in-situ hybridisation? (3)

Answer 17

the probes can be chemically tagged in several ways. A common approach is to make them radioactive. Because we cannot see radioactivity, hybridized probes are detected by laying the brain tissue on a sheet of special film that is sensitive to radioactive emissions. After exposure to the tissue, the film is developed like a photograph, and negative images of the ra- dioactive cells are visible as clusters of small white dots

It is also possible to use digital electronic imaging devices to detect the radioactivity. This technique for viewing the distribution of radioactivity is called autoradiography.

An alternative is to label the probes with brightly colorful fluorescent molecules that can viewed directly with an appropriate microscope. Fluorescence in situ hybridization is also known as FISH.

Question 18

How can it be shown that a neurotransmitter is actually released upon stimulation in the PNS?

Answer 18

In some cases, a specific set of cells or axons can be stimulated while taking samples of the fluids bathing their synaptic targets. The biological activity of the sample can then be tested to see if it mimics the effect of the intact synapses, and then the sample can be chemically analyzed to reveal the structure of the active molecule.

Question 19

Why does this method of examining the release of neurotransmitters not possible in the CNS? What did researchers then have to be content with doing? Describe this process

Answer 19

most regions of the central nervous system (CNS) contain a diverse mixture of inter- mingled synapses using different neurotransmitters. Until recently, this often made it impossible to stimulate a single population of synapses containing only a single neurotransmitter. Researchers had to be content with stimulating many synapses in a region of the brain and collecting and measuring all the chemicals that were released.

One way to do this is to use brain slices that are kept alive in vitro. To stimulate release, the slices are bathed in a solution containing a high K􏰃 concentration. This treatment causes a large membrane depolarization, thereby stimulating transmitter release from the axon terminals in the tissue. Because transmitter release requires the entry of Ca2􏰃 into the axon terminal, it must also be shown that the release of the neurotransmitter candidate from the tissue slice after depolarization occurs only when Ca2􏰃 ions are present in the bathing solution

Question 20

Name and describe a newer method of determining whether a neurotransmitter is released from a terminal

Answer 20

New methods such as optogenetics now make it possible to activate just one specific type of synapse at a time. Genetic methods are used to induce one particular population of neurons to express light-sensitive proteins, and then those neurons can be stimulated with brief flashes of light that have no effect on the surrounding cells. Any transmitters released are likely to have come from the optogenetically selected type of synapse.

Question 21

To assess the postsynaptic actions of a transmitter candidate, describe two methods that are sometimes used

Answer 21

microiontophoresis; Most neurotransmitter candidates can be dissolved in solutions that will cause them to acquire a net electrical charge. A glass pipette with a very fine tip, just a few micrometers across, is filled with the ionized solution. The tip of the pipette is carefully positioned next to the postsynaptic membrane of the neuron, and the transmitter candidate is ejected in very small amounts by passing electrical current through the pipette.

Neurotransmitter candidates can also be ejected from fine pipettes with pulses of high pressure. A microelectrode in the postsynaptic neuron can be used to measure the effects of the transmitter candidate on the membrane potential

Question 22

When can the molecule and transmitter considered to be the same chemical with microiontophoresis?

Answer 22

If iontophoretic or pressure application of the molecule causes electrophysiological changes that mimic the effects of transmitter released at the synapse, and if the other criteria of localization, synthesis, and release are met, then the molecule and the transmitter are usually considered to be the same chemical.

Question 23

What is meant by a receptor subtype?

Answer 23

As a rule, no two neurotransmitters bind to the same receptor; however, one neurotransmitter can bind to many different receptors. Each of the different receptors a neurotransmitter binds to is called a receptor subtype.

Question 24

Researchers have tried almost every method of biological and chemical analysis to study the different receptor subtypes of the various neurotransmitter systems. Three approaches have proved to be particularly useful. Name these

Answer 24

ligand-binding methods,

Neuropharmacological analysis of synaptic transmission

Molecular analysis of receptor proteins

Question 25

Much of what we know about receptor subtypes was first learned using Which of these techniques?

Answer 25

neuropharmacological analysis

Question 26

Describe basis of neuropharmacological analysis using nicotine and muscarine

Answer 26

Skeletal muscle and heart muscle respond differently to various cholinergic drugs. Nicotine, derived from the tobacco plant, is a receptoragonist in skeletal muscle but has no effect in the heart. On the other hand, muscarine, derived from a poisonous species of mushroom, has little or no effect on skeletal muscle but is an agonist at the cholinergic receptor subtype in the heart. (Recall that ACh slows the heart rate; muscarine is poisonous because it causes a precipitous drop in heart rate and blood pressure.) Thus, two ACh receptor subtypes can be distinguished by the actions of different drugs.

In fact, the receptors were given the names of their agonists: nicotinic ACh receptors in skeletal muscle and muscarinic ACh receptors in the heart. Nicotinic and muscarinic receptors also exist in the brain, and some neurons have both types of receptors.

Question 27

Different drugs were also used to distinguish several subtypes of glutamate receptors what names were given to them and what functions do they carry out?

Answer 27

Glutamate receptors mediate much of the synaptic excitation in the CNS. Three subtypes are AMPA receptors, NMDA receptors, and kainate receptors

Question 28

What were these glutamate receptors named after?

Answer 28

Their chemical agonists. The neurotransmitter glutamate activates all three receptor subtypes, but AMPA acts only at the AMPA receptor, NMDA acts only at the NMDA receptor, and so on.

Question 29

Describe how researchers discovered endorphins in the brain via ligand binding

Answer 29

Some researchers hypothesized that opiates might be agonists at specific receptors in neuronal membranes. They radioactively labeled opiate compounds and applied them in small quantities to neuronal membranes that had been isolated from different parts of the brain. If appropriate receptors existed in the membrane, the labeled opiates should bind tightly to them. This is just what they found. The radioactive drugs labeled specific sites on the membranes of some, but not all, neurons in the brain.

Following the discovery of opioid receptors, the search was on to identify endogenous opioids, or endorphins, the naturally occurring neurotransmitters that act on these receptors. Two peptides called enkephalins were soon isolated from the brain, and they eventually proved to be opioid neurotransmitters.

Question 30

Therefore what is the ligand-binding method?

Answer 30

Any chemical compound that binds to a specific site on a receptor is called a ligand for that receptor (from the Latin meaning “to bind”). The technique of studying receptors using radioactively or nonradioactively labeled ligands is called the ligand-binding method. Notice that a ligand for a receptor can be an agonist, an antagonist, or the chemical neurotransmitter itself.

Question 31

What has Ligand-binding techniques been enormously important for carrying out?

Answer 31

Ligand-binding methods have been enormously important for mapping the anatomical distribution of different neurotransmitter receptors in the brain.

Question 32

There has been an explosion of information about neurotransmitter receptors in recent decades, thanks to modern methods for studying protein molecules. What two groups have these methods enabled us to divide neurotransmitter receptors into?

Answer 32

transmitter-gated ion channels and G-protein-coupled (metabotropic) receptors

discovered through molecular analysis

Question 33

Evolution is conservative and opportunistic, and it often puts common and familiar things to new uses.

Explain how this is true in terms of the evolution of neurotransmitters

Answer 33

For the most part, they are similar or identical to the basic chemicals of life, the same substances that cells in all species, from bacteria to giraffes, use for metabolism. Amino acids, the building blocks of protein, are essential to life. Most of the known neurotransmitter molecules are either (1) amino acids, (2) amines derived from amino acids, or (3) peptides constructed from amino acids.

Question 34

Name an exception to these possibilities for neurotransmitters and what it is derived from

Answer 34

ACh is one exception, but it is derived from acetyl CoA, a ubiquitous product of cellular respiration in mitochondria, and choline, which is important for fat metabolism throughout the body.

Question 35

What is meant by Dale’s principle?

Answer 35

Amino acid and amine transmitters are generally each stored in and released by different sets of neurons. The convention established by Dale classifies neurons into mutually exclusive groups by neurotransmitter (cholinergic, glutamatergic, GABAergic, and so on). The idea that a neuron has only one neurotransmitter is often called Dale’s principle.

Question 36

How well does Dale’s principle hold up?

Answer 36

Many peptide-containing neurons violate Dale’s principle because these cells usually release more than one neurotransmitter: an amino acid or amine and a peptide, however most neurons seem to release only a single amino acid or amine neurotransmitter.

Question 37

When two or more transmitters are released from one nerve terminal, what are they called?

Answer 37

co-transmitters

Question 38

What neurotransmitter is the neurotransmitter at the neuromuscular junction and where is it synthesised?

Answer 38

Acetylcholine (ACh) is the neurotransmitter at the neuromuscular junction and is therefore synthesized by all the motor neurons in the spinal cord and brain stem.

Question 39

What is required for synthesis of acetylcholine?

Answer 39

ACh synthesis requires a specific enzyme, choline acetyltransferase (ChAT)

Question 40

In which neuons can ChAT be produced?

Answer 40

Only cholinergic neurons contain ChAT, so this enzyme is a good marker for cells that use ACh as a neurotransmitter.

Question 41

Where is ChAT produced in the neuron and where does it synthesise ACh?

Answer 41

Like nearly all presynaptic proteins, ChAT is manufactured in the soma and transported to the axon terminal.

ChAT synthesizes ACh in the cytosol of the axon terminal, and the neurotransmitter is concentrated in synaptic vesicles by the actions of a vesicular ACh transporter.

Question 42

Rewinding a bit, apart from distinguishing receptor subtypes by their agonsists (as with nicotine and muscarine) how else can recptor subtypes be distinguished? Give an example in regards to ACh

Answer 42

Another way to distinguish receptor subtypes is to use selective antagonists. The South American arrow-tip poison curare inhibits the action of ACh at nicotinic receptors (thereby causing paralysis), and atropine, derived from belladonna plants (also known as deadly nightshade), antagonizes ACh at muscarinic receptors

Question 43

describe how ChAT synthesises ACh

Answer 43

ChAT transfers an acetyl group from acetyl CoA to choline. The source of choline is the extracellular fluid, where it exists in low micromolar concentrations. Choline is taken up by the cholinergic axon terminals via a specific transporter that requires the cotransport of Na+ to power the movement of choline.

Question 44

What is meant by a rate-limiting step and how is it relevant to the synthesis of ACh?

Answer 44

Because the availability of choline limits how much ACh can be synthesised in the axon terminal, the transport of choline into the neuron is said to be the rate-limiting step in ACh synthesis.

Question 45

What is often prescribed for certain diseases in which a deficit in cholinergic synaptic transmission has been noted?

Answer 45

Dietary supplements of choline are sometimes prescribed to boost ACh levels in the brain.

Question 46

What else do cholinergic neurons manufacture which is used in the process of neurotransmission? Is this a good marker for cholinergic synapses

Answer 46

Cholinergic neurons also manufacture the ACh degradative enzyme acetylcholinesterase (AChE). AChE is secreted into the synaptic cleft and is associated with cholinergic axon terminal membranes.

However, AChE is also manufactured by some noncholinergic neurons, so this enzyme is not as useful a marker for cholinergic synapses as ChAT.

Question 47

What function does AChE perform during neurotransmission

Answer 47

AChE degrades ACh into choline and acetic acid. This happens very quickly because AChE has one of the fastest catalytic rates among all known enzymes.

Question 48

What happens to the choline after the ACh is broken down by the AChE

Answer 48

AChE degrades ACh into choline and acetic acid (Figure 6.11b). This happens very quickly because AChE has one of the fastest catalytic rates among all known enzymes. Much of the resulting choline is taken up by the cholinergic axon terminal via a choline transporter and reused for ACh synthesis

Question 49

Why is AChE the target of many nerve gases and insecticides?

Answer 49

Inhibition of AChE prevents the breakdown of ACh, disrupting transmission at cholinergic synapses on skeletal muscle and heart muscle. Acute effects include marked decreases in heart rate and blood pressure; however, death from the irreversible inhibition of AChE typically results from respiratory paralysis.

Question 50

What are catecholamines?

Answer 50

The amino acid tyrosine is the precursor for three different amino neurotransmitter that contain a chemical structure called a catechol, collectively called catecholamines.

Question 51

What are the three catecholamines?

Answer 51

• Dopamine (DA)
• Norepinephrine (NE)
• Epinephrine/Adrenaline

Question 52

What enzyme do all catecholaminergic neurons contain?

Answer 52

The actions of catecholamines in the synaptic cleft are terminated by selective uptake of the neurotransmitters back into the axon terminal via Na+ dependent transporters.
• Amphetamine and cocaine block catecholamine uptake