Chemical Signaling by Neurotransmitters and Hormones – Neurotransmitter Synthesis, Release, and Inactivation
As previously mentioned, neurotransmitters are chemical substances released by neurons to communicate with other cells. Scientists first thought that only a few chemicals were involved in neurotransmission, but well over 100 chemicals have now been identified. As there are many thousands of chemicals present in any cell, how do we know whether a particular substance qualifies as a neurotransmitter? Verifying a chemical’s status as a neurotransmitter can be a difficult process, but here are some of the important criteria:
1. The presynaptic cell should contain the proposed substance along with a mechanism for manufacturing it.
2. A mechanism for inactivating the substance should also be present.
3. The substance should be released from the axon terminal upon stimulation of the neuron.
4. There should be receptors for the proposed substance on the postsynaptic cell.
5. Direct application of the proposed substance or an agonist drug that acts on its receptors should have the same effect on the postsynaptic cell as stimulating the presynaptic neuron (which presumably would release the substance from the axon terminals).
6. Applying an antagonist drug that blocks the receptors should inhibit both the action of the applied substance and the effect of stimulating the presynaptic neuron.
Even if all criteria have not yet been met for a suspected neurotransmitter, there is often sufficient evidence to make a strong case for transmitter candidacy.
Neurotransmitters encompass several different kinds of chemical substances
Despite the great number of neurotransmitters, most of them conveniently fall into several chemical classes. A few neurotransmitters are categorized as amino acids/ Amino acids serve numerous functions: they are the individual building blocks contained within proteins, they also play other metabolic roles, including their role as neurotransmitters. Several other transmitters are monoamines, which are grouped together because each possesses a single (hence “mono”) amine group. Monoamine transmitters are derived from amino acids by a series of biochemical reactions that include removal of the acidic part (-COOH) of the molecule. Consequently, we say that the original amino acid is a precursor because it precedes the amine in the biochemical pathway.One important neurotransmitter that is neither an amino acid nor a monoamine is ACh. Together with acetylcholine, the amino acid and monoamine neurotransmitters are sometimes called “classical” transmitters because they were generally discovered before the other categories.
Besides the classical transmitters, there are several other types of neurotransmitters. The largest group of “non-classical” neurotransmitters are the neuropeptides, whose name simply means peptides found in the nervous system. Peptides are small proteins, typically made up of 3 to 40 amino acids instead of the 100+ amino acids found in most proteins. Neuropharmacologists are very interested in the family of neuropeptides called endorphins and enkephalins, which stimulate the same opioid receptors that are activated by heroin and other abused opiate drugs. Another important neuropeptide is corticotropin-releasing factor (CRF), which is now believed to play a role in anxiety. A few transmitters are considered lipids, which is the scientific term for fatty substances. For example, in a future post we discuss a substance called anandamide, a lipid made in the brain that acts like marijuana (or, more specifically, A9- tetrahydrocannabinol [THC], which is the major active ingredient in marijuana). Finally, the most recently discovered and intriguing group of neurotransmitters are the gaseous transmitters. Later in this post we discuss nitric oxide, the best known of these unusual transmitters, and we will see that these substances break some of the normal rules followed by other transmitter molecules.
When scientists first discovered the existence of neurotransmitters, it was natural to assume that each neuron only made and released one transmitter substance, suggesting a simple chemical coding of cells in the nervous system. Much research over the past 20 years has shattered that initial assumption. We now know that many neurons make and release two, three, or occasionally even more, different transmitters. Some instances of transmitter coexistence within the same cell involve one or more neuropeptides along with a classical transmitter. In such cases, the neuron has two different types of synaptic vesicles: small vesicles that contain only the classical transmitter and large vesicles that contain the neuropeptide along with the classical transmitter.
Classical transmitters and neuropeptides are synthesized by different mechanisms
How and where in the nerve cell are neurotransmitters manufactured? Except for the neuropeptides, transmitters are synthesized by enzymatic reactions that can occur anywhere in the cell. Typically, the enzymes required for producing a neurotransmitter are shipped out in large amounts to the axon terminals, so the terminals are an important site of transmitter synthesis. The neuropeptides are different, however. Their precursors are protein molecules, within which the peptides are embedded. The protein precursor for each type of peptide must be made in the cell body, which is the site of almost all protein synthesis in the neuron. The protein is then packaged into large vesicles along with enzymes that will break down the precursor and liberate the neuropeptide. These vesicles are transported to the axon terminals so that release occurs from the terminals, as with the classical transmitters. On the other hand, new neuropeptide molecules can only be generated in the cell body, not in the terminals. An important consequence of this difference is that replenishment of neuropeptides is slower than for small-molecule transmitters. When neurotransmitters are depleted by high levels of neuronal activity, small molecules can be resynthesized rapidly within the axon terminal. In contrast, neuropeptides cannot be replenished until large vesicles containing the peptide have been transported to the terminal from their site of origin within the cell body.
Chemicals that don’t act like typical neurotransmitters are sometimes called neuromodulators
Some investigators use the term neuromodulators to describe substances that don’t act exactly like typical neurotransmitters. For example, a neuromodulator might not have a direct effect itself on the postsynaptic cell. Instead, it might alter the action of a standard neurotransmitter by enhancing, reducing, or prolonging the transmitter’s effectiveness. Peptides that are co-released with a classical transmitter sometimes exhibit this kind of modulatory effect. Neuromodulators are also sometimes characterized as diffusing beyond the synapse to influence cells farther away. No matter which criteria you use, though, the dividing line between neurotransmitters and neuromodulators is vague. For example, a particular chemical may sometimes act within the synapse, but in other circumstances it may act at a distance from its site of release. Therefore, we will refrain from talking about neuromodulators and instead use the term neurotransmitter throughout the remainder of the website.
Neurotransmitter release involves the exocytosis and recycling of synaptic vesicles
Synaptic transmission involves a number of processes that occur within the axon terminal and the postsynaptic cell. We will begin our discussion of these processes with a consideration of neurotransmitter release from the terminal. When a neuron fires an action potential, the depolarizing current sweeps down the length of the axon and enters all of the axon terminals. This wave of depolarization has a very important effect within the terminals: it opens large numbers of voltage-sensitive calcium (Ca2+) channels, causing a rapid influx of Ca2+ ions into the terminals. The resulting increase in Ca2+ concentration within the terminals is the direct trigger for neurotransmitter release.
Exocytosis You already know that the neurotransmitter molecules destined to be released are stored within synaptic vesicles, yet these molecules must somehow make their way past the membrane of the axon terminal and into the synaptic cleft. This occurs through a remarkable process known as exocytosis. Exocytosis is a fusion of the vesicle membrane with the membrane of the axon terminal, which exposes the inside of the vesicle to the outside of the cell. In this way, the vesicle is opened and its transmitter molecules are allowed to diffuse into the synaptic cleft. In fact, transmitter release doesn’t occur just anywhere along the terminal, but only at specialized regions near the postsynaptic cell, which stain darkly in the electron micrograph. These release sites are called active zones. For exocytosis to take place, a vesicle must be transported to an active zone by a mechanism that isn’t yet fully understood. There, the vesicle must “dock” at the active zone, much like a boat docking at a pier. This docking step is carried out by a cluster of proteins, some located in the vesicle membrane and others residing in the membrane of the axon terminal. Docking is followed by a step called “priming,” which readies the vesicle for exocytosis once it receives the Ca2+ signal. Indeed, the Ca2+ channels that open in response to the membrane depolarization are concentrated in the active zones near the sites of vesicle docking, so the protein machinery is exposed to particularly high concentrations of Ca2+ when the channels open. One or more proteins that are sensitive to Ca2+ then cause the vesicle and terminal membranes to fuse, which allows the vesicle to open and the transmitter to be released.
Discussing the various proteins involved in vesicle docking and fusion is beyond the scope of this website, but it’s nevertheless interesting to briefly consider how these proteins have been discovered. One method has been to analyze the effects of various drugs or naturally occurring toxins that affect the release process. For example, botulism poisoning results from a bacterial toxin (botulinum toxin) that blocks transmitter release at neuromuscular junctions, thus causing paralysis. Researchers have found that this blockade of release is due to enzymes within the toxin that attack some of the proteins that are required for the exocytosis process. Another important method has been to use genetic mutants of the fruit fly Drosophila melanogaster. Using genetic engineering techniques, researchers knocked out the gene for a protein they suspected was important for exocytosis. The fruit fly larvae exhibited no transmitter release at the neuromuscular junction when their motor nerves were stimulated; consequently, the larvae were virtually paralyzed and couldn’t even hatch from their egg cases (Deitcher et al, 1998).
Endocytosis When a synaptic vesicle fuses with the axon terminal to release its transmitter contents, the vesicle membrane is temporarily added to the membrane of the terminal. If this process were never reversed, we can imagine that the terminal membrane would grow larger and larger as more and more vesicle membrane was added to it. In reality, a process called endocytosis quickly retrieves the vesicle membrane from the terminal membrane. New vesicles are then rapidly formed and refilled with neurotransmitter molecules so that they can participate again in transmitter release. This continuous release and re-formation of vesicles is termed vesicle recycling. It is worth noting that recycling only occurs with the small vesicles containing classical transmitters, but not with the larger neuropeptide-containing vesicles. You’ll recall that neuropeptide precursor proteins must be packaged into the large vesicles in the cell body; therefore, recycling of such vesicles cannot occur at the axon terminal.
Several mechanisms control the rate of neurotransmitter release by nerve cells
Neurotransmitter release is regulated by several different mechanisms. The most obvious is the rate of cell firing. When a neuron is rapidly firing action potentials, it will release much more transmitter than when it is firing at a slow rate. A second factor is the probability of transmitter release from the terminal. It might seem odd that an action potential could enter a terminal and open Ca2+ channels but not release any transmitter. Yet many studies have shown that synapses in different parts of the brain vary widely in the probability that even a single vesicle will undergo exocytosis in response to an action potential. Estimated probabilities range from less than 0.1 (10%) to 0.9 (90%) or greater for different populations of synapses. We don’t yet know why these probabilities can vary so much, but it is clearly an important factor in the regulation of neurotransmitter release.
A third factor in the rate of transmitter release is the presence of autoreceptors on axon terminals or cell bodies and dendrites. An autoreceptor on a particular neuron is a receptor for the same neurotransmitter released by that neuron (“auto-” in this case means “self”). Neurons may possess two different kinds of autoreceptors: terminal autoreceptors and somatodendritic autoreceptors. Terminal autoreceptors are so named because they are located on axon terminals. When they are activated by the neurotransmitter, their main function is to inhibit further transmitter release. This function is particularly important when the cell is firing rapidly and there are high levels of neurotransmitter in the synaptic cleft. Think of the thermostat (“autoreceptor”) in your house, which shuts off the furnace (“release mechanism”) when the level of heat (“neurotransmitter”) gets too high. Somatodendritic autoreceptors are also descriptively named, since they are autoreceptors found on the cell body (soma) or dendrites. When these autoreceptors are activated, they slow the rate of cell firing, which ultimately causes less neurotransmitter release, as fewer action potentials reach the axon terminals to stimulate exocytosis.
Researchers can use drugs to stimulate or block specific autoreceptors, thereby influencing the release of a particular neurotransmitter for experimental purposes. For example, administration of a low dose of the drug apomorphine to rats or mice selectively activates the terminal autoreceptors for DA. This results in less DA release, an overall reduction in dopaminergic transmission, and reduced locomotor activity in the animals. A different drug, whose name is abbreviated 8-OH-DPAT, activates the somatodendritic autoreceptors for 5-HT and powerfully inhibits the firing of serotonergic neurons. The behavioral effects of 8-OH-DPAT administration include increased appetite and altered responses on several tasks used to assess anxiety.
Finally, you’ll recall from our earlier discussion that in addition to autoreceptors, axon terminals may also have receptors for other transmitters released at axoaxonal synapses. Such receptors have come to be known as heteroreceptors, to distinguish them from autoreceptors. Heteroreceptors also differ from autoreceptors in that they may either enhance or reduce Ae amount of transmitter being released from the axon terminal.
Neurotransmitters are inactivated by reuptake and by enzymatic breakdown
Any mechanical or biological process that can be turned on also must have a mechanism for termination (imagine the problem you would have with a car in which the ignition could not be turned off once the car had been started). Thus, it is necessary to terminate the synaptic signal produced by each instance of transmitter release so that the postsynaptic cell is free to respond to the next release. This termination is accomplished by removing neurotransmitter molecules from the synaptic cleft. How is this done?
One mechanism is enzymatic breakdown within or near the synaptic cleft. This mechanism is very important for the classical neurotransmitter ACh, for the lipid and gaseous transmitters, and also for the neuropeptide transmitters. An alternative mechanism is for the neurotransmitter to be removed from the synaptic cleft by a transport process involving specialized proteins called transporters located on the cell membrane. This mechanism is important for amino acid transmitters like glutamate and GABA and also for amine transmitters such as DA, NE, and 5-HT. Transport out of the synaptic cleft is sometimes accomplished by the same cell that released the transmitter, in which case it is called reuptake. In other cases, the transmitter may be taken up either by the postsynaptic cell or by nearby glial cells (specifically astrocytes). Some important psychoactive drugs work by blocking neurotransmitter transporters. Cocaine, for example, blocks the transporters for DA, 5-HT, and NE. Many antidepressant drugs block the 5-HT transporter, the NE transporter, or both. Since these transporters are so important for clearing the neurotransmitter from the synaptic cleft, it follows that when the transporters are blocked, neurotransmitter molecules remain in the synaptic cleft for a longer period of time and neurotransmission is enhanced at those synapses.
When neurotransmitter transporters are active, some transmitter molecules removed from the synaptic cleft are reused by being packaged into recycled vesicles. However, other transmitter molecules are broken down by enzymes present within the cell. Thus, uptake and metabolic breakdown are not mutually exclusive processes. Many transmitter systems use both mechanisms. Finally, it is important to keep in mind the distinction between autoreceptors and transporters. Even though both may be present on axon terminals, they serve different functions. Terminal autoreceptors modulate transmitter release, but they don’t transport the neurotransmitter. Transporters take up the transmitter from the synaptic cleft, but they are not autoreceptors.
Synapses are specialized structures that mediate chemical communication between nerve cells. Synapses can be classified as axodendritic, axosomatic, or axoaxonic, depending on which part of the postsynaptic cell is receiving input from the presynaptic axon terminal. Axodendritic and axosomatic synapses affect the firing of the postsynaptic cell, whereas axoaxonic synapses either stimulate or inhibit neurotransmitter release from terminals of the postsynaptic cell. Connections between neurons and muscle cells are called neuromuscular junctions, and they have many features in common with ordinary synapses.
The chemical substances released at synapses and neuromuscular junctions are called neurotransmitters. The initial criteria for determining whether a substance qualifies as a neurotransmitter include (1) the presence of the substance in axon terminals, along with a mechanism for synthesis of the substance; (2) the presence of a mechanism for inactivating the substance; (3) release of the substance upon nerve stimulation; and (4) the presence of appropriate receptors on the postsynaptic cell. Pharmacologically, (5) application of the substance or an agonist drug to the postsynaptic cell should mimic the effect of nerve stimulation, whereas (6) applying an antagonist drug should block the effects of both nerve stimulation and the substance itself. Most neurotransmitters fall into one of several broad chemical categories: amino acid transmitters, monoamine transmitters, lipid transmitters, neuropeptide transmitters, and gaseous transmitters. Acetylcholine is an important neurotransmitter that doesn’t fall into any of these categories. Many instances are known where two or more different neurotransmitters are synthesized and released from the same neuron.
Except for the neuropeptides, the axon terminals are the most critical site for the synthesis of most neurotransmitters. However, neuropeptides are formed from large precursor proteins that must be produced in the cell body and then transported down the axon to the terminals. Most neurotransmitters, including neuropeptides, are stored in synaptic vesicles. Within each axon terminal, a few vesicles are docked at specialized places called active zones. When an action potential invades the terminal, Ca2+ channels open in response to the membrane depolarization. This triggers a process known as exocytosis, which involves fusion of the vesicle membrane with the terminal membrane, followed by release of neurotransmitter molecules into the synaptic cleft. The vesicle membrane is subsequently removed from the axon terminal by the process of endocytosis, and new vesicles are generated. This continuous process of vesicle release and re-formation is called vesicle recycling.
Neurotransmitter release is controlled by several factors. First, faster firing by the neuron leads to increased release. Second, when an action potential reaches the axon terminal, it may or may not lead to vesicle exocytosis. The probability of release varies widely at different synapses throughout the brain. Third, transmitter release can be inhibited by the action of auto receptors located either on the terminal (called terminal autoreceptors) or on the membrane of the cell body and dendrites (somatodendritic autoreceptors).
Finally, there are several mechanisms for terminating the action of neurotransmitters. One mechanism is enzymatic breakdown, which is important for ACh, lipid and gaseous transmitters, and neuropeptides. Another mechanism, which is used by amino acid and monoamine transmitters, is transport out of the synaptic cleft either by the axon terminal that released the transmitter (reuptake) or by nearby glial cells. Even when reuptake occurs, however, enzymatic metabolism within the cell is still needed to prevent the neurotransmitter from building up to excessive levels.