Serotonin Synthesis, Release and Inactivation
he National Institutes of Health declared the 1990s to be the “Decade of the Brain,” to highlight the stunning advances in neuroscience being made at that time. If the 1990s was the decade of the brain for neuroscientists generally, then just as surely it was the “Decade of Serotonin” for psychopharmacologists. Serotonin, or, more technically speaking, 5-hydroxytryptamine (5-HT), has been featured in the popular culture as the culprit in just about every human malady or vice, including depression, anxiety, obesity, impulsive aggression and violence, and even drug addiction. Can a single neurotransmitter really have such far-reaching behavioral consequences? The answer is not a simple one—5-HT probably does influence many different behavioral and physiological systems, yet the ability of this chemical to either destroy us (if imbalanced) or to cure all that ails us (if brought back into equilibrium) has unfortunately been oversold by a sensationalist media aided and abetted by a few publicity-seeking scientists. In this post, we learn about the neurochemistry, pharmacology, and functional characteristics of this fascinating neurotransmitter.
Serotonin synthesis is regulated by the activity of tryptophan hydroxylase and the availability of the serotonin precursor tryptophan
Serotonin is synthesized from the amino acid tryptophan, which comes from protein in our diet. There are two steps in the biochemical pathway. The first step is catalyzed by the enzyme tryptophan hydroxylase, which converts tryptophan to 5-hydroxytryptophan (5- HTP). 5-HTP is then acted upon by aromatic amino acid decarboxylase (AADC) to form 5-HT.
Many features of this pathway are similar to the pathway described in the previous post involving the formation of dopamine from the amino acid tyrosine. Just as the initial step in the synthesis of DA (that is, tyrosine to DOPA) is the rate-limiting step, the conversion of tryptophan to 5-HTP is rate-limiting in the 5-HT pathway. Furthermore, just as tyrosine hydroxylase is only found in neurons that synthesize catecholamines, tryptophan hydroxylase similarly is a specific marker for neurons that make 5-HT (these are called serotonergic neurons). Another important point is that the second enzyme in the pathway, AADC, is the same for both catecholamines and 5-HT.
Serotonin synthesis in the brain can be stimulated by giving animals a large dose of tryptophan, but 5-HTP administration is even more effective because it is converted so rapidly and efficiently to 5-HT. There is also an interesting link between food intake and 5-HT synthesis that was first discovered many years ago by John Fernstrom and Richard Wurtman (1972). Imagine a group of rats that has been fasted overnight and then fed a protein-rich meal. The level of tryptophan in their blood goes up, and thus you would probably expect brain 5-HT to rise as well, since an injection of pure tryptophan produces such an effect. Surprisingly, however, Fernstrom and Wurtman found that consumption of a protein-rich meal did not cause increases in either tryptophan or 5-HT in the brain, even though tryptophan levels in the bloodstream were elevated. The researchers explained this result by showing that tryptophan competes with a group of other amino acids (called large neutral amino acids) for transport from the blood to the brain across the blood-brain barrier. Consequently, it’s the ratio between the amount of tryptophan in the blood and the overall amount of its competitors that counts. Most proteins contain larger amounts of these competitor amino acids than tryptophan, and thus when these proteins are consumed, the critical ratio either stays the same or even goes down.
Even more surprising was an additional finding of Fernstrom and Wurtman. When the researchers fed previously fasted rats a diet low in protein but high in carbohydrates, that experimental treatment led to increases in brain tryptophan and 5-HT levels. How could this be the case? You might already know that eating carbohydrates (starches and sugars) triggers a release of the hormone insulin from the pancreas. One important function of this insulin response is to stimulate the uptake of glucose from the bloodstream into various tissues, where it can be metabolized for energy. But glucose is not the only substance acted on by insulin. The hormone also stimulates the uptake of most amino acids from the bloodstream; tryptophan, however, is relatively unaffected. Because of this difference, we can see that a low-protein, high-carbohydrate meal will increase the ratio of tryptophan to competing amino acids, allowing more tryptophan to cross the blood-brain barrier and more 5-HT to be made in the brain.
Do the dietary effects observed in rats also occur in humans eating typical meals? Wurtman and colleagues (2003) recently addressed this issue by measuring the plasma ratio of tryptophan to large neutral amino acids in subjects eating either a high-carbohydrate, low-protein breakfast (consisting of waffles, maple syrup, orange juice, and coffee with sugar) or a high-protein, low-carbohydrate breakfast (consisting of turkey ham, Egg Beaters, cheese, grapefruit, and butter). As predicted, the high-carbohydrate, low-protein meal did increase the ratio of tryptophan to large neutral amino acids, whereas this ratio was decreased by the high-protein, low-carbohydrate meal. However, the average increase following the high-carbohydrate, low-protein meal was only about 14%, which may not have much effect on brain 5-HT levels.
Pharmacological depletion of 5-HT has been widely used to assess the role of this neurotransmitter in various behavioral functions. One method often used in rodent studies is to administer the drugpara-chlorophenylalanine (PCPA), which selectively blocks 5-HT synthesis by irreversibly inhibiting tryptophan hydroxylase. One or two high doses of PCPA can reduce brain 5-HT levels in rats 80 to 90% for as long as 2 weeks, until the serotonergic neurons make new molecules of tryptophan hydroxylase that haven’t been exposed to the inhibitor. Because PCPA can cause adverse side effects in humans, researchers have developed an alternative approach that has been particularly valuable for studying the role of 5-HT in mood and mood disorders. Based in part on the rat studies of Fernstrom and Wurtman, this method involves the administration of an amino acid “cocktail” containing a large quantity of amino acids except for tryptophan. This cocktail leads to a temporary depletion of brain 5-HT for two reasons: (1) the surge of amino acids in the bloodstream stimulates protein synthesis by the liver, which reduces the level of plasma tryptophan below its starting point; and (2) the large neutral amino acids in the cocktail inhibit entry of the remaining tryptophan into the brain. The 5-HT depletion produced by this method is not nearly as great nor as long-lasting as that produced by PCPA.
However, several studies have shown that giving the amino acid cocktail to previously depressed patients often causes a reappearance of depressive symptoms. In one case, 15 women who had suffered from repeated episodes of major depression but who were recovered at the time of the study were given either a tryptophan-free or tryptophan- containing amino acid mixture under double-blind conditions (Smith et al., 1997). Whereas the tryptophan-containing mixture had no effect on mood or depressive symptoms, the tryptophan-free mixture led to significant increases in depression ratings for 10 of the subjects as well as an overall increase in self-reported feelings of sadness. Such findings implicate 5-HT in mood regulation and further suggest that in patients successfully treated with antidepressant medications, symptom improvement may depend on continued activity of the serotonergic system.
The processes that regulate storage, release, and inactivation are similar for serotonin and the catecholamines
Serotonin is transported into synaptic vesicles using the same vesicular transporter, VMAT2 (vesicular monoamine transporter), found in dopaminergic and noradrenergic neurons. As with the catecholamines, storage of 5-HT in vesicles plays a critical role in protecting the transmitter from enzymatic breakdown in the nerve terminal. Consequently, the VMAT blocker reserpine depletes serotonergic neurons of 5-HT, just as it depletes catecholamines in dopaminergic and noradrenergic cells.
Serotonergic auto receptors control 5-HT release in the same way as the DA and NE autoreceptors discussed in the previous post. Terminal autoreceptors directly inhibit 5- HT release, whereas other autoreceptors on the cell body and dendrites of the serotonergic neurons (somatodendritic autoreceptors) indirectly inhibit release by slowing the rate of firing of the neurons. Somatodendritic autoreceptors are of the 5-HT1A subtype, whereas the terminal autoreceptors are either of the 5-HT1B or 5-HT1D subtype, depending on the species (see later discussion of 5-HT receptors).
Release of 5-HT can be directly stimulated by a family of drugs based on the structure of amphetamine. These compounds include para-chloroamphetamine, which is mainly used experimentally; fenfluramine, which at one time was prescribed for appetite suppression in obese patients; and 3,4-methylenedioxymethamphetamine (MDMA), which is a recreational and abused drug. Besides their acute behavioral effects, these drugs (particularly para-chloroamphetamine and MDMA) can also exert toxic effects on the serotonergic system.
When we examine the processes responsible for inactivation of 5-HT after its release, there are again many similarities to the catecholamine systems. After 5-HT is released, it is rapidly removed from the synaptic cleft by a reuptake process. Analogously to DA and NE, this mechanism involves a protein on the nerve terminal known as the 5-HT transporter. This protein turns out to be a key target of drug action. For example, the introduction of fluoxetine (better known as Prozac) in late 1987 spawned a whole new class of antidepressant drugs based on the idea of inhibiting 5-HT reuptake. These compounds are, therefore, called selective serotonin reuptake inhibitors (SSRIs). Certain abused drugs such as cocaine and MDMA likewise interact with the 5-HT transporter, but they are not selective in their effects because they also influence the DA transporter.
You will recall that DA and NE are metabolized by two different enzymes, monoamine oxidase (MAO) and catechol-O-methyltrans- ferase (COMT). Since 5-HT is not a catecholamines, it is not affected by COMT. However, its breakdown is catalyzed by MAO to yield the metabolite 5-hydroxyindoleacetic acid (5-HIAA). The level of 5-HIAA in the brains of animals or in the cerebrospinal fluid of humans or animals is often used as a measure of the activity of serotonergic neurons. This is based on research showing that when these neurons fire more rapidly, they make more 5-HT and there is a corresponding increase in the formation of 5-HIAA.
The neurotransmitter 5-HT is synthesized from the amino acid tryptophan in two biochemical reactions. The first and rate-limiting reaction is catalyzed by the enzyme tryptophan hydroxylase. Under appropriate conditions, the synthesis of brain 5-HT in rats can be enhanced by the consumption of a high-carbohydrate, low-protein meal. Administration of an amino acid mixture lacking tryptophan has been used to temporarily deplete 5-HT in human studies. Like the other transmitters previously discussed, 5-HT is stored in synaptic vesicles for subsequent release. Serotonin release is inhibited by autoreceptors located on the cell body, dendrites, and terminals of serotonergic neurons. The terminal autoreceptors are of either the 5-HT1B or 5-HT1D subtype, depending on the species, whereas the somatodendritic autoreceptors are of the 5-HT1A subtype. Serotonergic transmission is terminated by reuptake of 5-HT from the synaptic cleft. This process is mediated by the 5-HT transporter, which is an important target of several antidepressant drugs. Serotonin is ultimately metabolized by MAO to form the major breakdown product 5-HIAA.