Historical Aspects of Serotonin

Since 1868, serum (sero-) from blood clots was known to possess a substance that caused blood vessels to constrict, increasing their smooth muscle tone (-tonin). Subsequent physiologic studies of this vasoconstrictive activity vacillated between some unknown substance and epinephrine as the cause. Eventually, the issue was clarified when it was observed that the serum constricted frog vascular and rabbit intestinal preparations, whereas epinephrine caused only relaxation of the gut. Because no evidence of epinephrine was found in the blood plasma, it was assumed that vasoconstriction was caused by a substance in the coagulated blood, and by the early 1900s the source of that substance was identified with platelets.

Janeway and associates²⁰ did a thorough investigation of the vasoconstrictive substance and noted that it was not present in uncoagulated or citrated blood, that it was definitely associated with platelets, that it was soluble in water more than ether or chloroform, and that the factor did not depend on the clot formation but on the disintegration of the platelets in the clot. The substance itself was eventually isolated and named serotonin by Rapport and colleagues in 1948.³⁶ Shortly after this, Rapport and colleagues identified the agent as 5-HT, and Hamlin and Fischer¹⁸ reported synthesizing it in 1951.

Meanwhile, in Italy, in a separate series of studies, Erspamer and Asero⁸ isolated a substance from the mucosa of rabbit stomach and found that it was abundant in the enterochromaffin cells of the gut, could be extracted with alcohol and acetone, was an amine that affected smooth muscle, and was deactivated by deamination. Erspamer and Asero named it enteramine. By 1952, serotonin and enteramine had been chemically identified as 5-HT, eventually leading to international wrangling over the naming of 5-HT. It was argued that “enteramine” was inaccurate because the substance was found in places other than the gut, and “serotonin” was equally inadequate from the points of origin and pharmacologic action. In 1986, when the International Serotonin Club was organized, American researchers prevailed over the European contingent in naming the substance serotonin by arguing that serotonin was the most widely accepted name, 5-hydroxytryptamine was too long, and 5-HT was only an abbreviation (but one used here).

5-HT and Pain

Stimulation of the periaqueductal gray (PAG) was shown to modulate nociception on a spinal level.²⁸ This effect is known as stimulation-produced analgesia (SPA). Although a number of areas have been studied in animals, human studies of necessity have been limited. In humans, stimulation of the midbrain region of the PAG and areas slightly more rostral in the periventricular gray matter of the hypothalamus are known to produce SPA. Neurosurgeons were able to show SPA in humans by stimulating the equivalent human midbrain sites. Researchers had determined that electrical stimulation of brainstem PAG produced analgesia in animals. Although the exact boundaries of the responsive area were not clearly defined, the sites most responsive to SPA were: ventral to the midbrain cerebral aqueduct; in the PAG; sites lateral to this structure; the rostroventral medulla (RVM), including the midline nucleus raphe magnus (NRM) and reticular formations; the hypothalamus; the frontal lobe; and the spinal cord. Areas outside of the midbrain have not been systematically studied.

Most of the projections from the RVM/PAG are tryptaminergic. Injection of morphine in the PAG also has a similar antinociceptive effect and is thought to be mediated by activation of a raphe-spinal pathway. Other studies have implicated descending 5-HT fibers and other non–5-HT–containing fibers in this process. Increased production of 5-HT in the bulbospinal 5-HT neurons supports the role of 5-HT in modulation of pain in these pathways. Studies of the raphe pathways have confirmed that with such stimulation there is a concomitant increase in 5-hydroxyindoleacetic acid (5-HIAA), a major metabolite of 5-HT, in the dorsal horn, implicating activation and degradation of 5-HT in the process.

Anatomic Distribution

5-HT is a biogenic monoamine and is widely distributed throughout the plant and animal kingdom. In mammals, the highest concentrations are found in the enterochromaffin cells of the gastrointestinal mucosa, central nervous system (CNS), and blood platelets. The structure of 5-HT is shown in Figure 23-1. Its most notable features are the hydroxyl group on position 5 of the indole nucleus and the primary amine nitrogen that can accept a proton, making the compound hydrophilic and unable to pass the blood-brain barrier easily. Rapport and colleagues³⁶ found the substance in the brain, indicating that it must be synthesized and perform some unidentified function there. It was subsequently assumed that 5-HT was associated with psychiatric disorders such as depression and schizophrenia when it was shown that the psychedelic drug lysergic acid diethylamide (LSD) antagonized 5-HT function. 5-HT is now known to be involved in many behavioral and psychiatric disorders, such as schizophrenia, obsessive-compulsive disorder, depression, and anxiety, and drugs that have an effect on the 5-HT system have been beneficial in treating these disorders (see Chapter 12).

FIGURE 23-1 Biosynthesis and metabolism of serotonin. MAO, Monoamine oxidase.

Despite earlier suggestions that 5-HT was a neurotransmitter synthesized in the brain, the actual localization of 5-HT neurons was not determined for at least 10 more years. By using lesioning and fractionation techniques, 5-HT was grossly associated with specific neuronal elements, but it was impossible to observe the relationship directly until fluorescence histochemical techniques were developed. This process had inherent problems, however, that made identification a significant challenge. Dahlström and Fuxe,⁷ using immunocytochemical techniques, localized 5-HT–associated neurons in nine discrete clusters of cells along the midline of the upper brainstem and pons. These 5-HT–containing cell bodies, designated B1 to B9, corresponded for the most part to the dorsal raphe nuclei. Only approximately 40% to 50% of the dorsal raphe nuclei are serotonergic neurons, and some serotonergic nuclei are found outside the midline raphe nuclei area, although the major brain concentration is in the dorsal raphe nuclei.

Additional studies have shown that the lateral and dorsolateral pontine tegmentum, which contain many noradrenergic neurons, are also involved in nociceptive modulation when stimulated, and these sites send projections to the PAG, the RVM, and the spinal cord. The projections from the lateral and dorsolateral pons are noradrenergic and possess important α₂-adrenergic receptors. In animal studies, norepinephrine (NE) applied directly to the spinal cord blocks response to nociception through selective inhibition of the nociceptive dorsal horn neurons (see Chapter 20). Lesioning the white matter of the dorsolateral funiculus of the spinal cord blocks the inhibitory effect of SPA and confirms the existence of a descending modulatory pathway that travels through the dorsolateral funiculus. Further studies of the dorsolateral funiculus projections to the spinal cord have found that most of the brainstem projections arise in the RVM and dorsolateral pons, with few projections from the PAG. This finding implies that the PAG projections must be relayed through the RVM. This has been confirmed by studies showing that the major neuronal input to the RVM is from the PAG and adjacent structures, and lesioning or blocking RVM cells eliminates the analgesic effect obtained from PAG stimulation.

Anti–5-HT antibody labeling has identified 5-HT in all dorsal horn laminae, but the highest densities are found in laminae I, II, IV, V, and X. The RVM projections terminate mainly in laminae I, II, and V. These areas are important for pain because this is where the central terminals of afferent nociceptors and cell bodies of second-order neurons are found. This dorsal horn area is the major “switchboard” for pain, and stimulation of the PAG and RVM modulates nociceptive activity here (see Chapter 20).

Immunocytochemical studies have also found 5-HT reactive cells in the area postrema, the caudal locus coeruleus, and around the interpeduncular nucleus. Through lesioning studies, it has been observed that the caudal clusters project mainly to the medulla and spinal cord, the rostral clusters project to the telencephalon and diencephalon, and the more centrally located clusters project rostrally and inferiorly. In general, 5-HT cells send axons through virtually every part of the CNS, however, and more recent findings indicate a lack of pattern to this innervation.

Transmission of sensory and particularly nociceptive messages by afferent fibers entering the dorsal horn of the spinal cord is under control of pathways originating in the ventromedial medulla. It had been observed that neurons from the medullary raphe nuclei and particularly the NRM project predominantly to the dorsal horn, including the superficial laminae and the area around the central canal, and are involved in a descending inhibitory pathway for modulation of nociceptive input. Because the area was found to have an abundance of 5-HT–containing neurons, researchers postulated that 5-HT was a descending pain modulatory system neurotransmitter. 5-HT–containing neurons are located in the rostroventromedial medulla and caudal pons, and particularly in the NRM, the nucleus paragigantocellularis, and the ventral portion of the nucleus gigantocellularis. More recent studies have described other descending projections from the bulbomesencephalon to the spinal cord that do not contain 5-HT and are more numerous within the medulla and caudal pons, indicating that descending modulation is not limited to 5-HT fibers.²⁴

Immunocytochemical studies of antibodies directed against 5-HT have shown that two distinct types of 5-HT neurons innervate the cerebral cortex of many mammals. The studies have found fine axons with small varicosities originating from the dorsal raphe nuclei and beaded axons with large spherical varicosities originating from the median raphe nuclei. Apparently the two types of axons have different regional and laminar distributions and exhibit different sensitivities to neurotoxic drugs such as 3,4-methylenedioxymethamphetamine, commonly referred to as “ecstasy.” The fine axons seem to be more sensitive to the neurotoxic effects, with loss of functions that may be long-term or permanent. Cooper and associates⁶ suggested that laboratory animal findings may relate to humans’ use of the drug because the doses commonly used by recreational drug users are similar to what are used in animal studies. Ecstasy users have shown a 26% decrease in 5-HIAA, the 5-HT metabolite. The decrease in metabolite may indicate a decrease of 5-HT function in the brain related to loss of some 5-HT neurons. The functional distinction between these two types of neurons generally remains unclear, however.

Synthesis, Storage, and Fate

5-HT is synthesized from the amino acid l-tryptophan (see Figure 23-1). Although platelets contain large amounts of 5-HT, it only accumulates rather than being synthesized there. Synthesis in the CNS involves active transport of tryptophan through the blood-brain barrier. Tryptophan is derived primarily from the diet, and its elimination from the diet can profoundly decrease brain 5-HT. In addition, the active transport of tryptophan is affected by its concentration in the blood and the relative concentration of other amino acids that are transported by the same active transport mechanism. l-Tryptophan is converted in serotonergic neurons containing the enzyme tryptophan hydroxylase (l-tryptophan-5-monooxygenase).

The initial synthesis step is hydroxylation of tryptophan at the 5 position to form 5-hydroxytryptophan (see Figure 23-1). Tryptophan hydroxylase, the enzyme responsible for this reaction, occurs in low concentrations in most tissues, including the brain, and has proved to be difficult to isolate.

Tryptophan hydroxylase has a rate-limiting requirement for oxygen. In addition, mounting evidence suggests that the system adjusts to the amount of tryptophan available. It has been shown that drug treatments affecting the 5-HT system are soon counteracted by a built-in feedback mechanism involving regulation of the synthesis of 5-HT. Short-term treatment with lithium salts initially increases tryptophan uptake, resulting in increased amounts of tryptophan being converted to 5-HT; however, with long-term treatment, increased uptake is still measured, but the synthesis of 5-HT from the increased tryptophan returns to pretreatment levels.

5-Hydroxytryptophan is rapidly decarboxylated to form 5-HT by the aromatic enzyme l-amino acid decarboxylase, which is the same enzyme that catalyzes the decarboxylation of l-dopamine in catecholamine neurons (see Figure 23-1). Because the rate of the reaction is so rapid and requires less substrate than the initial reaction, the action of tryptophan hydroxylase in the first step is regarded as the rate-limiting step in the synthesis of 5-HT, and drugs targeting the action of the decarboxylase have not been shown to be effective.

The synthesis of 5-HT is markedly increased with the electrical stimulation of serotonergic soma. This is the result of enhanced conversion of tryptophan to 5-HT and depends on extracellular Ca⁺⁺. Because, as discussed previously, the rate-limiting step is the action of tryptophan hydroxylase on tryptophan, it is likely that Ca⁺⁺ affects the Ca⁺⁺-dependent phosphorylation of the enzyme, increasing its availability.

5-HT Receptors and Pain

In 1957, Gaddum and Picarelli¹¹ reported two separate 5-HT receptors in peripheral smooth muscle preparations studied in vitro. Since then, there has been an exponential development of information relating to 5-HT receptor types and functions, and numerous receptor subtypes have been identified and cloned more recently. Nevertheless, the complete picture of how 5-HT and its receptors modulate pain remains obscure. There are now seven main family groups of receptors, but current understanding attributes most of the 5-HT actions relative to pain to the families designated 5-HT₁, 5-HT₂, 5-HT₃, 5-HT₄, and 5-HT₇. Each receptor family is operationally and structurally distinct, each having its own separate transducing system.

Although other more recently identified 5-HT receptors also show characteristics indicative of separate classes or families of 5-HT receptors, not all receptors for 5-HT are fully included in the classification system at this time because of a lack of needed characterization through cDNA cloning and amino acid sequencing of their proteins or data concerning their operational and transductional characteristics. This situation applies to the 5-HT_1E, 5-HT_1F, 5-HT₅, and 5-HT₆ receptors, which have been cloned and their amino acid sequence defined, although their operational and transductional characteristics are unclear, and the final nomenclature is unsettled. Fourteen different 5-HT receptor subtypes have been identified.

As mentioned, seven classes or families of 5-HT receptors have been identified and designated 5-HT₁, 5-HT₂, 5-HT₃, 5-HT₄, 5-HT₅, 5-HT₆, and 5-HT₇. Each class has subtypes (with the exception of 5-HT₃), and all have been identified in the brain. Localization of the receptors in the CNS and spinal cord is variable, and not all 5-HT receptors are found in all locations. Within the spinal cord itself there is variability in the location of different receptors. Because 5-HT₁, 5-HT₂, and 5-HT₃ receptors have the highest distribution in the dorsal horn, it is assumed that they are involved in sensory processing. In general, the 5-HT₁ family is assumed to be inhibitory, and the other classes are thought to be excitatory; however, 5-HT₂ and 5-HT₃ receptors have been linked to an antinociceptive response as measured by some animal models. Much work is still to be done before the 5-HT–modulating system is fully understood.

The 5-HT_1D receptor, also called the terminal autoreceptor, is found on the presynaptic button. It turns off the release of serotonin in response to the presence of serotonin in the synaptic cleft. The receptor is also found on postsynaptic neurons and has been implicated in migraine pathophysiology. The 5-HT_1A receptor, also known as a somatodendritic autoreceptor, is found primarily on the dendrites and cell bodies of the serotonin neurons but performs a similar function in regulating the release of serotonin. When these receptors are activated or stimulated by serotonin or drugs that mimic serotonin action, the receptor causes a blockade of serotonin release. When the receptors are blocked by receptor blocking medications, the receptor is no longer able to inhibit serotonin release. Serotonin release is also modulated by an α₂-adrenergic heteroreceptor on the terminus of the serotonin neurons. This receptor is activated by NE resulting in the inhibition of serotonin release.

Physiologic Function and Drug Intervention

The 5-HT system modulates activity in diverse regions of the brain and spinal cord. It is suggested that the system coordinates various sensory and motor patterns associated with behavioral states. 5-HT activity is highest during waking and lowest during sleep. Descending neurons are involved in pain modulation in the dorsal horn and motor activity in the ventral horn. 5-HT activity is absent during rapid eye movement sleep when physical movement is limited, although the animal is in a state of heightened internal arousal. The increased 5-HT activity during waking periods aids in enhancing motor neuron excitability.

The raphe nuclei 5-HT neurons display spontaneous discharge activity of one to five spikes per second, releasing 5-HT into the presynaptic cleft. The neurons possess negative feedback autoreceptors that limit the amount of discharge and release of 5-HT. The autoreceptors seem to function only when the discharge and release of 5-HT reaches levels greater than the normal background activity inherent in the neurons. Dysfunction of the autoreceptor regulation has been associated with some forms of neuropathy, and the autoreceptor may provide a therapeutic target for medications. Although in some areas microelectrophoretically administered 5-HT can have an excitatory effect on the discharge rate, the most common response in 5-HT–containing neuron tracts is inhibition of the discharge rate.

5-HT released from neurons has presynaptic and postsynaptic receptor effects. There are many options for affecting the availability of 5-HT directly, either by inhibiting the processes that decrease its availability or by enhancing the processes that make it available (Figure 23-2). Figure 23-2 shows sites of interaction with known drugs that influence the 5-HT system. Of all the options, reuptake blockade of 5-HT by TCAs is the most common mechanism of 5-HT active medications prescribed for the treatment of depression and chronic pain; however, the issue of availability of 5-HT to help modulate pain has yet to be settled.

FIGURE 23-2 Possible drug sites for influencing 5-HT neurotransmission. The sites of potential drug action on the 5-hydroxytryptamine (5-HT) neuron are enumerated from 1 to 6. Site 1 represents the modulation of enzymatic action forming the 5-HT precursor and ultimately 5-HT from tryptophan. Site 2 is a potential target for drugs that affect the storage vesicles of 5-HT. Reserpine and tetrabenazine are known to interrupt the storage and cause release of 5-HT. Site 3 targets the release mechanism itself; however, there are currently no known agents that act to interrupt or increase the action of the transporter proteins that carry the 5-HT molecule across the membrane. Site 4 involves the reuptake mechanism that brings 5-HT back into the intracellular environment to be repackaged in the vesicles. Numerous drugs inhibit the reuptake of 5-HT (see Chapter 12). Site 5 refers to the 5-HT autoreceptor. Blockade of this receptor site allows more presynaptic release of 5-HT. Site 6 focuses on the action of monoamine oxidase that converts the free 5-HT to the metabolic product 5-HIAA. Monoamine oxidase (MAO) inhibitors such as phenelzine block this action. SSRIs, Selective serotonin reuptake inhibitors; TCAs, tricyclic antidepressants.

Ergot Derivatives

In the Middle Ages, epidemics of a gangrenous disorder known as “holy fire” or “St. Anthony’s fire” were afflicting communities in Europe. The condition was so named because of the attendant burning experienced by the sufferer. The disorder soon became associated with the grain of rye that had been contaminated with the ergot fungus Claviceps purpurea. In 1918, the ergot alkaloid ergotamine was isolated from the fungus and was found to have sympatholytic activity (see Chapter 7). Shortly thereafter, it was proposed for use as a therapeutic agent for migraine.

Ergotamine has a complex mode of action involving a variety of receptor activities, not only with 5-HT receptors, but also with dopamine and NE receptors. The vasoconstrictive effect, the most notable characteristic of this medication, can become problematic with overuse, leading to claudication of extremities. The major vasoconstrictive activity is noted in the carotid circulation; the cephalic arteriovenous anastomoses; the pulmonary, cerebral, temporal, and coronary arteries; and the blood pressure. These effects are short-lived, although constriction of leg arteries can last 8 hours. Chronic overuse becomes a problem, and clinicians need to monitor users carefully.

Ergot derivatives are nonselective partial agonists and antagonists at 5-HT receptors, having high affinity for the 5-HT_1B, 5-HT_1D, 5-HT_1F, and 5-HT₂ receptors and low to moderate affinity for the 5-HT_1C and 5-HT₃ receptors. It is currently believed that their primary mode of action in alleviating migraine attacks is through their action on the 5-HT_1B/1D receptors, inhibiting neurogenic inflammation and nociceptor activity. Table 23-1 lists some 5-HT receptors and related medications and some indications for the drugs.

Triptans

The new family of triptan antimigraine drugs represents the most dramatic advance in the understanding and treatment of migraine. They are classified as 5-HT_1B/1D receptor agonists. Discovery of the first of these drugs, sumatriptan, came after the 5-HT_1B/1D receptor was linked with migraine. When it became apparent that the receptor had an inhibitory G protein–linked action on the host vascular and nociceptor neurons, agonist agents were sought that could act with the receptor to stop the attack. Sumatriptan was originally thought to act primarily on nociceptor 5-HT_1D receptors, but action was also noted on dilated dural blood vessels. Sumatriptan-sensitive sites of action in the pain transmission pathway have been identified centrally and suggested as additional putative antimigraine targets for brain-penetrant triptan derivatives. Sumatriptan has selective affinity for 5-HT receptor subtypes, 5-HT_1B and 5-HT_1D. More recent development of 5-HT receptor subtype–specific compounds and antibodies has allowed a more precise identification of the vascular and neuronal sites of action, providing a basis for a more targeted therapeutic approach.

Moskowitz^29,30 developed the concept of neurovascular inflammation in the trigeminovascular system as a migraine mechanism. He observed that sumatriptan’s action on peripheral neuronal 5-HT_1D receptors blocked subsequent release of neuropeptides such as SP and CGRP that were responsible for the development of neurovascular inflammation with concomitant swelling of the dural blood vessels. Moskowitz then proposed that the blood vessel dilation and plasma extravasation noted during the migraine attack was an epiphenomenon of the migraine and not the cause of the migraine, as had been proposed by Graham and Wolff.¹⁷

The 5-HT_lB receptor was discovered in humans after the discovery of the 5-HT_1D receptor. It is expressed on human brain blood vessels where it induces contraction of dural blood vessels. An untoward observation of the effect of sumatriptan is the induced contraction of human coronary arteries, which also is most likely mediated by the 5-HT_1B receptors. Although the cardiac effect is not severe, it is a concern and has led to some more recent efforts to find a compound that would not have an agonistic effect on the coronary 5-HT_1B contractile receptors.

Centrally located 5-HT receptor targets in the trigeminal nucleus caudalis have also become the focus of research as potential targets for intervention in acute treatment of migraine. 5-HT_1B and 5-HT_1D receptors have been identified in the nucleus caudalis. On the basis of protein and mRNA localization studies, presynaptic 5-HT_1B/1D and postsynaptic 5-HT_1F receptors have been identified. These central 5-HT receptors may be potential sites of action for the new generation of brain-penetrant tripta/>