Chapter 13: 5-Hydroxytryptamine (Serotonin) and Dopamine

History. In the 1930s, Erspamer began to study the distribution of enterochromaffin cells, which stained with a reagent for indoles. The highest concentrations were found in GI mucosa, followed by platelets and the CNS. Soon thereafter, Page and colleagues isolated and chemically characterized a vasoconstrictor substance released from platelets in clotting blood. This substance, named serotonin, was shown to be identical to the indole isolated by Erspamer. Subsequent discovery of biosynthetic and degradative pathways for 5-HT and clinical presentation of patients with carcinoid tumors of intestinal enterochromaffin cells spurred interest in 5-HT. The gross effects of 5-HT, produced in excess in malignant carcinoid, gave some indication of the physiologic and pharmacologic actions of 5-HT, as did the identification of several naturally occurring and semi-synthetic tryptamine-like substances that were hallucinogenic and induced behavioral effects similar to those observed in carcinoid patients. In the mid-1950s, the discovery that the pronounced behavioral effects of reserpine are accompanied by a profound decrease in brain 5-HT led to the proposal that serotonin may function as a neurotransmitter in the mammalian CNS.

Sources and Chemistry. 5-HT [3-(β-aminoethyl)-5-hydroxyindole] is present in vertebrates, tunicates, mollusks, arthropods, coelenterates, fruits, and nuts. It is also a component of venoms, including those of the common stinging nettle and of wasps and scorpions. Numerous synthetic or naturally occurring congeners of 5-HT have pharmacological activity (see Figure 13–1 for chemical structures). Many of the N- and O-methylated indoleamines, such as N,N-dimethyltryptamine, are hallucinogens. Because these compounds are behaviorally active and might be synthesized by known metabolic pathways, they have long been considered candidates for endogenous psychotomimetic substances, potentially responsible for some psychotic behaviors. Another close relative of 5-HT, melatonin (5-methoxy-N-acetyltryptamine), is formed by sequential N-acetylation and O-methylation (Figure 13–2). Melatonin, not to be confused with melanin, is the principal indoleamine in the pineal gland, where it may be said to constitute a pigment of the imagination. Its synthesis is controlled by external factors including environmental light. Melatonin induces pigment lightening in skin cells and suppresses ovarian functions; it also serves a role in regulating biological rhythms and shows promise in the treatment of jet lag and other sleep disturbances (Cajochen et al., 2003).

The enzyme that converts L-5-hydroxytryptophan to 5-HT, aromatic L-amino acid decarboxylase (AADC), is widely distributed and has a broad substrate specificity. A long-standing debate about whether L-5-hydroxytryptophan decarboxylase and l-DOPA decarboxylase are identical enzymes was clarified when cDNA cloning confirmed that a single gene product decarboxylates both amino acids. 5-Hydroxytryptophan is not detected in the brain because it is rapidly decarboxylated. The synthesized product, 5-HT, is accumulated in secretory granules by a vesicular monoamine transporter (VMAT2); vesicular 5-HT is released by exocytosis from serotonergic neurons. In the nervous system, the action of released 5-HT is terminated via neuronal uptake by a specific 5-HT transporter. The 5-HT transporter (SERT [SLC6A4]) is localized in the membrane of serotonergic axon terminals (where it terminates the action of 5-HT in the synapse) and in the membrane of platelets (where it takes up 5-HT from the blood). This uptake system is the means by which platelets acquire 5-HT, since they lack the enzymes required for 5-HT synthesis. The 5-HT transporter and other monoamine transporters have been cloned (Chapters 5, 8, 12). The amine transporters are distinct from VMAT2, which concentrates amines in intracellular storage vesicles and is a nonspecific amine carrier, whereas the 5-HT transporter is specific. The 5-HT transporter is regulated by phosphorylation and subsequent internalization (for a review, see Steiner et al., 2008), providing a mechanism for dynamic regulation of serotonergic transmission.

Since 5-HIAA formation accounts for nearly 100% of the metabolism of 5-HT in brain, the turnover rate of brain 5-HT is estimated by measuring the ratio of 5-HIAA/5-HT. 5-HIAA from brain and peripheral sites of 5-HT storage and metabolism is excreted in the urine along with small amounts of 5-hydroxytryptophol sulfate or glucuronide conjugates. The usual range of urinary excretion of 5-HIAA by a normal adult is 2-10 mg daily. Larger amounts are excreted by patients with malignant carcinoid, providing a reliable diagnostic test for the disease. Ingestion of ethanol results in elevated amounts of NADH₂ (Chapter 23), which diverts 5-hydroxyindole acetaldehyde from the oxidative route to the reductive pathway (Figure 13–2) and tends to increase the excretion of 5-hydroxytryptophol and correspondingly reduce the excretion of 5-HIAA.

Of the two isoforms of MAO (A and B) (see Chapter 8 and Bortolato et al., 2008 for a review), MAO-A preferentially metabolizes 5-HT and norepinephrine; clorgyline is a specific inhibitor of this enzyme. MAO-B prefers β-phenylethylamine and benzylamine as substrates; low-dose selegiline is a relatively selective inhibitor of MAO-B. Dopamine and tryptamine are metabolized equally well by both isoforms. Neurons contain both isoforms of MAO, localized primarily in the outer membrane of mitochondria. MAO-B is the principal isoform in platelets, which contain large amounts of 5-HT.

Other minor pathways of metabolism of 5-HT, such as sulfation and O- or N-methylation, have been suggested. The latter reaction could lead to formation of an endogenous psychotropic substance, 5-hydroxy-N,N-dimethyltryptamine (bufotenine; Figure 13–1). However, other methylated indoleamines such as N,N-dimethyltryptamine and 5-methoxy-N,N-dimethyltryptamine are far more active hallucinogens and are more likely candidates to be endogenous psychotomimetics.

History of 5-HT Receptor Subtypes. In 1957, Gaddum and Picarelli proposed the existence of two 5-HT receptor subtypes, M and D receptors. M receptors were believed to be located on parasympathetic nerve endings, controlling the release of acetylcholine, whereas D receptors were thought to be located on smooth muscle. Subsequent studies in both the periphery and brain were consistent with the notion of multiple subtypes of 5-HT receptors (for a review, see Sanders-Bush and Airey, 2008), now known to be members of the superfamilies of GPCRs and ligand-gated ion channel receptors. The current classification scheme (Hoyer et al., 1994) is based on pharmacological properties, second-messenger function, and deduced amino acid sequence, and includes seven subfamilies of 5-HT receptors (Table 13–1).

The radioligand-binding studies of Peroutka and Snyder (1979) provided the first definitive evidence for two distinct recognition sites for 5-HT. 5-HT₁ receptors had a high affinity for [³H]5-HT, while 5-HT₂ receptors had a low affinity for [³H]5-HT and a high affinity for [³H]spiperone. Subsequently, high affinity for 5-HT was used as a primary criterion for classifying a receptor subtype as a member of the 5-HT₁ receptor family. This classification strategy proved to be invalid; for example, a receptor expressed in the choroid plexus was named the 5-HT_1C receptor because it was the third receptor shown to have a high affinity for 5-HT. However, based on its pharmacological properties, second-messenger function, and deduced amino acid sequence, the 5-HT_1C receptor clearly belonged to the 5-HT₂ receptor family and was subsequently renamed the 5-HT_2C receptor. Evidence suggests that the 5-HT_1Dβ receptor is the human homolog of the 5-HT_1B receptor originally characterized and subsequently cloned from rodent brain. Although the rat 5-HT_1B receptor and the human 5-HT_1D receptor show > 95% amino-acid sequence homology, they have distinct pharmacological properties. The rat 5-HT_1B receptor has an affinity for β adrenergic antagonists (e.g., pindolol and propranolol) that is two to three orders of magnitude higher than that of the human 5-HT_1D receptor. This difference appears to be due to a single amino acid difference (D→R) in the seventh transmembrane span. Many 5-HT receptor knockout mice have been created that shed light on the role of specific receptors (Table 13–2); however, most of these models cannot distinguish between developmental events versus changes related to the absence of the receptor in the adult mouse.

Additional Cloned 5-HT Receptors. Two other cloned receptors, 5-HT₆ and 5-HT₇, are linked to activation of adenylyl cyclase. Multiple splice variants of the 5-HT₇ receptor have been found, although functional distinctions are not clear. The absence of selective agonists and antagonists has foiled definitive studies of the role of the 5-HT₆ and 5-HT₇ receptors. Circumstantial evidence suggests that 5-HT₇ receptors play a role in the relaxation of smooth muscle in the GI tract and the vasculature. The atypical antipsychotic drug clozapine has a high affinity for 5-HT₆ and 5-HT₇ receptors; whether this property is related to the broader effectiveness of clozapine compared to conventional antipsychotic drugs is not known (Chapter 16). Two subtypes of the 5-HT₅ receptor have been cloned; although the 5-HT_5A receptor has been shown to inhibit adenylyl cyclase, functional coupling of the cloned 5-HT_5B receptor has not yet been described.

Platelets. Platelets differ from other formed elements of blood in expressing mechanisms for uptake, storage, and endocytotic release of 5-HT. 5-HT is not synthesized in platelets, but is taken up from the circulation and stored in secretory granules by active transport, similar to the uptake and storage of serotonin by serotonergic nerve terminals. Thus, Na⁺-dependent transport across the surface membrane of platelets, via the 5-HT transporter, is followed by VMAT2-mediated uptake into dense core granules creating a gradient of 5-HT as high as 1000:1 with an internal concentration of 0.6 M in the dense core storage vesicles. Measuring the rate of Na⁺-dependent 5-HT uptake by platelets provides a sensitive assay for 5-HT uptake inhibitors.

Main functions of platelets include adhesion, aggregation, and thrombus formation to plug holes in the endothelium; conversely, the functional integrity of the endothelium is critical for platelet action. A complex local interplay of multiple factors, including 5-HT, regulates thrombosis and hemostasis (Chapters 30 and 33). When platelets make contact with injured endothelium, they release substances that promote platelet aggregation, and secondarily, they release 5-HT (Figure 13–4). 5-HT binds to platelet 5-HT_2A receptors and elicits a weak aggregation response that is markedly augmented by the presence of collagen. If the damaged blood vessel is injured to a depth where vascular smooth muscle is exposed, 5-HT exerts a direct vasoconstrictor effect, thereby contributing to hemostasis, which is enhanced by locally released autocoids (thromboxane A₂, kinins, and vasoactive peptides). Conversely, 5-HT may interact with endothelial cells to stimulate production of NO and antagonize its own vasoconstrictor action, as well as the vasoconstriction produced by other locally released agents.

Cardiovascular System. The classical response of blood vessels to 5-HT is contraction, particularly in the splanchnic, renal, pulmonary, and cerebral vasculatures. 5-HT also induces a variety of responses by the heart that are the result of activation of multiple 5-HT receptor subtypes, stimulation or inhibition of autonomic nerve activity, or dominance of reflex responses to 5-HT (Kaumann and Levy, 2006). Thus, 5-HT has positive inotropic and chronotropic actions on the heart that may be blunted by simultaneous stimulation of afferent nerves from baroreceptors and chemoreceptors. Activation of 5-HT₃ receptors on vagus nerve endings elicits the Bezold-Jarisch reflex, causing extreme bradycardia and hypotension. The local response of arterial blood vessels to 5-HT also may be inhibitory, the result of the stimulation of endothelial NO production and prostaglandin synthesis and blockade of norepinephrine release from sympathetic nerves. On the other hand, 5-HT amplifies the local constrictor actions of norepinephrine, angiotensin II, and histamine, which reinforce the hemostatic response to 5-HT. Presumably, these constrictor effects result, at least in part, from direct action of 5-HT on vascular smooth muscle. In response to IV injection of 5-HT, these disparate effects on blood pressure may be temporally distinct.

GI Tract. Enterochromaffin cells in the gastric mucosa are the site of the synthesis and most of the storage of 5-HT in the body and are the source of circulating 5-HT. These cells synthesize 5-HT from tryptophan, by means of TPH1, and store 5-HT and other autacoids, such as the vasodilator peptide substance P and other kinins. Basal release of enteric 5-HT is augmented by mechanical stretching, such as that caused by food, and by efferent vagal stimulation. Released 5-HT enters the portal vein and is subsequently metabolized by MAO-A in the liver. 5-HT that survives hepatic oxidation may be captured by platelets or rapidly removed by the endothelium of lung capillaries and inactivated. 5-HT released from enterochromaffin cells also acts locally to regulate GI function (Gershon and Tack, 2007). Motility of gastric and intestinal smooth muscle may be either enhanced or inhibited by at least six subtypes of 5-HT receptors (Table 13–3). The stimulatory response occurs at nerve endings on longitudinal and circular enteric muscle (5-HT₄ receptors), at postsynaptic cells of the enteric ganglia (5-HT₃ and 5-HT_1P receptors), and by direct effects of 5-HT on the smooth-muscle cells (5-HT_2A receptors in intestine, 5-HT_2B receptors in stomach fundus). In esophagus, 5-HT acting at 5-HT₄ receptors causes either relaxation or contraction, depending on the species. Abundant 5-HT₃ receptors on vagal and other afferent neurons and on enterochromaffin cells play a pivotal role in emesis (Chapter 46). Serotonergic nerve terminals have been described in the myenteric plexus. Enteric 5-HT triggers peristaltic contraction when released in response to acetylcholine, sympathetic nerve stimulation, increases in intraluminal pressure, and lowered pH.

CNS. A multitude of brain functions are influenced by 5-HT, including sleep, cognition, sensory perception, motor activity, temperature regulation, nociception, mood, appetite, sexual behavior, and hormone secretion. All of the cloned 5-HT receptors are expressed in the brain, often in overlapping domains. Although patterns of 5-HT receptor expression in individual neurons have not been extensively defined, it is likely that multiple 5-HT receptor subtypes with similar or opposing actions are expressed in individual neurons (Bonsi et al., 2007), leading to a tremendous diversity of actions.

The principal cell bodies of 5-HT neurons are located in raphe nuclei of the brainstem and project throughout the brain and spinal cord (Chapter 14). In addition to being released at discrete synapses, release of serotonin also seems to occur at sites of axonal swelling, termed varicosities, which do not form distinct synaptic contacts. 5-HT released at nonsynaptic varicosities is thought to diffuse to outlying targets, rather than acting on discrete synaptic targets. Such non-synaptic release with ensuing widespread effects is consistent with the idea that 5-HT acts as a neuromodulator as well as a neurotransmitter (Chapter 14).

Serotonergic nerve terminals contain the proteins needed to synthesize 5-HT from L-tryptophan (Figure 13–2). Newly formed 5-HT is rapidly accumulated in synaptic vesicles (through VMAT2), where it is protected from MAO. 5-HT released by nerve-impulse flow is reaccumulated into the pre-synaptic terminal by the 5-HT transporter, SERT (SLC6A4; Chapter 5). Pre-synaptic reuptake is a highly efficient mechanism for terminating the action of 5-HT released by nerve-impulse flow. MAO localized in postsynaptic elements and surrounding cells rapidly inactivates 5-HT that escapes neuronal reuptake and storage.

Electrophysiology. The physiological consequences of 5-HT release vary with the brain area and the neuronal element involved, as well as with the population of 5-HT receptor subtype(s) expressed (Bockaert et al., 2006). 5-HT has direct excitatory and inhibitory actions (Table 13–4), which may occur in the same preparation, but with distinct temporal patterns. For example, in hippocampal neurons, 5-HT elicits hyperpolarization mediated by 5-HT_1A receptors followed by a slow depolarization mediated by 5-HT₄ receptors.

5-HT_1A receptor–induced membrane hyperpolarization and reduction in input resistance results from an increase in K⁺ conductance. These ionic effects, which are blocked by pertussis toxin, are independent of cAMP, suggesting that 5-HT_1A receptors couple, by means of G_βγ subunits, to receptor-operated K⁺ channels (Andrade et al., 1986). Somatodendritic 5-HT_1A receptors on raphe cells also elicit a K⁺-dependent hyperpolarization. The G protein involved is pertussis toxin–sensitive, but the K⁺ current apparently is different from the current elicited at postsynaptic 5-HT_1A receptors in the hippocampus. The precise signaling mechanism involved in inhibition of neurotransmitter release by the 5-HT_1B/1D autoreceptor at synaptic terminals is not known, although inhibition of voltage-gated Ca⁺⁺ channels likely contributes.

Slow depolarization induced by 5-HT_2A receptor activation in areas such as the prefrontal cortex and nucleus accumbens involves a decrease in K⁺ conductance. A second, distinct mechanism involving Ca²⁺-activated membrane currents enhances neuronal excitability and potentiates the response to excitatory signals such as glutamate. The role of intracellular signaling cascades in these physiological actions of 5-HT_2A receptors has not been clearly defined. In areas where 5-HT₁ and 5-HT_2A receptors co-exist, the effect of 5-HT may reflect a combination of the two opposing responses: a prominent 5-HT₁ receptor–mediated hyperpolarization and an opposing 5-HT_2A receptor–mediated depolarization. When 5-HT_2A receptors are blocked, hyperpolarization is enhanced. In many cortical areas, 5-HT_2A receptors are localized on both GABAergic interneurons and pyramidal cells. Activation of interneurons enhances GABA (γ-aminobutyric acid) release, which secondarily slows the firing rate of pyramidal cells. Thus, there is the potential for the 5-HT_2A receptor to differentially regulate cortical pyramidal cells, depending on the specific target cells (interneurons versus pyramidal cells). Activation of 5-HT_2C receptors has been shown to depress a K⁺ current, an effect that may contribute to the excitatory response. The 5-HT₄ receptor, which is coupled to activation of adenylyl cyclase, also elicits a slow neuronal depolarization mediated by a decrease in K⁺ conductance. It is not clear why two distinct 5-HT receptor families linked to different signaling pathways can elicit a common neurophysiological action. Yet another receptor, the 5-HT_1P receptor, elicits a slow depolarization. This receptor, which couples to activation of adenylyl cyclase, is restricted to the enteric nervous system (Gershon and Tack, 2007). The unique pharmacological profile of the 5-HT_1P receptor is most consistent with the 5-HT₇ receptor.

The fast depolarization elicited by 5-HT₃ receptors reflects direct gating of an ion channel intrinsic to the receptor structure itself. The 5-HT₃ receptor–induced inward current has the characteristics of a cation-selective, ligand-operated channel. Membrane depolarization is mediated by simultaneous increases in Na⁺ and K⁺ conductance, comparable to the nicotinic cholinergic receptor. 5-HT₃ receptors have been characterized in the CNS and in sympathetic ganglia, primary afferent parasympathetic and sympathetic nerves, and enteric neurons. The pharmacological properties of 5-HT₃ receptors, which are different from those of other 5-HT receptors, suggest that multiple 5-HT₃ receptor subtypes may exist and may correspond to different combinations of subunits (Jensen et al., 2008).

Behavior. The behavioral alterations elicited by drugs that interact with 5-HT receptors are extremely diverse. Many animal behavioral models for initial assessment of agonist and antagonist properties of drugs depend on aberrant motor or reflex responses, such as startle reflexes, hind-limb abduction, head twitches, and other stereotypical behaviors. Operant behavioral paradigms, such as drug discrimination, provide models of specific 5-HT receptor activation and are useful for exploring the action of CNS-active drugs, including agents that interact with 5-HT. For example, investigations of the mechanism of action of hallucinogenic drugs have relied heavily on drug discrimination (Winter, 2009). The following discussion focuses on animal models that may relate to pathological conditions in humans and will not attempt to cover the voluminous literature dealing with 5-HT and behavior. See King and associates (2008), Lesch and coworkers (2003), Lucki (1998), and Swerdlow and colleagues (2000) for excellent reviews on this topic.

Sleep-Wake Cycle. Control of the sleep-wake cycle is one of the first behaviors in which a role for 5-HT was identified. Following pioneering work in cats (Jouvet, 1999), many studies showed that depletion of 5-HT with p-chlorophenylalanine, a tryptophan hydroxylase inhibitor, elicits insomnia that is reversed by the 5-HT precursor, 5-hydroxytryptophan. Conversely, treatment with L-tryptophan or with nonselective 5-HT agonists accelerates sleep onset and prolongs total sleep time. 5-HT antagonists reportedly can increase and decrease slow-wave sleep, probably reflecting interacting or opposing roles for subtypes of 5-HT receptors. One relatively consistent finding in humans and in laboratory animals is an increase in slow-wave sleep following administration of a selective 5-HT_2A/2C-receptor antagonist such as ritanserin.

Aggression and Impulsivity. Studies in laboratory animals and in humans suggest that 5-HT serves a critical role in aggression and impulsivity. Many human studies reveal a correlation between low cerebrospinal fluid 5-HIAA and violent impulsivity and aggression. As with many effects of 5-HT, pharmacological studies of aggressive behavior in laboratory animals are not definitive, but suggest a role for 5-HT. Two genetic studies have reinforced and amplified this notion. Knockout mice lacking the 5-HT_1B receptor exhibited extreme aggression (Saudou et al., 1994), suggesting either a role for 5-HT_1B receptors in the development of neuronal pathways important in aggression or a direct role in the mediation of aggressive behavior. A human genetic study identified a point mutation in the gene encoding MAO-A, which was associated with extreme aggressiveness and mental retardation (Brunner et al., 1993), and this has been confirmed in knockout mice lacking MAO-A (Cases et al., 1995).

Anxiety and Depression. The effects of 5-HT–active drugs in anxiety and depressive disorders, like the effects of selective serotonin reuptake inhibitors (SSRIs), strongly suggest a role for 5-HT in the neurochemical mediation of these disorders. Mutant mice lacking the 5-HT transporter display anxiety and a "depressive-like" phenotype (Fox et al., 2007). 5-HT-related drugs with clinical effects in anxiety and depression have varied effects in classical animal models of these disorders, depending on the experimental paradigm, species, and strain. For example, the anxiolytic buspirone (Chapter 15), a partial agonist at 5-HT_1A receptors, does not reduce anxiety in classical approach-avoidance paradigms that were instrumental in development of anxiolytic benzodiazepines. However, buspirone and other 5-HT_1A receptor agonists are effective in other animal behavioral tests used to predict anxiolytic effects. Furthermore, studies in 5-HT_1A receptor knockout mice suggest a role for this receptor in anxiety, and possibly depression. Agonists of certain 5-HT receptors, including 5-HT_2A and 5-HT_2C receptors (e.g., m-chlorophenylpiperazine), have anxiogenic properties in laboratory animals and in human studies. Similarly, these receptors have been implicated in the animal models of depression, such as learned helplessness.

An impressive finding in humans with depression is the abrupt reversal of the antidepressant effects of drugs such as SSRIs by manipulations that rapidly reduce the amount of 5-HT in the brain. These approaches include administration of p-chlorophenylalanine or a tryptophan-free drink containing large quantities of neutral amino acids (Delgado et al., 1990). Curiously, this kind of 5-HT depletion has not been shown to worsen or to induce depression in nondepressed subjects, suggesting that the continued presence of 5-HT is required to maintain the effects of these drugs. This clinical finding adds credence to somewhat less convincing neurochemical findings that suggest a role for 5-HT in the pathogenesis of depression.

Experimental strategies for evaluating the role of 5-HT depend on techniques that manipulate tissue levels of 5-HT or block 5-HT receptors. Until recently, manipulation of the levels of endogenous 5-HT was the more commonly used strategy, because the actions of 5-HT receptor antagonists were poorly understood.

Tryptophan hydroxylase is the rate-limiting enzyme in 5-HT synthesis, and manipulation of its catalytic flux provides useful ways to alter 5-HT levels. A diet low in tryptophan reduces the concentration of brain 5-HT; conversely, ingestion of a tryptophan load increases levels of 5-HT in the brain. An acute decrease in brain 5-HT can be achieved by oral administration of an amino acid mixture devoid of tryptophan, a valuable tool for human studies of the role of 5-HT. In addition, administration of a tryptophan hydroxylase inhibitor causes a profound depletion of 5-HT. The most widely used selective tryptophan hydroxylase inhibitor is p-chlorophenylalanine, which acts irreversibly to produce long-lasting depletion of 5-HT levels with no change in levels of catecholamines.

p-Chloroamphetamine and other halogenated amphetamines promote the release of 5-HT from platelets and neurons. A rapid release of 5-HT is followed by a prolonged and selective depletion of 5-HT in brain. The mechanism of 5-HT release involves reversal of 5-HT transporter, analogous to the mechanism of the catecholamine-releasing action of amphetamine. The halogenated amphetamines are valuable experimental tools and two of them, fenfluramine and dexfenfluramine, were used clinically to reduce appetite; the once popular diet drug regimen, "fen-phen," combined fenfluramine and phentermine. Fenfluramine (pondimin) and dexfenfluramine (redux) were withdrawn from the U.S. market in the late 1990s after reports of life-threatening heart valve disease and pulmonary hypertension associated with their use. This toxicity may be secondary to 5-HT_2B receptor activation (Roth, 2007). The mechanism for long-term depletion of brain 5-HT by the halogenated amphetamines remains controversial. A profound reduction in levels of 5-HT in the brain lasts for weeks and is accompanied by an equivalent loss of proteins (5-HT transporter and tryptophan hydroxylase) selectively localized in 5-HT neurons, suggesting that the halogenated amphetamines have a neurotoxic action. Despite these long-lasting biochemical deficits, neuroanatomical signs of neuronal death are not readily apparent. Another class of compounds, ring-substituted tryptamine derivatives such as 5,7-dihydroxytryptamine (Figure 13–1) produces unequivocal degeneration of 5-HT neurons. In adult animals, 5,7-dihydroxytryptamine selectively destroys serotonergic axon terminals; the remaining intact cell bodies allow eventual regeneration of axon terminals. In newborn animals, degeneration is permanent because 5,7-dihydroxytryptamine destroys serotonergic cell bodies as well as axon terminals.

The pathogenesis of migraine headache is complex, involving both neural and vascular elements (Mehrotra et al., 2008). Evidence suggesting that 5-HT is a key mediator in the pathogenesis of migraine includes:

• plasma and platelet concentrations of 5-HT vary with the different phases of the migraine attack

• urinary concentrations of 5-HT and its metabolites are elevated during most migraine attacks

• migraine may be precipitated by agents (e.g., reserpine and fenfluramine) that release 5-HT from intracellular storage sites

Consistent with the 5-HT hypothesis, 5-HT receptor agonists have become a mainstay for acute treatment of migraine headaches. Treatments for the prevention of migraines, such as β adrenergic antagonists and newer anti-epileptic drugs, have mechanisms of action that are, presumably, unrelated to 5-HT (Mehrotra et al., 2008).

The Triptans. The triptans are effective, acute anti-migraine agents. Their ability to decrease, rather than exacerbate, the nausea and vomiting of migraine is an important advance in the treatment of the condition. The selective pharmacological effects of the triptans at 5-HT₁ receptors has provided insights into the pathophysiology of migraine. Available compounds include almotriptan (axert), eletriptan (relpax), frovatriptan (frova), naratriptan (amerge), rizatriptan (maxalt, others), sumatriptan (imitrex, others), and zolmitriptan (zomig). Sumatriptan for migraine headaches is also marketed in a fixed-dose combination with naproxen (treximet).

History. Sumatriptan emerged from the first experimentally based approach to identify and develop a novel therapy for migraine. In 1972, Humphrey and colleagues initiated a project aimed at identifying novel therapeutic agents in the treatment of migraine with the goal of developing selective vasoconstrictors of the extracranial circulation, based on the theories of the etiology of migraine prevalent in the early 1970s (Humphrey et al., 1990). Sumatriptan, first synthesized in 1984, became available for clinical use in the United States in 1992; since then, several other triptans have been FDA-approved for clinical use. The second generation triptans have higher oral bioavailabilty.

Chemistry. The triptans are indole derivatives. Representative structures are given in Figure 13–5.

Pharmacological Properties. In contrast to ergot alkaloids (described later), the pharmacological effects of the triptans appear to be limited to the 5-HT₁ family of receptors, providing evidence that this receptor subclass plays an important role in the acute relief of a migraine attack. The triptans interact potently with 5-HT_1B and 5-HT_1D receptors and have a low or no affinity for other subtypes of 5-HT receptors, as well as α₁ and α₂ adrenergic, β adrenergic, dopaminergic, muscarinic cholinergic, and benzodiazepine receptors. Clinically effective doses of the triptans correlate well with their affinities for both 5-HT_1B and 5-HT_1D receptors, supporting the hypothesis that 5-HT_1B and/or 5-HT_1D receptors are the most likely receptors involved in the mechanism of action of acute anti-migraine drugs.

Mechanism of Action. There remains a controversy about the relative importance of vascular versus neurological dysfunction in migraine; thus the mechanism of the efficacy of 5-HT_1B/1D agonists in migraine is not resolved. One hypothesis implicates the capacity of these receptors to cause constriction of intracranial blood vessels including arteriovenous anastomoses. According to a prominent pathophysiological model of migraine, unknown events lead to the abnormal dilation of carotid arteriovenous anastomoses in the head and shunting of carotid arterial blood flow, producing cerebral ischemia and hypoxia. Based on this model, an effective anti-migraine agent would close the shunts and restore blood flow to the brain. Indeed, ergotamine, dihydroergotamine, and sumatriptan share the capacity to produce this vascular effect with a pharmacological specificity that mirrors the effects of these agents on 5-HT_1B and 5-HT_1D receptor subtypes. An alternative hypothesis concerning the significance of one or more 5-HT₁ receptors in migraine pathophysiology relates to the observation that both 5-HT_1B and 5-HT_1D receptors serve as presynaptic autoreceptors, modulating neurotransmitter release from neuronal terminals (Figure 13–5). 5-HT₁ agonists may block the release of pro-inflammatory neuropeptides at the level of the nerve terminal in the perivascular space, which could account for their efficacy in the acute treatment of migraine.

Absorption, Fate, and Excretion. When given subcutaneously, sumatriptan reaches its peak plasma concentration in ~12 minutes. Following oral administration, peak plasma concentrations occur within 1-2 hours. Bioavailability following the subcutaneous route of administration is ~97%; after oral administration or nasal spray, bioavailability is only 14-17%. The elimination t_1/2 is ~1-2 hours. Sumatriptan is metabolized predominantly by MAO-A, and its metabolites are excreted in the urine.

Zolmitriptan reaches its peak plasma concentration 1.5-2 hours after oral administration. Its bioavailability is ~40% following oral ingestion. Zolmitriptan is converted to an active N-desmethyl metabolite, which has severalfold higher affinity for 5-HT_1B and 5-HT_1D receptors than does the parent drug. Both the metabolite and the parent drug have half-lives of 2-3 hours.

Naratriptan, administered orally, reaches its peak plasma concentration in 2-3 hours and has an absolute bioavailability of ~70%. It is the second longest acting of the triptans, having a t_1/2 of ~6 hours. Fifty percent of an administered dose of naratriptan is excreted unchanged in the urine, and ~30% is excreted as products of oxidation by CYPs.

Rizatriptan has an oral bioavailability of ~45% and reaches peak plasma levels within 1-1.5 hours after oral ingestion of tablets of the drug. An orally disintegrating dosage form has a somewhat slower rate of absorption, yielding peak plasma levels of the drug 1.6-2.5 hours after administration. The principal route of metabolism of rizatriptan is by oxidative deamination by MAO-A.

Plasma protein binding of the triptans ranges from ~14% (sumatriptan and rizatriptan) to 85% (eletriptan).

Adverse Effects and Contraindications. Rare but serious cardiac events have been associated with the administration of 5-HT₁ agonists, including coronary artery vasospasm, transient myocardial ischemia, atrial and ventricular arrhythmias, and myocardial infarction, predominantly in patients with risk factors for coronary artery disease. In general, however, only minor side effects are seen with the triptans in the acute treatment of migraine. After subcutaneous injection of sumatriptan, patients often experience irritation at the site of injection (transient mild pain, stinging, or burning sensations). The most common side effect of sumatriptan nasal spray is a bitter taste. Orally administered triptans can cause paresthesias; asthenia and fatigue; flushing; feelings of pressure, tightness, or pain in the chest, neck, and jaw; drowsiness; dizziness; nausea; and sweating.

The triptans are contraindicated in patients who have a history of ischemic or vasospastic coronary artery disease (including history of stroke or transient ischemic attacks), cerebrovascular or peripheral vascular disease, hemiplegic or basilar migraines, other significant cardiovascular diseases, or ischemic bowel diseases. Because triptans may cause an acute, usually small, increase in blood pressure, they also are contraindicated in patients with uncontrolled hypertension. Naratriptan is contraindicated in patients with severe renal or hepatic impairment. Rizatriptan should be used with caution in patients with renal or hepatic disease but is not contraindicated in such patients. Eletriptan is contraindicated in hepatic disease. Almotriptan, rizatriptan, sumatriptan, and zolmitriptan are contraindicated in patients who have taken a monoamine oxidase inhibitor within the preceding 2 weeks, and all triptans are contraindicated in patients with near-term prior exposure to ergot alkaloids or other 5-HT agonists.

Use in Treatment of Migraine. The triptans are effective in the acute treatment of migraine (with or without aura), but are not intended for use in prophylaxis of migraine. Treatment with triptans should begin as soon as possible after onset of a migraine attack. Oral dosage forms of the triptans are the most convenient to use, but they may not be practical in patients experiencing migraine-associated nausea and vomiting. Approximately 70% of individuals report significant headache relief from a 6-mg subcutaneous dose of sumatriptan. This dose may be repeated once within a 24-hour period if the first dose does not relieve the headache. An oral formulation and a nasal spray of sumatriptan also are available. The onset of action is as early as 15 minutes with the nasal spray. The recommended oral dose of sumatriptan is 25-100 mg, which may be repeated after 2 hours up to a total dose of 200 mg over a 24-hour period. When administered by nasal spray, from 5-20 mg of sumatriptan is recommended, repeatable after 2 hours up to a maximum dose of 40 mg over a 24-hour period. Zolmitriptan is given orally in a dose of 1.25-2.5 mg, which can be repeated after 2 hours, up to a maximum dose of 10 mg over 24 hours if the migraine attack persists. Naratriptan is given orally in a dose of 1-2.5 mg, which should not be repeated until 4 hours after the previous dose. The maximum dose over a 24-hour period should not exceed 5 mg. The recommended oral dose of rizatriptan is 5-10 mg. The dose can be repeated after 2 hours up to a maximum dose of 30 mg over a 24-hour period. The safety of treating more than 3 or 4 headaches over a 30-day period with triptans has not been established. No triptan should be used concurrently with (or within 24 hours of) an ergot derivative (described later) or another triptan.

History. Ergot is the product of a fungus (Claviceps purpurea) that grows on rye and other grains. The contamination of an edible grain by a poisonous, parasitic fungus spread death for centuries. As early as 600 B.C., an Assyrian tablet alluded to a "noxious pustule in the ear of grain." Written descriptions of ergot poisoning first appeared in the Middle Ages, describing strange epidemics in which the characteristic symptom was gangrene of the feet, legs, hands, and arms. In severe cases, extremities became dry and black, and mummified limbs separated off without loss of blood. Limbs were said to be consumed by the holy fire, blackened like charcoal with agonizing burning sensations, the disease was called holy fire or St. Anthony's fire in honor of the saint at whose shrine relief was said to be obtained. The relief that followed migration to the shrine of St. Anthony was probably real, for the sufferers received a diet free of contaminated grain during their sojourn at the shrine. The symptoms of ergot poisoning were not restricted to limbs. A frequent complication of it was a dramatic abortive effect of ergot ingested during pregnancy. The active principles of ergot were isolated and chemically identified in the early 20th century.

Chemistry. The ergot alkaloids can all be considered to be derivatives of the tetracyclic compound 6-methylergoline (Table 13–6). The naturally occurring alkaloids contain a substituent in the beta configurations at position 8 and a double bond in ring D. The natural alkaloids of therapeutic interest are amide derivatives of d-lysergic acid. The first pure ergot alkaloid, ergotamine, was obtained in 1920, followed by the isolation of ergonovine in 1932. Numerous semisynthetic derivatives of the ergot alkaloids have been prepared by catalytic hydrogenation of the natural alkaloids (e.g., dihydroergotamine). Another synthetic derivative, bromocriptine (2-bromo-α-ergocryptine), is used to control the secretion of prolactin, a property derived from its DA agonist effect. Other products of this series include lysergic acid diethylamide (LSD), a potent hallucinogen, and methysergide, a serotonin antagonist.

Absorption, Fate, and Excretion. The oral administration of ergotamine by itself generally results in low or undetectable systemic drug concentrations, because of extensive first-pass metabolism. The bioavailability after administration of rectal suppositories is greater. Ergotamine is metabolized in the liver by largely undefined pathways, and 90% of the metabolites are excreted in the bile. Despite a plasma t_1/2 of ~2 hours, ergotamine produces vasoconstriction that lasts for 24 hours or longer. Dihydroergotamine is eliminated more rapidly than ergotamine, presumably due to its rapid hepatic clearance.

Ergonovine and methylergonovine are rapidly absorbed after oral administration and reach peak concentrations in plasma within 60-90 minutes that are more than 10-fold those achieved with an equivalent dose of ergotamine. Although often given IV, an uterotonic effect in postpartum women can be observed within 10 minutes after oral administration of 0.2 mg of ergonovine. The t_1/2 of methylergonovine in plasma ranges between 0.5 and 2 hours.

Use in the Treatment of Migraine. The multiple pharmacological effects of ergot alkaloids have complicated the determination of their precise mechanism of action in the acute treatment of migraine. Based on the mechanism of action of sumatriptan and other5-HT_1B/1D receptor agonists, the actions of ergot alkaloids at 5-HT_1B/1D receptors likely mediate their acute anti-migraine effects.

The use of ergot alkaloids for migraine should be restricted to patients having frequent, moderate migraine or infrequent, severe migraine attacks. As with other medications used to abort an attack, ergot preparations should be administered as soon as possible after the onset of a headache. GI absorption of ergot alkaloids is erratic, perhaps contributing to the large variation in patient response to these drugs. Available preparations currently in the United States include sublingual tablets of ergotamine tartrate (ergomar) and a nasal spray and solution for injection of dihydroergotamine mesylate (migranal, D.H.E. 45, respectively). Ergotamine in fixed-dose combinations with caffeine is also marketed under the brand names of cafergot (oral tablets) and migergot (rectal suppositories) and as generic formulations (oral tablets). The recommended dose for ergotamine tartrate is 2 mg sublingually, which can be repeated at 30-minute intervals if necessary up to a total dose of 6 mg in a 24-hour period or 10 mg a week. Dihydroergotamine mesylate injections can be given intravenously, subcutaneously, or intramuscularly. The recommended dose is 1 mg, which can be repeated after 1 hour if necessary up to a total dose of 2 mg (intravenously) or 3 mg (subcutaneously or intramuscularly) in a 24-hour period or 6 mg in a week. The dose of dihydroergotamine mesylate administered as a nasal spray is 0.5 mg (one spray) in each nostril, repeated after 15 minutes for a total dose of 2 mg (4 sprays). The safety of > 3 mg over 24 hours or 4 mg over 7 days has not been established.

Adverse Effects and Contraindications of Ergot Alkaloids. Nausea and vomiting, due to a direct effect on CNS emetic centers, occur in ~10% of patients after oral administration of ergotamine, and in about twice that number after parenteral administration. This side effect is problematic, since nausea and sometimes vomiting are part of the symptomatology of a migraine headache. Leg weakness is common, and muscle pains that occasionally are severe may occur in the extremities. Numbness and tingling of fingers and toes are other reminders of the ergotism that this alkaloid may cause. Precordial distress and pain suggestive of angina pectoris, as well as transient tachycardia or bradycardia, also have been noted, presumably as a result of coronary vasospasm induced by ergotamine. Localized edema and itching may occur in an occasional hypersensitive patient, but usually do not necessitate interruption of ergotamine therapy. In the event of acute or chronic poisoning (ergotism), treatment consists of complete withdrawal of the offending drug and symptomatic measures to maintain adequate circulation by agents such as anticoagulants, low-molecular-weight dextran, and potent vasodilator drugs such as intravenous sodium nitroprusside. Dihydroergotamine has lower potency than does ergotamine as an emetic, vasoconstrictor, and oxytocic.

Ergot alkaloids are contraindicated in women who are, or may become, pregnant because the drugs may cause fetal distress and miscarriage. Ergot alkaloids also are contraindicated in patients with peripheral vascular disease, coronary artery disease, hypertension, impaired hepatic or renal function, and sepsis. Ergot alkaloids should not be taken within 24 hours of the use of the triptans, and should not be used concurrently with other drugs that can cause vasoconstriction.

Use of Ergot Alkaloids in Postpartum Hemorrhage. All of the natural ergot alkaloids markedly increase the motor activity of the uterus. After small doses, uterine contractions are increased in force or frequency, or both, but are followed by a normal degree of relaxation. As the dose is increased, contractions become more forceful and prolonged, resting tone is dramatically increased, and sustained contracture can result. Although this characteristic precludes their use for induction or facilitation of labor, it is quite compatible with their use postpartum or after abortion to control bleeding and maintain uterine contraction. The gravid uterus is very sensitive, and small doses of ergot alkaloids can be given immediately postpartum to obtain a marked uterine response, usually without significant side effects. In current obstetric practice, ergot alkaloids are used primarily to prevent postpartum hemorrhage. Although all natural ergot alkaloids have qualitatively the same effect on the uterus, ergonovine is the most active and also is less toxic than ergotamine. For these reasons ergonovine (ergotrate) and its semisynthetic derivative methylergonovine (methergine, others) have replaced other ergot preparations as uterine-stimulating agents in obstetrics.

Methysergide. Methysergide (sansert; 1-methyl-d-lysergic acid butanolamide) is a congener of methylergonovine (Table 13–6). Reflecting the fickle nature of the ergot compounds, methysergide is neither a selective antagonist nor always an antagonist. It interacts with 5-HT₁ receptors, but its therapeutic effects appear primarily to reflect blockade of 5-HT₂ receptors; it blocks 5-HT_2A and 5-HT_2C receptors, but has partial agonist activity in some preparations. Although methysergide is an ergot derivative, it has only weak vasoconstrictor and oxytocic activity.

Methysergide is without benefit when given during an acute migraine attack but has been used for the prophylactic treatment of migraine and other vascular headaches, including Horton's syndrome. A potentially serious complication of prolonged treatment is inflammatory fibrosis, giving rise to various syndromes that include retroperitoneal fibrosis, pleuropulmonary fibrosis, and coronary and endocardial fibrosis. Usually the fibrosis regresses after drug withdrawal, although persistent cardiac valvular damage has been reported. Because of this danger, other drugs are preferred for the prophylactic treatment of migraine (see the earlier discussion of migraine therapy). If methysergide is used chronically, treatment should be interrupted for 3 weeks or more every 6 months. Methysergide is not available in the U.S.

D-Lysergic Acid Diethylamide (LSD). Of the many drugs that are nonselective 5-HT agonists, LSD (see structure in Table 13–6) is the most remarkable. This ergot derivative profoundly alters human behavior, eliciting perception disturbances such as sensory distortion (especially visual) and hallucinations at doses as low as 1 μg/kg. The potent, mind-altering effects of LSD explain its abuse by humans (Chapter 24), as well as the fascination of scientists with the mechanism of action of LSD.

LSD was synthesized in 1943 by Albert Hoffman, who discovered its unique properties by accidental ingestion of the drug. The chemical precursor, lysergic acid, occurs naturally in a fungus that grows on wheat and rye but is devoid of hallucinogenic actions. LSD contains an indolealkylamine moiety embedded within its structure, and early investigators postulated that it would interact with 5-HT receptors. Indeed, LSD interacts with brain 5-HT receptors as an agonist/partial agonist. LSD mimics 5-HT at 5-HT_1A autoreceptors on raphe cell bodies, producing a marked slowing of the firing rate of serotonergic neurons. In the raphe, LSD and 5-HT are equi-effective; however, in areas of serotonergic axonal projections (such as visual relay centers), LSD is far less effective than is 5-HT. In an animal behavioral model thought to reflect the subjective effects of abused drugs, the discriminative stimulus effects of LSD and other hallucinogenic drugs appear to be mediated by activation of 5-HT_2A receptors (Winter, 2009). Recent studies of 5-HT_2A receptor knockout mice confirm the critical role for this receptor in LSD hallucinogenic effects (González-Maeso et al., 2007). LSD also interacts potently with many other 5-HT receptors, including cloned receptors whose functions have not yet been determined. On the other hand, the hallucinogenic phenethylamine derivatives such as 1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane are selective 5-HT_2A/2C receptor agonists. Current theories of the mechanism of action of LSD and other hallucinogens focus on 5-HT_2A receptor-mediated disruption of thalamic gating with sensory overload of the cortex (Geyer and Vollenweider, 2008). Progress in understanding the unusual properties of hallucinogens are arising from clinical investigations. For example, positron emission tomography imaging studies revealed that administration of the hallucinogen psilocybin (the active component of "shrooms") mimics the pattern of brain activation found in schizophrenic patients experiencing hallucinations. Consistent with animal studies, this action of psilocybin is blocked by pretreatment with a 5-HT_2A/2C antagonist.

8-Hydroxy-(2-N,N-Dipropylamino)-Tetraline (8-OH-DPAT). This prototypic 5-HT_1A-selective receptor agonist is a valuable experimental tool.

8-OH-DPAT is considered to be a 5-HT_1A selective agonist. It does not interact with other members of the 5-HT₁-receptor subfamily or with 5-HT₂, 5-HT₃, or 5-HT₄ receptors, although activation of the 5-HT₇ receptor has been reported. 8-OHDPAT reduces the firing rate of raphe cells by activating 5-HT_1A autoreceptors and inhibits neuronal firing in terminal fields (e.g., hippocampus) by direct interaction with postsynaptic 5-HT_1A receptors.

Buspirone (BUSPAR, others). A series of long-chain arylpiperazines, such as buspirone, gepirone, and ipsapirone, are selective partial agonists at 5-HT_1A receptors. Other closely related arylpiperazines act as 5-HT_1A-receptor antagonists. Buspirone, the first clinically available drug in this series, has been effective in the treatment of anxiety (Chapter 15). Buspirone mimics the antianxiety properties of benzodiazepines but does not interact with GABA_A receptors and or display the sedative and anticonvulsant properties of benzodiazpeines. The absence of sedative effects may explain why patients prefer the benzodiazepines to relieve anxiety.

m-Chlorophenylpiperazine (mCPP). The actions of mCPP in vivo primarily reflect activation of 5-HT_1B and/or 5-HT_2A/2C receptors, although this agent is not subtype selective in radioligand-binding studies in vitro.

mCPP, an active metabolite of the antidepressant drug trazodone, has been employed to probe brain 5-HT function in humans, where it alters a number of neuroendocrine parameters and elicits profound behavioral effects, with anxiety as a prominent symptom. Animal studies suggest a greater involvement of the 5-HT_2C receptor in the anxiogenic actions of mCPP. mCPP elevates cortisol and prolactin secretion, probably through a combination of 5-HT₁ and 5-HT_2A/2C receptor activation. It also increases growth hormone secretion, apparently by a 5-HT independent mechanism.

Laboratory studies have documented agonist-promoted receptor subsensitivity and down-regulation of 5-HT receptor subtypes, a compensatory response common to many neurotransmitter systems. However, an unusual adaptive process, antagonist-induced down-regulation of 5-HT_2A/5-HT_2C receptors, takes place in rats and mice after chronic treatment with receptor antagonists (Gray and Roth, 2001). The mechanism of this paradoxical regulation of 5-HT_2A/2C receptors has generated considerable interest, since many clinically effective drugs, including clozapine, ketanserin (not licensed for use in the U.S.), and amitriptyline, exhibit this unusual property. These drugs, as well as several other 5-HT_2A/2C-receptor antagonists, possess negative intrinsic activity, reducing constitutive (spontaneous) receptor activity as well as blocking agonist occupancy. This property of negative intrinsic activity or inverse agonism is frequently observed when constitutive (agonist-independent) activity can be measured; in the absence of constitutive activity, inverse agonists behave as competitive antagonists. Another group of 5-HT_2A/2C receptor antagonists acts in the classical manner, simply blocking receptor occupancy by agonists. Recent evidence for constitutive activity of the 5-HT_2C receptor in vivo (Aloyo et al., 2009) justifies refinement of drug development to target pre-existing constitutive neuronal activity as opposed to blockade of excess neurotransmitter action.

Chemical relatives of ketanserin such as ritanserin are more selective 5-HT_2A receptor antagonists with low affinity for α₁ adrenergic receptors. However, ritanserin, as well as most other 5-HT_2A receptor antagonists, also potently antagonize 5-HT_2C receptors. The physiological significance of 5-HT_2C-receptor blockade is unknown. M100,907 is the prototype of a new series of potent 5-HT_2A receptor antagonists, with high selectivity for 5-HT_2A versus 5-HT_2C receptors. Clinical trials of M100,907 in the treatment of schizophrenia have been inconclusive.

History. Dopamine (DA) was first synthesized in 1910. Later that year, Henry Dale characterized the biological properties of DA in the periphery, and described it as a weak, adrenaline-like substance. In the 1930s, DA was recognized as a transitional compound in the synthesis of NE and epinephrine, but was believed to be little more than a biosynthetic intermediate. Not until the early 1950s were stores of DA were found in tissues, suggesting DA had a signaling function of its own. By decade's end, Carlsson and Montagu had each identified DA stores in the brain. Soon thereafter, Hornykiewicz discovered the DA deficit in the parkinsonian brain, fueling interest in the role of DA in neurological diseases and disorders (see Hornykiewicz, 2002).

Sources and Chemistry. DA consists of a catechol moiety linked to an ethyl amine, leading to its classification as a catecholamine. DA is closely related to melanin, a pigment that is formed by oxidation of DA, tyrosine, or l-DOPA. Melanin exists in the skin and cuticle and gives the substantia nigra its namesake dark color. Both DA and l-DOPA are readily oxidized by non-enzymatic pathways to form cytotoxic reactive oxygen species and quinones. DA- and DOPA-quinones form adducts with α-synuclein, a major constituent of Lewy bodies in Parkinson disease (Chapter 22). DA is a polar molecule that does not readily cross the blood-brain barrier.

The neurochemical events that underlie DA neurotransmission are summarized in Figure 13–7. In dopaminergic neurons, synthesized DA is packaged into secretory vesicles (or into granules within adrenal chromaffin cells) by the vesicular monoamine transporter, VMAT2. This packaging allows DA to be stored in readily releasable aliquots and protects the transmitter from further anabolism or catabolism. By contrast, in adrenergic or noradrenergic cells, the DA is not packaged; instead, it is converted to NE by DA β-hydroxylase and, in adrenergic cells, further altered to epinephrine in cells expressing phenylethanolamine N-methyltransferase (Chapter 8). Synaptically released DA is subject to both transporter clearance and metabolism. The DA transporter (DAT) is not selective for DA; moreover, DA can also be cleared from the synapse by the NE transporter, NET. Reuptake of DA by the DA transporter is the primary mechanism for termination of DA action, and allows for either vesicular repackaging of transmitter or metabolism. The DA transporter is regulated by phosphorylation, offering the potential for DA to regulate its own uptake.

The DA transporter is predominantly localized perisynaptically so that DA is cleared at a distance from its release site, suggesting that high concentrations of DA are released into the synapse, thus necessitating a spatially distant transporter. In addition to clearing synaptic neurotransmitter, the DA transporter is a site of action for cocaine and methamphetamine, which have distinct mechanisms to increase extracellular DA. The DA transporter is also the molecular target for some neurotoxins, including 6-hydroxydopamine and 1-methyl-4-phenylpyridium (MPP+), the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Following uptake into dopaminergic neurons, MPP+ and 6-hydroxydopamine elicit intra- and extracellular DA release, ultimately resulting in neuronal death. This selective dopaminergic degeneration mimics Parkinson disease, and serves as an animal model for this disorder.

DOPAC can be further metabolized by catechol-O-methyltransferase (COMT) to form homovanillic acid (HVA). Both DOPAC and HVA, as well as DA, are excreted in the urine. Levels of DOPAC and HVA are reliable indicators of DA turnover; ratios of these metabolites to DA in cerebral spinal fluid serve as accurate representations of brain dopaminergic activity. In addition to metabolizing DOPAC, COMT also utilizes DA as a substrate to generate 3-methoxytyramine, which is subsequently converted to HVA by MAO. In humans, HVA is the principal metabolite of DA. COMT in the periphery also metabolizes l-DOPA to 3-O-methyldopa, which then competes with l-DOPA for uptake into the CNS. Consequently, l-DOPA given in the treatment of Parkinson disease must be co-administered with peripheral COMT inhibitors to preserve l-DOPA and allow sufficient entry into the CNS (Chapter 22).

History of DA Receptor Subtypes. In 1971, Kebabian and Greengard determined that DA caused an increase in neostriatal cyclic AMP production, presumably by activating a DA-sensitive adenylyl cyclase enzyme. Subsequently, the DA-induced increase in cyclic AMP was attributed to a D1 receptor while a second category of DA receptors that did not raise cyclic AMP levels was termed the D2 receptor. Differences in pharmacological responsiveness gave rise to the hypothesis that there were multiple subtypes of DA receptors beyond the D1 and D2. This idea proved to be correct. The D1 subfamily consists of the D₁ and D₅ receptor subtypes (initially classified as D_1A and D_1B); both are GPCRs that couple to G_s to stimulate cellular cyclic AMP production couple, but they differ in their pharmacological profiles and primary amino acid sequences. The D2 subfamily contains the D₂, D₃, and D₄ receptors. All reduce intracellular cyclic AMP production by coupling to G_i/o proteins, but they diverge in amino acid sequence and pharmacology. The D1 and D2 families can be further differentiated by their anatomical distribution and by structural differences—the D1-like receptors have a relatively long carboxyl terminus and a short third intracellular loop.

Reports of D₁ receptors coupling to G_q raised the possibility that a third D1-like receptor might exist; there also are D₁-D₂ receptor heterodimers that couple to the G_q signaling cascade (Lee et al., 2004). The presence of a functionally distinct heterodimer with a unique dopaminergic pharmacology is a novel concept that could shed new light on the actions of DA and drugs acting on the dopaminergic system. In addition to forming protein complexes with other DA receptors, the D₁ receptor has been shown to interact with a variety of other proteins, including A₁ adenosine receptors, NMDA receptors, Na⁺,K⁺-ATPase, calnexin, and caveolin (for a review of DA receptor protein-protein interactions, see Hazelwood et al., 2009). The potential significance of these interactions with respect to DA receptor function and potential pharmacotherapies has not been fully realized.

D₂ receptor signaling through G_βγ also regulates a variety of cellular functions, including inwardly rectifying K⁺ channels, N-type Ca²⁺ channels, arachidonic acid levels (via PLA₂), extracellular signal-regulated kinase, and IP₃-sensitive Ca²⁺ stores and L-type Ca²⁺ channels. The D₂ receptor has the ability to activate G_i/o proteins independent of agonist, a property known as constitutive activity (Strange, 1999). As with the D₁ receptor, the D₂ DA receptor can also form stable complexes with other proteins, including the AMPA receptor, Na⁺,K⁺-ATPase, calnexin, and the DA transporter (Hazelwood et al., 2009).

Heart and Vasculature. At low concentrations, circulating DA primarily stimulates vascular D₁ receptors, causing vasodilation and reducing cardiac afterload. The net result is a decrease in blood pressure and an increase in cardiac contractility. As circulating DA concentrations rise, DA is able to activate β adrenergic receptors to further increase cardiac contractility. At very high concentrations, circulating DA activates α adrenergic receptors in the vasculature, thereby causing vasoconstriction; thus, high concentrations of DA increase blood pressure. Clinically, DA administration is used to treat severe congestive heart failure, sepsis, or cardiogenic shock. It is only administered intravenously and is not considered a long-term treatment.

Kidney. DA is a paracrine/autocrine transmitter in the kidney and binds to both D1-like and D2-like receptors. Renal DA primarily serves to increase natriuresis, though it can also increase renal blood flow and glomerular filtration. Under basal sodium conditions, DA regulates Na⁺ excretion by inhibiting the activity of various Na⁺ transporters, including the apical Na⁺-H⁺ exchanger and the basolateral Na⁺,K⁺-ATPase. DA can also influence the renin–angiotensin system: activation of D₁ receptors increases renin secretion, whereas DA, acting on D₃ receptors, reduces renin secretion. Abnormalities in the DA system and its receptors have been implicated in human hypertension (Jose et al., 1998). In some cases, poor regulation of natriuresis occurs due to constitutive desensitization of the D₁ receptor and uncoupling of the receptor from the signal transduction machinery. Although multiple polymorphisms exist in the DA receptors, none has been consistently associated with human hypertension.

Pituitary Gland. DA is the primary regulator of prolactin secretion from the pituitary gland. DA is released from the hypothalamus into the hypophyseal portal blood supply, directly infusing lactotrophs in the pituitary with high concentrations of DA. DA acts on lactotroph D_2S and D_2L receptors to decrease prolactin secretion (Chapter 38).

Catecholamine Release. Both D₁ and D₂ receptors modulate the release of NE and epinephrine. The D₂ receptor provides tonic inhibition of epinephrine release from chromaffin cells of the adrenal medulla, and of norepinephrine release from sympathetic nerve terminals. In contrast, the D₁ receptor responds to high-frequency DA stimulation to promote the release of catecholamines from the adrenal medulla. This D₁ receptor stimulation is thought to contribute to the "fight or flight" response.

CNS. DA in the brain projects via four main pathways (Figure 13–9)—mesolimbic, mesocortical, nigrostriatal and tuberoinfundibular—to regulate a variety of functions. The physiological processes under dopaminergic control include reward, emotion, cognition, memory, and motor activity. Dysregulation of the dopaminergic system is critical in a number of disease states, including Parkinson disease, Tourette's syndrome, bipolar depression, schizophrenia, attention-deficit hyperactivity disorder, and addiction/substance abuse.

The mesolimbic pathway is associated with reward and, less so, with learned behaviors. Dysfunction in this pathway is associated with addiction, schizophrenia, and psychoses (including bipolar depression), and learning deficits. The mesocortical pathway is important for "higher-order" cognitive functions including motivation, reward, emotion, and impulse control. It is also implicated in psychoses, including schizophrenia, and in attention-deficit hyperactivity disorder. The mesolimbic and mesocortical pathways are sometimes grouped together as mesolimbocortical. The nigrostriatal pathway is a key regulator of movement (Chapter 22). Impairments in this pathway are evident in Parkinson disease and underlie detrimental movement side effects associated with dopaminergic therapy, including tardive dyskinesia. DA released in the tuberoinfundibular pathway is carried by the hypophyseal blood supply to the pituitary, where it regulates prolactin secretion.

Electrophysiology. DA receptors regulate multiple distinct voltage-gated ion channels that impact firing of action potentials and neural transmission. D1-like receptor activation modulates Na⁺, as well as N-, P- and L-type Ca²⁺ currents, via a PKA-dependent pathway. D2 receptors regulate K⁺ currents. DA also modulates the activity of ligand-gated ion channels, including NMDA and AMPA receptors. As such, DA is not a classical excitatory or inhibitory neurotransmitter, but rather acts as a modulator of neurotransmission. A lack of subtype-specific ligands, and overlapping expression patterns, makes investigation of individual DA receptors difficult. As a consequence, most studies have investigated D1 and D2 families of receptors (with family-specific ligands) in discrete brain regions, especially the striatum and prefrontal cortex.

Dopaminergic neurons are strongly influenced by excitatory glutamate and inhibitory GABA input. In general, glutamate inputs enable burst-like firing of dopaminergic neurons, resulting in high concentrations of synaptic DA. GABA inhibition of DA neurons causes a tonic, basal level of DA release into the synapse (Goto et al., 2007). Interestingly, DA release also modulates GABA and glutamate neurons, thus providing an additional level of interaction and complexity between DA and other neurotransmitters. Strong phasic or slow tonic release of DA, and the subsequent activation of DA receptors, has differential effects on the induction of long-term potentiation (LTP) and long-term depression (LTD). In the striatum, phasic activation of DA neurons and stimulation of D1 receptors favors LTP induction, while tonic DA release with concomitant activation of both D1- and D2-like receptors favors LTD (Gerdeman et al., 2003).

Locomotion: Models of Parkinson Disease (PD). In the early 1980s, several young people in California developed rapid-onset parkinsonism. All of the affected individuals had injected a synthetic analog of meperidine that was contaminated with 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP). MPTP is metabolized by MAO-B to the neurotoxic MPP⁺, which is selectively taken into dopaminergic neurons by the DA transporter. Once inside the cell, MPP⁺ causes intra- and extracellular DA release, which is oxidized to form quinones and reactive oxygen species that cause in neuronal death. Because of the high specificity of MPP⁺ for the DA transporter, neuronal death is largely restricted to the substantia nigra and ventral tegmental area, resulting in a phenotype remarkably similar to Parkinson's disease. 6-Hydroxydopamine (6-OHDA) is similar to MPTP in both mechanism of action and utility as an animal model. In contrast to MPTP, however, 6-OHDA does not cross the blood-brain barrier and it is not specific to dopaminergic neurons (it is also a substrate for NET, the neuronal NE transporter). Thus, in animal models of Parkinson disease, 6-OHDA must be injected intracranially and co-administered with a blocker of NET. Lesioning animals with MPTP or 6-OHDA results in tremor, grossly diminished locomotor activity, and rigidity. As with Parkinson disease, these motor deficits are alleviated with l-DOPA therapy or dopaminergic agonists. These neurotoxins are valuable tools for studying potential neuroprotective agents and novel treatment strategies, such as neural grafting and stem cell transplantation; their effects underscore the singular importance of the dopaminergic system in locomotor activity and Parkinson disease.

Other pharmacological agents are also known to alter locomotor activity via dopaminergic actions, including cocaine and amphetamine. These drugs of abuse bind to DAT and inhibit reuptake of synaptic DA. Amphetamine is a substrate for the transporter, blocks it competitively, and enters the neuron, where it displaces (releases) DA from vesicular stores, causing an efflux of DA from the neuronal cytosol into the synapse, possibly by reversal of the DA transporter. The accumulation of synaptic DA increases stimulation of DA receptors and results in heightened locomotor activity. Mice lacking DAT are hyperactive, and do not display increased locomotion in response to cocaine or amphetamine treatment.

Targeted disruption of the DA receptor genes has revealed specific information about the receptor subtypes that mediate locomotor activity. Mice lacking the D₁ receptor show generalized alterations in locomotor activity, although there is not good agreement about the specific motor phenotype. Studies with D₁ receptor knockout mice indicate that the D₁ receptor, but not the D₅ receptor, is primarily responsible for the increase in locomotor activity that occurs following administration of D1-family agonists. D₂ receptor knockout mice display marked reductions in locomotor activity, initiation of movement, and rearing behaviors. This reduction in motor activity is also present in mice that are specifically lacking only the D_2L receptor, indicating that the D_2S isoform plays a lesser role in regulation of movement and is not able to compensate for the lack of D_2L receptor. The D₃ and D₄ receptor knockout animals display unique locomotor alterations in response to novel environments. However, these changes may be related to novelty-seeking behavior rather than actual motor impairments.

Reward: Implications in Addiction. In general, drugs of abuse cause increased DA levels in the nucleus accumbens, an area critical for rewarded behaviors. This role for mesolimbic DA in addiction has led to numerous studies on abused drugs in DA receptor knockout mice. Studies of D₁ receptor knockout mice show a reduction in the rewarding properties of ethanol, suggesting that the rewarding and reinforcing properties of ethanol are dependent, at least in part, on the D₁ receptor. D₂ receptor knockout mice also display reduced preference for ethanol consumption. Morphine also lacks rewarding properties in D₂ knockout mice when measured by conditioned place-preference or self-administration paradigms. However, mice lacking the D₂ receptor exhibit enhanced self-administration of high doses of cocaine. These data implicate a complex and drug-specific role for the D₂ receptor in rewarding and reinforcing behaviors. The D₃ receptor, highly expressed in the limbic system, has also been implicated in the rewarding properties of several drugs of abuse. However, D₃ knockout mice display drug-associated place preference similar to wild-type mice following amphetamine or morphine administration. Recently developed D₃-selective ligands implicate a role for the D₃ receptor in motivation for drug-seeking and in drug relapse, rather than in the direct reinforcing effects of the drugs (Heidbreder, 2008).

Cognition, Learning and Memory. Mice lacking the D₁ receptor display deficits in multiple forms of memory. D₁ knockout mice have impaired spatial memory using the Morris water maze as a read-out. Deficits in prefrontal cortex-dependent working memory occur in D₁ knockout mice, in agreement with pharmacological evidence that cortical working memory can be modulated with D₁ agonists and antagonists (Williams and Castner, 2006). Amphetamine administration disrupts prepulse inhibition in wildtype animals but not in D₂ knockout animals. Prepulse inhibition is unaltered in mice lacking only the D_2L isoform of the receptor, indicating that the long and short isoforms of the receptor may serve different purposes in sensorimotor gating and higher order processing. These findings imply an important role for the D₂ receptor in disorders with defects in sensorimotor gating, most notably schizophrenia. Indeed, many of the antipsychotic drugs used in the treatment of schizophrenia are high affinity antagonists for the D₂ receptor.

DA Receptor Agonists and Parkinson Disease. Parkinson disease (PD), a neurodegenerative disorder of unknown etiology, is characterized by extensive degeneration of dopaminergic neurons within the substantia nigra, resulting in tremor, rigidity, and bradykinesia. While the principal pharmacotherapy for PD is l-DOPA, limitations to its therapeutic effects (Chapter 22) have generated intense interest in developing alternative therapies for PD, with the intent of either delaying the usage of l-DOPA or alleviating l-DOPA side effects and restoring its efficacy. One treatment strategy is the use of DA receptor agonists, which act directly on the depleted nigrostriatal dopaminergic system and have fewer undesirable side effects. DA agonists can be used in conjunction with lower doses of l-DOPA in a combined therapy approach. Two general classes of dopaminergic agonists are used in the treatment of PD: ergots and non-ergots. The pharmacological properties of these drugs are described below; their use in the management of PD is described in Chapter 22.

D1/D2 Receptor Agonists: Ergot Allkaloids. Ergot derivatives are nonselective compounds that act on several different neurotransmitter systems, including DA, 5-HT, and adrenergic receptors. In the U.S., bromocriptine (parlodel) and pergolide (permax) are approved for the treatment of PD (see Table 13–6 for structures); however, their use has fallen out of favor because of the associated risk for serious cardiac complications. Bromocriptine is a potent D₂ receptor agonist and a weak D₁ antagonist. Pergolide is a partial agonist of D₁ receptors and a strong D2-family agonist with high affinity for both D₂ and D₃ receptor subtypes. Ergot derivatives are commonly reported to cause unpleasant side effects, including nausea, dizziness, and hallucinations. Pergolide was removed from the U.S. market as therapy for PD after it was associated with an increased risk for valvular heart disease.

Ergot Alkaloids in the Treatment of Hyperprolactinemia. Despite the contraindications for PD, ergot-based DA agonists are still used in the treatment of hyperprolactinemia. Like bromocriptine, cabergoline (dostinex) is a strong agonist at D₂ receptors, and has lower affinity for D₁, 5-HT, and α adrenergic receptors. The therapeutic utility of bromocriptine and cabergoline in hyperprolactinemia is derived from their properties as DA receptor agonists: they activate D₂ receptors in the pituitary to reduce prolactin secretion. The risk of valvular heart disease in ergot therapy is associated with higher doses of drug (necessary for PD treatment), but not with the lower doses used in treating hyperprolactinemia. The use of bromocriptine and cabergoline in the management of hyperprolactinemia is described in Chapter 38.

D1/D2 Receptor Agonists (Non-Ergot Alkaloids). Apomorphine (apokyn), a derivative of morphine, is approved for the treatment of PD. Apomorphine binds with the order of potence D₄>D₂>D₃>D₅. Apomorphine also binds with lower affinity to D₁, α-adrenergic, 5-HT_1A, and 5-HT₂ receptors. The therapeutic benefits of apomorphine in PD are likely due to its higher affinity and efficacy at DA receptors, especially D2-family receptors. Apomorphine is most commonly used in combination with l-DOPA to surmount the sudden "off" periods that can occur after long-term l-DOPA treatment.

Rotigotine is offered in a transdermal patch (neupro) for the treatment of PD. Rotigotine preferentially binds to D₂ and D₃ receptors and has much lower affinity for D₁ receptors. In addition, rotigotine is an agonist at 5-HT_1A and 5-HT₂ receptors, and an antagonist at α₂ adrenergic receptors. Due to problems with patch release, rotigotine is currently not sold in the U.S., but is available in Europe.

D2 Family Receptor Agonists (Non-Ergot Alkaloids). Based on the role of DA in PD and the initial therapeutic utility of ergot compounds, more selective D2 dopaminergic agonists were developed for PD treatment. Pramipexole (mirapex) and ropinirole (requip) are agonists at all D2 family receptors and, notably, bind with highest affinity to the D₃ receptor subtype. Interestingly, pramipexole is reported to have neuroprotective properties when administered before MPTP or 6-OHDA in animal models of PD (Joyce and Millan, 2007); agonist activity at the D₃ receptor may be the underlying neuroprotective mechanism.

Ropinirole in the Treatment of Restless Leg Syndrome (RLS). In addition to its utility in the treatment of PD, ropinirole has also been FDA-approved as pharmacotherapy for RLS. Mild dopaminergic hypofunction has been noted in patients with RLS. Somnolence and other side effects reported for PD therapy are less pronounced in the treatment of RLS, presumably due to the lower dose required for RLS therapy.

D₄ Receptor Agonists and Attention Deficit-Hyperactivity Disorder (ADHD). The D₄ receptor is known to be significant in ADHD; an association has been reproducibly demonstrated between the seven-repeat D₄ VNTR variant and patients with ADHD. Recent preclinical testing has uncovered potential therapeutic benefits of the selective D₄ agonist, A-412997, in cognitive tasks and animal models of ADHD. Animals treated with A-412997 showed cognitive improvements well below hyperlocomotive doses and, importantly, A-412997 did not show potential for abuse at any dose tested (Woolley et al., 2008). This is in marked contrast to the currently available drug therapies for ADHD treatment that have high abuse potential. While all published studies to date are preclinical, D₄-selective agonists show significant promise for the next generation of ADHD therapy.

DA Receptor Antagonists and Schizophrenia. DA receptor antagonists are a mainstay in the pharmacotherapy of schizophrenia. While many neurotransmitter systems likely contribute to the complex pathology of schizophrenia (Chapter 16), DA dysfunction is considered the basis of this disorder. The DA hypothesis of schizophrenia has its origins in the characteristics of the drugs used to treat this disorder: all antipsychotic compounds used clinically have high affinity for DA receptors. Moreover, psychostimulants that increase extracellular DA levels can induce or worsen psychotic symptoms in schizophrenic patients. The advent of neuroimaging techniques for visualization of DA in human brain regions has led to new insights into the role of specific DA systems. DA hyperfunction in subcortical regions, most notably the striatum, has been associated with the positive symptoms of schizophrenia that respond well to antipsychotic treatment. In contrast, the prefrontal cortex of schizophrenic patients exhibits dopaminergic hypofunction, which has been associated with the more treatment-refractory negative/cognitive symptoms. The drugs currently used to treat schizophrenia are classified as either typical (also referred to as first generation) or atypical (second generation) antipsychotics. This nomenclature stems from the high efficacy and lack of extrapyramidal side effects observed with atypical antipsychotics. Some of the newer drugs in development do not fit into this classification scheme, including the D1-selective agonist, dihydrexidine. Dihydrexidine shows great promise to improve the refractory negative symptoms of schizophrenia, and may be especially useful in combination therapy with current antipsychotics that preferentially reduce positive symptoms by antagonizing D2 receptors (Mu et al., 2007).

Antipsychotic Agents

Typical Antipsychotics. The first antipsychotic drug used to treat schizophrenia was chlorpromazine (thorazine). Despite its affinity for a wide array of neurotransmitter receptors, the antipsychotic properties of chlorpromazine were attributed to its antagonism of DA receptors, especially the D₂ receptor. More D₂-selective ligands were developed to improve the antipsychotic properties, including haloperidol (haldol) and similar D₂-selective drugs. While all typical antipsychotics markedly improve positive symptoms, they are not very beneficial in the treatment of negative or cognitive symptoms. As promising as these drugs initially seemed, their debilitating side effects limited their utility and gave rise to the development of the next generation of antipsychotic drugs, the atypical agents.

Atypical Antipsychotics. This class of antipsychotic drugs originated with clozapine (fazaclo) and is distinguished by a vastly improved side effect profile over the typical antipsychotics. The lack of extrapyramidal side effects has been attributed to a much lower affinity for the D₂ receptor compared to the typical antipsychotics. Atypical agents are also less likely to stimulate prolactin production. Clozapine has higher affinity for the D₄ receptor, which is highly expressed in the limbic system. Most atypical antipsychotics are low affinity antagonists at the D₂ receptor and high affinity antagonists or inverse agonists at the 5-HT_2A receptor. While the precise role of 5-HT_2A receptor binding in the efficacy of clozapine remains unclear, this dual DA-5-HT receptor blockade shaped the development of antipsychotics for several decades.

Aripiprazole (abilify) has gained recent attention for causing even fewer side effects than earlier atypical antipsychotics. Aripiprazole diverges from the traditional atypical profile in two ways: first, it has higher affinity for D₂ receptors than for 5-HT_2A receptors; secondly, and perhaps more importantly, it is a partial agonist, rather than an antagonist, at D₂ receptors. As a partial agonist, aripiprazole may diminish the subcortical DA hyperfunction by competing with DA for receptor binding, while simultaneously enhancing dopaminergic neurotransmission in the prefrontal cortex by acting as an agonist. The dual mechanism afforded by a partial agonist may thus treat both the positive and negative symptoms associated with schizophrenia.

D₃ Receptor Antagonists and Drug Addiction. Although much work remains to determine clinical utility, D₃-selective antagonists show promise in the treatment of addiction (Heidbreder, 2008). This interest stems from the high expression of the D₃ receptor in the limbic system, the reward center of the brain. Animal studies of highly D₃-selective antagonists BP-897 and SB-277011A have suggested a role for the D₃ receptor in the motivation to abuse drugs and in the potential for drug-abuse relapse.