CHAPTER 6: Widely Projecting Systems: Monoamines, Acetylcholine, and Orexin

• The monoamine neurotransmitters (dopamine [DA], norepinephrine [NE], epinephrine, serotonin, and histamine), the related small molecule neurotransmitter, acetylcholine (ACh), and the neuropeptides, orexin A and B, have an unusual but functionally significant organization in the brain. Their cell bodies are restricted to a small number of nuclei in the brainstem, hypothalamus, and basal forebrain, but their axons project widely throughout the central nervous system. This widely projecting organization permits each of these neurotransmitters to modulate activity in diverse circuits, sometimes in a coordinated fashion.

• For example, these systems play critical roles in sleep, arousal, and attention, and in survival responses to relevant stimuli.

• Widely projecting DA neurons have their cell bodies in the midbrain, within the substantia nigra pars compacta and the ventral tegmental area. DA is also produced by neurons in the arcuate nucleus of the hypothalamus, and by local circuit neurons in the retina. Midbrain DA neurons project widely to the forebrain and influence motivation, motor behavior, and multiple forms of memory. DA released from the hypothalamus suppresses synthesis and release of prolactin by the anterior pituitary.

• NE is synthesized in nuclei within the medulla and pons, the most prominent of which is the locus ceruleus (LC). The LC provides virtually all of the NE to the cerebral cortex. NE regulates arousal, attention, vigilance, and memory. Descending NE fibers modulate afferent pain signals.

• Serotonin (5HT or 5-hydroxytryptamine) is synthesized by neurons within the raphe nuclei of the midbrain. Their axons project very widely in the brain to influence diverse circuits involved in arousal, sensory processing, mood, and emotions.

• ACh is the neurotransmitter at the neuromuscular junction (nerve–muscle synapses). In the brain it is produced by widely projecting neurons with cell bodies in the brainstem and in the basal forebrain that project to the cerebral cortex and hippocampus. It is also produced by interneurons in the striatum. ACh in the forebrain influences many processes including motivation, learning, and memory.

• Histamine is produced by neurons in the tuberomammillary nucleus that lies within the posterior hypothalamus. These neurons project throughout the brain to regulate arousal. Inactivity of histamine neurons promotes sleep. Peripherally, histamine in the stomach promotes secretion of gastric acid via H₂ receptors; histamine released from mast cells is involved in allergic responses mediated via H₁ receptors.

• Orexin A and B (also known as hypocretin 1 and 2) are related neuropeptides that regulate sleep and wakefulness by interacting with monoaminergic and cholinergic neurons that in turn regulate arousal, emotion, motivation, and feeding. Orexin neurons have cell bodies in the lateral hypothalamus, and project widely throughout the brain. Among the major recipients of orexinergic projections are the LC, a source of NE, and the tuberomammillary nucleus of the hypothalamus, the source of histamine.

• All receptors for DA, NE, and histamine and 12 of the 13 5HT receptors are G protein–coupled receptors. The 5HT₃ receptor is a ligand-gated ion channel. ACh receptors are divided into two major classes: nicotinic receptors, which are ligand-gated ion channels, and muscarinic receptors, which are G protein–coupled receptors. Like most neuropeptide receptors, both orexin receptors, OX₁ and OX₂ receptors, are G protein–coupled receptors.

• The monoamines, DA, NE, and 5HT, share a common vesicular monoamine transporter (VMAT2) that loads them into synaptic vesicles to be stored for release. DA, NE, and 5HT each has a specific plasma membrane transporter in their respective presynaptic terminals that terminates their synaptic actions after release by reuptake into the terminals. The monoamines are metabolized by monoamine oxidase (MAO), which exists in two forms, MAO_A and MAO_B. The catecholamines, DA and NE, are also metabolized by catechol-O-methyltransferase.

• Degeneration of dopaminergic neurons in the substantia nigra causes Parkinson disease; dopaminergic ventral tegmental area neurons play a central role in normal reward-related behavior and in addiction and also in attention and working memory.

• DA acts on two related families of G protein–linked receptors, the D₂ family (D₂, D₃, and D₄ receptors) that are coupled to G_i/G_o and the beta arrestin pathway and the D₁ family (D₁ and D₅ receptors) that are coupled to G_s and the closely related G_olf.

• Proteins within dopaminergic synapses are targets for several significant classes of drugs. The psychostimulants, cocaine, amphetamine, and methylphenidate, are indirect DA agonists that interact with DA transporters. Parkinson disease is treated with the DA precursor L-dihydroxyphenylalanine (L-dopa), D₂ receptor agonists, and monoamine oxidase inhibitors (MAOIs). All currently used antipsychotics are D₂ receptor antagonists.

• NE interacts with α- and β-adrenergic receptors. Many antidepressants (which are also anxiolytic) increase synaptic NE or 5HT levels by blocking their plasma membrane transporters; others increase synaptic NE or 5HT by blocking metabolism (MAOIs). β-Adrenergic antagonists may dampen memories encoded under strong emotion including trauma.

• Proteins within 5HT synapses are targets for the treatment of depression and anxiety. Drugs that block the serotonin transporter are efficacious in obsessive–compulsive disorder (OCD). Many second-generation antipsychotic drugs, such as clozapine and many others, block the 5HT_2A receptor, which may help limit Parkinson-like side effects due to concomitant blockade of D₂ DA receptors. Hallucinogens, such as LSD, are partial agonists at 5HT_2A receptors.

• The five different muscarinic ACh receptors can be divided into two families based on the subtype of G protein to which they couple.

• Nicotinic ACh receptors (nAChRs) are pentamers constructed of subsets of 12 different subunits. Stimulation of nAChRs at the neuromuscular junction triggers muscle contractions. nAChRs are also found in the brain, where, among other roles, they are targets of the addictive drug, nicotine.

The survival and reproduction of free-living animals requires that they obtain nutrition and hydration, find safety, succeed in mating, and avoid predators and other dangers. Such behaviors require regulation of sleep–wake cycles in response, for example, to the waking times of predators or prey and the predominant environment (such as dark–light cycles and diurnal temperature variation). Successful survival also requires that arousal, vigilance, and attention be regulated appropriately in response to salient stimuli, and that behaviors be selected and executed in accordance with adaptive goals. Likewise, success demands that an organism engage in goal-directed behaviors when the risks and costs warrant it and disengage when the costs are too high. An organism must be able to record in memory the circumstances under which significant threats or rewards are found and learn motor programs that will maximize the speed and efficiency of escape from threats or the attainment of rewarding goals. Many circuits and neurotransmitters are involved in such behaviors, but the neurotransmitters discussed in this chapter play the central coordinating roles. Chapters on arousal and sleep (Chapter 13), higher cognitive function (Chapter 14), mood and emotion (Chapter 15), and reinforcement and addiction (Chapter 16) will develop many of these topics in greater detail, but will depend on the basic information presented in this chapter.

An important goal of neuroscience, often described under the rubric of “connectomics,” is to map the connectivity of animal and human brains, with the ultimate goal being the elucidation of functional maps. Maps of functional activity will depend not only on the locations and connectional patterns of cells, synapses, and circuits but also on the types and strength of synaptic connections, and thus on the “chemical neuroanatomy” that reflects the precise distribution of neurotransmitters, receptors, and their intracellular signaling mechanisms. At the simplest level of generalization, a diversity of important circuits in the brain underlies the detailed information processing and accurate point-to-point communication that permits the brain to create rich sensory representations of the world and to learn, plan, and execute effective motor programs. Such circuits generally depend on excitatory and inhibitory amino acid neurotransmitters acting on rapidly and transiently responsive ligand-gated ion channels (Chapter 5) to achieve spatially and temporally precise communication. Overlaying such circuits are diverse neurotransmitter systems that “fine-tune” their activity in response to salient stimuli, homeostatic needs, and emotional states. These neurotransmitters generally, but not exclusively, produce slower forms of synaptic transmission acting via G protein–linked receptors.

Neurotransmitters exerting such modulatory functions may act at varying distances from their targets. They may act locally, as in the case of cannabinoids and purines (Chapter 8); they may act at intermediate or longer distances, as is the case for some neuropeptides (Chapter 7). A small number of neurotransmitters exhibit a striking organization: they are synthesized by a relatively small number of neurons located within the brainstem, hypothalamus, or basal forebrain. These neurons project very widely throughout the brain, and in some cases descend also into the spinal cord, with some individual axons innervating an astoundingly large number of targets. Such architecture is not consistent with precise information transfer, but rather with the ability to coordinate the responses of many neurons in response to global state changes or significant stimuli. Neurotransmitters with such widely projecting organization include the monoamines (so-called because they contain a single amine group; 6–1), the functionally closely related neurotransmitter acetylcholine (ACh), and the peptide orexin (also called hypocretin). The monoamines include three catecholamines: dopamine (DA), norepinephrine (NE), and epinephrine (E), which are produced from a shared biosynthetic pathway, as well as serotonin (5-hydroxytryptamine or 5HT), and histamine. These neurotransmitters have sometimes been referred to as neuromodulators because their actions via G protein–linked receptors alter the responses of neurons to excitatory and inhibitory amino acids. We discourage the use of the term neuromodulator because many neurotransmitters, including glutamate, GABA, ACh, and serotonin, have both G protein–linked receptors and receptors that are ligand-gated ion channels. Function is determined by the receptor and its associated effector molecules rather than by the neurotransmitter.

DA illustrates the remarkable organization of widely projecting neurotransmitter systems: there are approximately 100 billion neurons in the human brain, but only about 500,000 produce DA. The cell bodies of most of these neurons lie in two contiguous regions of the midbrain, the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA). This small number of midbrain DA neurons innervates extensive terminal fields within the forebrain. Neurons from the SNc densely innervate the dorsal striatum where they play a critical role in learning and executing motor programs (Chapter 14). Neurons from the VTA innervate the ventral striatum (nucleus accumbens), olfactory bulb, amygdala, hippocampus, orbital and medial prefrontal cortex, and cingulate cortex 6–1. VTA DA neurons play a critical role in motivation, reward-related behavior, attention, and multiple forms of memory (Chapters 14 and 16). This organization of the DA system, wide projection from a limited number of cell bodies, permits coordinated responses to potent new rewards. Thus, acting in diverse terminal fields, DA confers motivational salience on the reward itself or associated cues (nucleus accumbens shell region), updates the value placed on different goals in light of this new experience (orbital prefrontal cortex), helps consolidate multiple forms of memory such as the location and cues of the reward (amygdala and hippocampus), and encodes new motor programs that will facilitate the ability to obtain this reward in the future (nucleus accumbens core region and dorsal striatum). In this example, DA modulates the processing of sensorimotor information in diverse neural circuits to maximize the ability of the organism to obtain future rewards.

Synthetic and Degradative Pathways

Catecholamine biosynthesis

Catecholamines are molecules that contain a catechol nucleus with an ethylamine group attached at the 1 position 6–2. 5HT is an indolamine with a hydroxy group at the 5 position and a terminal amine group on the carbon chain 6–3. The catecholamine neurotransmitters, NE, DA, and E, are sequential products of a single biosynthetic pathway that originates with the amino acid tyrosine 6–4. 5HT and melatonin are synthesized from the amino acid tryptophan 6–5. The catecholamine neurotransmitters and serotonin are discussed together because they have similar widely projecting anatomic organizations, interact with each other functionally, have related mechanisms of clearance from synapses and some shared pathways of metabolism, and are jointly targeted by several important classes of drugs, including psychostimulants (DA, NE, and 5HT), tricyclic antidepressants (TCAs; NE and 5HT), serotonin and NE reuptake inhibitor antidepressants (NE and/or 5HT), and monoamine oxidase inhibitor (MAOI) antidepressants (DA, NE, and 5HT).

Catecholamine biosynthesis begins with dietary tyrosine, which is actively transported into the brain (or peripheral sympathetic neurons; Chapter 9). It is hydroxylated within neurons at the 3 position by the enzyme tyrosine hydroxylase (TH) to form dihydroxyphenylalanine (dopa; known as levodopa orL-dopa). TH requires Fe²⁺ as a cofactor, as well as molecular oxygen and tetrahydrobiopterin (a hydrogen donor). An inhibitor of TH, α-methylparatyrosine (AMPT), has been used historically as an experimental tool to study catecholamine function; more recent alternatives include mice genetically engineered to lack TH or other enzymes in the biosynthetic pathway.

In dopaminergic neurons, one additional enzyme in this pathway is expressed, L-aromatic amino acid decarboxylase (AADC), which converts dopa to DA. AADC is a cytoplasmic enzyme that requires pyridoxal phosphate, a cofactor derived from vitamin B₆. AADC was originally known as dopa decarboxylase until it was recognized that it decarboxylates other substrates, including 5-hydroxytryptophan, the precursor of serotonin 6–5.

In noradrenergic neurons, an additional enzyme, dopamine-β-hydroxylase (DBH), is expressed that catalyzes the conversion of DA to NE. DBH requires Cu²⁺ and ascorbic acid (vitamin C) as cofactors. DBH is associated with synaptic vesicles that store NE. In the adrenal medulla and in brainstem neurons that produce E, an additional enzyme, phenylethanolamine-N-methyltransferase (PNMT), is expressed that converts NE to E. S-Adenosyl-L-methionine (SAM), a methyl donor, is a required cofactor for this step.

TH, the rate-limiting enzyme in catecholamine synthesis, is regulated by multiple mechanisms. Increased catecholamine release leads to increased TH activity that results from regulation at the transcriptional, translational, and posttranslational levels. Rapid activation of TH activity occurs via its phosphorylation at four serine residues in the N terminus of the protein by several protein kinases, including protein kinase A, Ca²⁺/calmodulin-dependent protein kinase II (CaM-kinase II), and protein kinase C. It is believed that such phosphorylation induces a conformational change in the protein that results in a higher affinity for its tetrahydrobiopterin cofactor and a lower affinity for catecholamines that trigger end-product inhibition of TH. The end result is an increase in the TH catalytic activity. Longer-term changes in TH activity can occur through transcriptional regulation of the TH gene by extracellular stimuli. Stimuli that upregulate TH expression include chronic environmental stress and drugs such as caffeine, nicotine, and morphine; drugs that downregulate TH expression include many antidepressants (which initially increase synaptic concentrations of NE and/or 5HT).

The ability of tyrosine to penetrate the blood–brain barrier depends on an active transport process. With normal dietary consumption of tyrosine, both active transport and TH activity are fully saturated. Thus, the administration of supplemental tyrosine does not produce significant increases in catecholamine synthesis in the central nervous system (CNS). However, increased catecholamine synthesis can be achieved by peripheral administration of L-dopa, which bypasses this rate-limiting enzymatic step and penetrates the blood–brain barrier, so long as its peripheral metabolism is blocked. For this reason, L-dopa is used in the treatment of Parkinson disease (Chapter 18).

Serotonin biosynthesis

5HT is synthesized from the amino acid tryptophan 6–5, which is actively transported across the blood–brain barrier and hydroxylated by tryptophan hydroxylase (TPH) to produce 5HT. This product is then decarboxylated to form 5HT by AADC, the same enzyme involved in the biosynthesis of catecholamines. In the pineal gland, additional enzymatic steps convert 5HT to melatonin, which is discussed further in Chapter 13.

TPH is the rate-limiting enzyme for 5HT biosynthesis. There are two closely related genes: TPH1, predominantly expressed in the periphery, and TPH2, expressed preferentially in the brain. TPH is subject to short-term and long-term regulatory processes similar to those described for TH, a related amino acid hydroxylase. Like TH, TPH requires molecular oxygen and tetrahydrobiopterin as cofactors, and can be activated by protein kinase A and CaM-kinase II. Although genetic variations in TPH2 have been linked weakly with depression, such findings are not significant in large genome-wide association studies.

An additional member of the amino acid hydroxylase family is phenylalanine hydroxylase, which converts phenylalanine to tyrosine. Mutations of this enzyme that decrease its catalytic activity result in phenylalaninemias (eg, phenylketonuria) that, among other ill effects, can damage the developing brain and cause generalized intellectual disability. Interference with the metabolism of phenylalanine causes the buildup of oxidized derivatives such as phenylketones, which exert toxic effects on neurons. Individuals with such mutations can prevent phenylketonuria by eliminating phenylalanine from the diet, a remarkable example of preventing a genetic disease with an environmental intervention.

Levels of 5HT in the brain can be altered by several means. Drugs such as p-chlorophenylalanine (PCPA), for example, can irreversibly inhibit TPH to produce a long-lasting depletion of 5HT. Experimental manipulation of tryptophan intake also can reduce levels of 5HT in the brain. Individuals who are asked to follow a low-tryptophan diet and subsequently are challenged with a beverage containing other amino acids but lacking tryptophan typically experience not only a dramatic reduction in blood tryptophan levels but also a substantial reduction of 5HT in the brain. In nonhuman primates, where direct measures are possible, a 90% reduction can be achieved. Among patients who have recovered from depression, tryptophan depletion induces a return of depressive symptoms in those who were successfully treated with a selective serotonin reuptake inhibitor (SSRI); however, depressive symptoms do not occur in healthy individuals or in those who were treated with antidepressants that influence NE reuptake. Thus, depletion of 5HT most likely does not cause depression; instead, patients treated with SSRIs may experience withdrawal symptoms on tryptophan depletion that include transient return of depressive symptoms (Chapter 15).

Degradation of catecholamines and serotonin

The most significant mechanism by which the synaptic actions of catecholamines, 5HT, and histamine are terminated is by reuptake into the nerve terminal via neurotransmitter-specific transporters expressed on the plasma membranes of presynaptic terminals. In addition, these monoamines are enzymatically catabolized by monoamine oxidase (MAO). The catecholamines, but not 5HT or histamine, are also metabolized by catechol-O-methyltransferase (COMT).

MAO has both intracellular and extracellular forms. The intracellular form is associated with the outer membrane of mitochondria; given that mitochondria are plentiful in presynaptic terminals, the primary action of MAO is to metabolize catecholamines, 5HT, and histamine after they are taken up into presynaptic terminals. However, the extracellular form may also act to metabolize neurotransmitter while in the synapse. Two major forms of MAO have been described: MAO_A and MAO_B. These forms are derived from distinct genes on the X chromosome and differ with regard to several biochemical properties, including their substrate specificity, cellular localization, and regulation by pharmacologic agents. MAO_A mRNA is expressed almost exclusively in noradrenergic neurons, such as those in sympathetic ganglia and locus ceruleus (LC; described below and shown in 6–7). MAO_B mRNA is detected predominantly in serotonergic and histaminergic neurons. There are conflicting reports about the expression of MAO genes and protein in dopaminergic neurons, although the evidence favors expression of MAO_A.

Both enzymes oxidize monoamines but differ somewhat in their affinity for substrates. MAO_A displays a strong affinity for NE and 5HT, even though it is not expressed in serotonergic neurons. The function of MAO_B may be not to oxidize 5HT, but rather to metabolize DA or trace amines that might act as false neurotransmitters, such as β-phenylethylamine, for which it has highest affinity. Extracellular 5HT appears to be oxidized by MAO_A derived from sources other than 5HT neurons.

MAOIs, such as phenelzine and tranylcypromine, are used to treat depression and anxiety disorders; the MAOIs selegiline and rasagiline are used to treat Parkinson disease (Chapters 15 and 18). However, clinical use of MAOIs as antidepressants and antianxiety agents has been limited by their side effects 6–1. Unsuccessful efforts to link naturally occurring variations in MAO genes with risk for certain complex behaviors are discussed in 6–2.

Catecholamines are also catabolized by COMT. Peripherally the major isoform is soluble, but, in the brain, a longer, membrane-bound isoform predominates, which is found in catecholamine synapses. COMT methylates catecholamines using S-adenosyl-methionine as a methyl donor. COMT inhibitors, such as entacapone and tolcapone, increase levels of catecholaminergic neurotransmitter in synapses and prolong receptor activation. In general, COMT appears to play a far smaller role in terminating the synaptic action of DA and NE than their specific membrane transporters, but in the prefrontal cortex, where the dopamine transporter (DAT) is expressed at relatively low levels, COMT may exert a more significant effect.

The major products that emerge from the enzymatic breakdown of catecholamines by MAO and COMT are shown in 6–4. Historically, these metabolites were investigated as indirect measures of brain catecholaminergic function in depression and schizophrenia. They were measured in cerebrospinal fluid, blood, and urine; however, interpretation of metabolite levels was significantly confounded by activity of the sympathetic nervous system and adrenal medulla and by many other factors. Thus, their usefulness as markers of CNS catecholamine function proved quite limited. 5HT metabolites were also historically investigated. After 5HT is oxidized by MAO, aldehyde dehydrogenase acts to produce 5-hydroxyindoleacetic acid (5-HIAA) as an end product 6–5. Reduction of 5-HIAA in cerebrospinal fluid has been reported to correlate with impulsive violence in some circumstances, most notably among individuals who have attempted suicide by violent means. Despite considerable research, the significance of these findings remains unclear. Overall, it may have been naïve to believe that major neuropsychiatric disorders reflected global abnormalities in levels of one neurotransmitter, rather than disorders of more specific neural circuits that could be influenced by DA, NE, or 5HT.

Functional Anatomy

Dopamine

The overall anatomy of DA in the brain is shown in 6–1. Historically, the first recognition of the importance of DA in the brain came from the investigation of Parkinson disease, which results from degeneration of SNc DA neurons. Death of these neurons results in denervation of the neostriatum (composed of the caudate and putamen in the human brain and the dorsal striatum in the rat and mouse brains), resulting in a movement disorder characterized by tremor, rigidity, abnormal gait, and difficulty initiating voluntary movement (Chapter 18). The major strategy for treatment is DA replacement, but DA is polar and cannot penetrate the blood–brain barrier. Instead, L-dopa is administered. After it crosses the blood–brain barrier it is taken up by dopaminergic nerve terminals via DAT and subsequently converted into DA by AADC 6–6. However, because AADC also resides in peripheral tissues, a significant fraction of L-dopa is decarboxylated into DA before it can be transported into the brain. Among other side effects, this produces nausea because there are DA receptors in the area postrema of the medulla, an area of the brain that controls nausea and vomiting, and which lies outside the blood–brain barrier so that it can sample the systemic environment (Chapter 2). L-Dopa is thus coadministered with an AADC inhibitor such as carbidopa that cannot penetrate into the brain.

Given the role of DA receptors in vomiting, D₂ antagonists are effective antiemetic medications. However, due to their risk of causing Parkinson-like side effects, alternatives are often used. 5HT₃ antagonists such as ondansetron, and neurokinin 1 (NK₁) receptor antagonists (the NK₁ receptor recognizes the neuropeptide, substance P; Chapter 7) such as aprepitant, are effective antiemetics and lack the side effects of D₂ receptor antagonists.

VTA projections to the limbic forebrain, as noted earlier, are required for reward-related behaviors. VTA DA projections also form the substrate on which certain drugs produce addiction, which is described in detail in Chapter 16. Under normal circumstances VTA DA neurons fire prior to behaviors that are elicited by cues predictive of reward. In order to coordinate responses to such cues, the VTA receives inputs from diverse brain regions. These include reciprocal inputs with regions involved in valuation of rewards and also inputs from other widely projecting systems involved in arousal, attention, and memory. Different subregions of the VTA receive glutamatergic inputs from the prefrontal cortex, orexinergic inputs from the lateral hypothalamus (see 6–25), cholinergic and also glutamatergic and GABAergic inputs from the laterodorsal tegmental nucleus and pedunculopontine nucleus 6–18, noradrenergic inputs from the LC 6–7, serotonergic inputs from the raphe nuclei 6–10, and GABAergic inputs from the nucleus accumbens and ventral pallidum. The activity of VTA DA neurons is also suppressed by activation of the lateral habenula, which sends glutamatergic inputs to GABAergic neurons in the vicinity of the VTA, which normally inhibit VTA DA neurons.

DA has multiple actions in the prefrontal cortex. It promotes the “cognitive control” of behavior: the selection and successful monitoring of behavior to attain chosen goals. Aspects of cognitive control in which DA plays a role include working memory, the ability to hold information “on line” in order to guide actions, suppression of prepotent behaviors that compete with goal-directed actions, and control of attention and thus the ability to overcome distractions (Chapter 14). Cognitive control is impaired in several disorders, including attention deficit hyperactivity disorder (ADHD), which is treated with psychostimulants, a term used to describe indirect DA agonists such as methylphenidate and amphetamines that block DAT or cause reverse transport of DA into synapses. Cognitive control is also deficient in addiction and a range of poorly described conditions known as impulse control disorders. Working memory is impaired in schizophrenia, to a lesser degree in some nonpsychotic relatives of individuals with schizophrenia, and in patients with bipolar disorder with psychotic symptoms (Chapter 17). Antipsychotic drugs do not have a therapeutic benefit for working memory or for any of the other cognitive impairments of schizophrenia.

Antipsychotic drugs produce their therapeutic effects (diminishing psychotic symptoms, eg, delusions and hallucinations) by blocking D₂ receptors, presumably those that reside in the terminal fields of VTA DA neurons in subcortical structures in the limbic forebrain. The mechanism by which reducing dopaminergic stimulation of these structures ameliorates psychotic symptoms is unclear. Because antipsychotic drugs block D₂ receptors in the caudate and putamen as well as in the neurons where they exert therapeutic effects, these drugs produce side effects that are similar to the symptoms of Parkinson disease. (These are often called extrapyramidal side effects to distinguish striatally based motor systems from the corticospinal motor system, the fibers of which descend in a brainstem structure called the “pyramids.”) Because these are “on-target” side effects, that is, side effects that arise from the same molecular target—the D₂ DA receptor—that is required for their therapeutic effects, attempts to mitigate the side effects must depend on stimulation or blockade of other receptors. Antipsychotic drug–induced Parkinson-like effects are not treated with L-dopa or indirect DA agonists because D₂ DA receptors are already blocked and, were DA or DA agonists to successfully compete for D₂ DA receptors, the likely result would be worsening of psychotic symptoms. Instead extrapyramidal side effects are treated with anticholinergic drugs (ie, drugs that block muscarinic cholinergic receptors). Clozapine and second-generation antipsychotic drugs have less tendency to produce these side effects than first-generation drugs partly as a result of lower affinity for D₂ receptors, but also as a result of having other receptor actions, including antagonism of 5HT_2A receptors, antagonism of muscarinic cholinergic receptors, or some combination (Chapter 17).

The tuberoinfundibular DA system, arising in the arcuate nucleus of the hypothalamus, inhibits prolactin synthesis and release from the anterior pituitary. Antipsychotic drugs, which antagonize D₂ receptors, elevate levels of prolactin. Conversely, D₂ receptor agonists, such as bromocriptine, can be used to suppress hyperprolactemia, which is most commonly caused by prolactin-secreting pituitary adenomas (Chapter 10).

Norepinephrine

NE is produced by neurons contained within multiple nuclei of the pons and medulla 6–7. The LC, which is located on the floor of the fourth ventricle in the rostral pons, contains more than 50% of all noradrenergic neurons in the brain; it innervates both the forebrain (eg, it provides virtually all of the NE to the cerebral cortex) and regions of the brainstem and spinal cord. Yet in the human brain, the LC contains only about 12,500 neurons per side, illustrating its remarkably wide projections. The other noradrenergic neurons in the brain occur in loose collections of cells in the brainstem, including the lateral tegmental regions. These neurons project largely within the brainstem and spinal cord.

NE, along with 5HT, ACh, histamine, and orexin, is a critical regulator of the sleep–wake cycle and of levels of arousal (Chapter 13). Not surprisingly, the LC receives significant input from other widely projecting systems involved in regulating sleep and arousal, such as orexin-containing neurons 6–25. LC neurons fire at a basal (tonic) rate during the waking state; the firing rate decreases during slow-wave sleep, and the LC does not fire during paradoxical sleep, the rodent equivalent of rapid eye movement (REM) sleep in humans. Transient increases in LC firing (phasic firing) are correlated with the onset of sensory stimulation; the highest rates of firing are associated with stimuli that portend threat. In addition to increasing arousal, the LC influences diverse aspects of attention and vigilance. In response to threat, LC firing may also increase anxiety, by releasing NE in the amygdala and other regions of the limbic forebrain. Stimulation of β-adrenergic receptors (β-ARs) or α₁-adrenergic receptors in the amygdala results in enhanced memory for information encoded under strong emotion, facilitating the recall of stimuli that predict danger. However, this mechanism likely contributes to posttraumatic stress disorder (PTSD) in humans. β-Adrenergic (eg, propranolol) and α₁-adrenergic (eg, prazosin) receptor antagonists have been investigated as interventions to treat PTSD, but their efficacy appears to be modest. Part of the challenge is that administration generally follows a traumatic experience; the drugs might be more effective if taken before—but this would require something implausible under most circumstances, prediction of trauma (Chapter 15). In the laboratory, administration of α₂-adrenergic receptor antagonists, such as yohimbine, which increase firing of LC neurons by blocking inhibitory autoreceptors 6–8, induces fear and anxiety in laboratory animals and in humans. Opiates, which bind to μ opioid receptors, inhibit LC firing and are thus anxiolytic, among the many other actions of these drugs. With chronic administration, cellular adaptations within LC neurons lead to tolerance and dependence. With cessation of opiate administration, very high rates of LC firing contribute to the opiate physical withdrawal syndrome. These mechanisms are discussed fully in Chapter 16.

Electrophysiologic studies in monkeys suggest that the LC may have relatively specific roles in arousal and attention-related behaviors, that is, survival-related behaviors, including exploring the environment for sources of reward (eg, food, water, or safety), exploiting these resources, and disengaging with satiety or when the source of reward is depleted or otherwise becomes problematic. The LC receives inputs from the orbitofrontal cortex, which is involved in valuation of rewards, and which responds both to anticipation of rewards and to satiety. It also receives inputs from the anterior cingulate cortex, which is involved in monitoring task performance, and providing information as to whether performance is successfully approaching the selected goal. It has been argued that phasic LC firing optimizes task performance (exploitation), while tonic firing returns when task utility wanes and correlates with disengagement from the task and a search for alternative behaviors (exploration). In response to these altered firing patterns, NE released in the prefrontal cortex and other brain regions would modulate task-related behaviors. If correct, this model of LC function would contribute to our understanding of ADHD and other disorders of “top-down” cognitive control. In ADHD, there is impaired control of engagement and disengagement with tasks, as well as impaired ability to resist distractions. Noradrenergic projections from the LC thus interact with dopaminergic projections from the VTA to regulate cognitive control. Drugs that increase synaptic NE 6–9 by blocking the NE transporter (NET), such as the antidepressants desipramine or atomoxetine, exhibit some efficacy in treating ADHD. Psychostimulants have greater efficacy in most patients, however, probably because they increase DA as well as NE. Indeed stimulants act not only on DAT and NET but also on the serotonin transporter (SERT), although it has not been shown that 5HT makes a therapeutic contribution to treatment of ADHD. This model also suggests limits to current selective NE reuptake inhibitor or stimulant treatment because these produce sustained elevations in synaptic NE and DA, rather than optimizing the phasic and tonic firing patterns of monoamine neurons.

Epinephrine

E occurs in only a small number of central neurons, all located in the medulla. E is involved in visceral functions, including control of respiration. It is also produced by the adrenal medulla.

Serotonin

It has been estimated that there are several hundred thousand serotonergic neurons in the human brain. These neurons are confined almost exclusively to discrete nuclei in the brainstem raphe (raphe refers to their midline location; 6–10). The most caudal clusters of the raphe innervate the medulla as well as the spinal cord. The dorsal and median raphe are located in the midbrain and innervate much of the rest of the CNS by means of numerous and sometimes diffuse projection pathways. The dorsal raphe forms the ventral-most portion of the periaqueductal gray. The median raphe is located ventral to the dorsal raphe in roughly the same anterior–posterior position of the midbrain. Although these two nuclei have overlapping terminal fields, the dorsal raphe preferentially innervates the cerebral cortex, thalamus, striatal regions (caudate–putamen and nucleus accumbens), and dopaminergic nuclei of the midbrain (eg, the substantia nigra and VTA), while the median raphe innervates the hippocampus, septum, and other structures of the limbic forebrain. Projections from these and other raphe nuclei are so extensive that virtually every neuron in the brain may be contacted by a serotonergic fiber.

The function of serotonergic projections has been challenging to study partly because they are remarkably extensive, partly because the raphe nuclei contain a mixed population of neurons of which only a minority produce 5HT (making physiologic recordings difficult), and partly because of the large number of 5HT receptors for which truly selective antagonists have only recently become available. Well-formulated, testable hypotheses on the overall effects of 5HT are also difficult to construct because some of the actions of serotonin mediated by different receptors produce effects in opposite directions. Nevertheless, 5HT affects numerous functions including sleep, arousal, attention, processing of sensory information, and important aspects of emotion (likely including aggression) and mood regulation.

Storage, Release, and Reuptake

Most biosynthesis of catecholamines and 5HT does not occur in the cell bodies of their neurons; rather, the synthetic enzymes are transported to nerve terminals, where transmitter synthesis predominantly takes place (6–6, 6–9, and 6–11). DA is synthesized in the cytoplasm and is packaged in storage vesicles by means of the vesicular monoamine transporter protein (VMAT). In noradrenergic terminals, DA is converted to NE by DBH, which also is located in storage vesicles. DA, NE, and 5HT are all transported by the same VMAT protein, which spans the vesicle membrane. Vesicular storage not only permits rapid release of neurotransmitter in response to action potentials but also protects a reservoir of neurotransmitter from metabolism by MAO. VMAT2 is the form of VMAT that operates in the brain, while VMAT1 is the form found in adrenal medulla.

Reserpine, a compound initially derived from Rauwolfia, a plant used in Indian herbal medicine, disrupts catecholamine and 5HT reuptake into storage vesicles by blocking VMAT; the monoamine transmitters are then subject to metabolism by MAO. Based on its ability to deplete NE, reserpine was once used as an antihypertensive agent. Based on its ability to deplete DA, it can act as an antipsychotic drug. Indeed, it was used empirically for this purpose in the 1950s. Clinical use of reserpine was abandoned, however, because of its side effects. Tetrabenazine is another VMAT inhibitor that depletes monoamine stores. It is now approved for the treatment of chorea in Huntington Disease.

Plasma membrane transporters

As mentioned above, the DA, NE, and 5HT transporters (DAT, NET, and SERT, respectively) are related transmembrane proteins that move neurotransmitter from the synapse into the cytoplasm of the presynaptic terminal, where it is either reloaded into vesicles by VMAT2 or degraded by MAO. Each of the plasma membrane transporters has 12 hydrophobic membrane-spanning domains 6–12. The rapid reuptake of synaptic transmitter by these transporters has several consequences that are vitally important to signaling among neurons. First, reuptake limits the duration of presynaptic and postsynaptic receptor activation. Second, it limits the diffusion of transmitter molecules to other synapses. Third, it permits recycling and reuse of unmetabolized transmitter. The critical role played by these transporters (compared with the action of MAO or COMT) is illustrated by DAT knockout mice. These mice exhibit extreme hyperactivity, and exhibit other evidence of a hyperdopaminergic state, despite multiple compensatory adaptations in enzyme and receptor function that help to counter the elevated synaptic levels of DA.

NET, DAT, and SERT are targets for two major classes of psychotropic drugs, antidepressants and psychostimulants 6–2. The TCAs and newer serotonin–norepinephrine reuptake inhibitors (SNRIs) block NET, SERT, or both with differing selectivity. The norepinephrine reuptake inhibitors (NRIs) and SSRIs are selective for NET or SERT, respectively. Less commonly, antidepressants such as bupropion may also block DAT (Chapter 15). The most significant clinical difference between SSRIs, SNRIs, and NRIs versus TCAs is that the TCAs are also antagonists of α₁-adrenergic receptors, H₁ histamine receptors, and muscarinic cholinergic receptors resulting in postural hypotension (due to α₁ receptor blockade), sedation (due to H₁, α₁, and muscarinic cholinergic receptor blockade), and dry mouth, failure of pupillary accommodation, constipation, and urinary retention (due to muscarinic antagonism) (Chapter 9). While the newer antidepressants also have significant side effects (eg, sexual dysfunction), they are safer in overdose and far better tolerated. The clinical utility of these antidepressants in the treatment of depression and chronic pain syndromes is discussed further in Chapters 11 and 15.

Psychostimulants interact with DAT, NET, and SERT. Cocaine blocks these three transporters and thereby blocks reuptake of all three neurotransmitters after normal vesicular release. Amphetamines are a family of related drugs that have a more complex mode of action. They enter monoaminergic nerve terminals via DAT, NET, and SERT, and disrupt the action of VMAT2 to cause leakage of neurotransmitter out of synaptic vesicles. This causes cytoplasmic levels of the neurotransmitters to rise, which triggers the “reverse transport” of the neurotransmitters by DAT, NET, and SERT into the synapse. The rewarding and addictive actions of the psychostimulants result primarily from their actions on DAT (Chapter 16).

Halogenated amphetamines such as fenfluramine are selective for SERT, and stimulate rapid “reverse transport” of 5HT into the synapse. Fenfluramine was prescribed alone or in combination with phentermine, a sympathomimetic drug with amphetamine-like actions, to suppress appetite. However, fenfluramine was removed from the market because it caused cardiac valvular disease and primary pulmonary hypertension in some patients, an action mediated via its peripheral effects on 5HT and the activation of 5HT_2B receptors. (In blood, platelets are a significant source of 5HT.) There is evidence that the weight loss caused by these agents is mediated via activation of 5HT_2C receptors in the medial hypothalamus (Chapter 10).

The brain expresses low levels of additional types of plasma membrane transporters, which may also contribute to the removal of monoamines from the synapse, although this remains speculative. There is evidence that certain members of the SLC29 (equilibrative nucleoside transporter) family, discussed in Chapter 8, can transport DA and 5HT nearly as effectively as DAT and SERT. Organic cation transporters (OCTs), which are expressed in discrete brain areas, are reported to transport E and histamine. While the physiologic role of OCTs requires further investigation, they may be involved in maintenance of salt and fluid homeostasis.

Receptors

Catecholamine receptors

All receptors for DA and NE belong to the G protein–coupled receptor (GPCR) superfamily. E does not have its own receptors, but utilizes the same receptors as NE. As described in Chapter 4, binding of neurotransmitters to GPCRs initiates a conformational change in the receptor such that it activates a G protein, which in turn is coupled to regulation of an ion channel or second messenger–generating enzyme. Although DA and NE each has only one plasma membrane transporter, each has numerous receptors encoded by different genes. Each of these receptors has unique pharmacologic properties and localization, features that can be exploited in the design of drugs. There are more receptors than G proteins; thus, there is some convergence of actions at the level of intracellular signaling (eg, D₁ DA receptors, β₁- and β₂-adrenergic receptors, and 5HT₄ serotonin receptors activate the stimulatory G protein G_s), but specificity of action is maintained by the cell types on which these receptors are expressed, the location of the receptors on these cells (eg, distal or proximal dendrites, dendritic spines or shafts, perikarya, or presynaptic terminals), and their interactions with other receptor-activated signaling systems in the cells expressing them.

DA receptors 6–3 are divided into the D₁ family (D₁ and D₅ receptors, coupled to G_s or the related G_olf) and the D₂ family (D₂, D₃, and D₄, coupled to G_i/G_o and to beta-arrestin signaling). D₂ and D₃ receptors function as inhibitory presynaptic autoreceptors and as postsynaptic receptors (ie, heteroreceptors expressed on noncatecholamine neurons). D₂ receptors have two splice variants, D_2short and D_2long; however, distinct functional roles of these isoforms have not been identified. Numerous medications produce their clinical effects via actions on DA receptors. All antipsychotic drugs are antagonists of D₂ receptors (Chapter 17). By contrast, D₂ agonists (eg, bromocriptine) are useful in the treatment of Parkinson disease and hyperprolactinemia (Chapters 10 and 18). Pramipexole and ropinirole are agonists at D₂-like receptors, with some selectivity toward D₃ receptors, and show some promise in the treatment of Parkinson disease and restless leg syndrome.

Adrenergic receptors are divided into α and β families, both of which have multiple receptor subtypes 6–4. Each subtype responds in varying degrees to both NE and E. All β receptors are G_s-coupled, and most α₁ receptors are G_q-coupled; α₂ receptors, which are generally G_i-coupled, function as inhibitory autoreceptors and as postsynaptic receptors. Many medications on the market act via adrenergic receptors; most of their actions are in the autonomic nervous system, as discussed in Chapter 9.

The physiologic responses elicited by receptor stimulation vary not only among receptors but also for any given receptor, depending on the neuronal cell type involved. The latter phenomenon occurs because the ion channels regulated by GPCRs are separate molecules (as described in Chapters 2–4), unlike the situation for receptors that are ligand-gated channels, and different signaling molecules and ion channels are found in different cells types. For example, an ion channel that is inhibited by protein kinase A phosphorylation may predominate in one cell type, but a channel activated by protein kinase A phosphorylation might predominate in another, leading to very different responses to activation of the same receptor. Because the physiologic responses elicited by catecholamine receptor activation are numerous and complex, only general themes related to these responses are presented here.

The electrophysiologic consequences of activation of D₁-like receptors have been extremely difficult to pin down because of conflicting reports. Such inconsistencies likely reflect the heterogeneity of the cell types that have been studied, and also the complex cascades of protein kinases and protein phosphatases that are influenced by D₁-like receptors and that regulate many types of ion channels. The consequences of D₂ receptor activation appear to be more uniform, and frequently give rise to inhibitory responses, like most G_i/G_o-linked receptors, caused by the activation of inwardly rectifying K⁺ channels (Chapter 4).

β-ARs 6–13, coupled to G_s, lead to the activation of adenylyl cyclase, the synthesis of cAMP, and the activation of protein kinase A (Chapter 4). Such activation in turn leads to excitatory or inhibitory effects in neurons, depending on the protein kinase A substrates expressed in the particular cell type. In addition, cAMP can, independently of protein kinase A, activate cyclic nucleotide–gated channels called HCN (hyperpolarization- and cyclic nucleotide–activated) channels (Chapter 2). In the cerebral cortex and hippocampus, β-AR activation facilitates the excitation of pyramidal cells by blocking the activity of a Ca²⁺-activated K⁺ channel. In contrast, in cardiac muscle, β-AR activation leads to the phosphorylation and activation of voltage-gated Ca²⁺ channels, representing the mechanism by which NE released from sympathetic neurons and circulating E released from the adrenal medulla increase the force and rate of cardiac contraction.

The activation of α₁ receptors by means of G_q coupling triggers the phosphatidylinositol cascade, which can have multiple effects on neuronal excitability. In contrast, the activation of α₂ receptors, typically through G_i coupling, causes inhibitory responses in many cells; indeed α₂ receptors serve as autoreceptors on NE neurons as stated earlier.

The brain and certain peripheral tissues also express receptors for so-called “trace amines,” which refer to endogenous amines (eg, tryptamine) that are structurally related to catecholamines and present in mammalian brain at extremely low levels. Trace amines can activate trace amine–associated receptors (TAARs) that are G protein–coupled. There is some evidence that amphetamine and related drugs can bind to TAARs; however, the functional importance of trace amines and TAARs remains unknown.

Serotonin receptors

There are 13 known human 5HT receptors 6–5, 12 of which are GPCRs and 1, the 5HT₃ receptor, is ionotropic. Activation of the 5HT₃ receptor by 5HT opens a nonselective cation channel and triggers a rapid, transient depolarizing current that is carried by Na⁺ and K⁺. Like the nicotinic cholinergic and GABA_A receptors, 5HT₃ receptors are pentamers with subunits arrayed around a central pore. Several variants of the 5HT₃ have been reported, but little is known about their differing functions and pharmacology.

5HT_1A receptors function as somatodendritic autoreceptors because they reside on the cell bodies and dendrites of 5HT neurons; their activation reduces cell firing and inhibits the synthesis and release of 5HT. The activation of serotonergic autoreceptors expressed on presynaptic nerve terminals, 5HT_1B and 5HT_1D receptors, decreases local synthesis and release of transmitter. 5HT_1A, 5HT_1B, and 5HT_1D receptors are highly homologous, and signal by coupling to the inhibitory G protein, G_i. It is believed that their inhibitory effects on serotonergic neurons, like those of other G_i-linked receptors, are mediated by the activation of inwardly rectifying K⁺ channels, the inhibition of voltage-gated Ca²⁺ channels, and the inhibition of adenylyl cyclase. The actions of these autoreceptors are represented in 6–14. These various 5HT receptors are also expressed on nonserotonergic neurons where they mediate the postsynaptic actions of the neurotransmitter.

Pharmacology of serotonin receptors Extensive attention has been given to the development of agents that selectively target individual 5HT receptors either as probes of neural function or as putative drugs. Partial 5HT_1A receptor agonists, for example, buspirone and gepirone, have been used to treat generalized anxiety disorder (GAD). They lack the risks of dependence, sedation, or abuse that characterize benzodiazepines, but exhibit only modest efficacy (Chapter 15). Sumatriptan and a large number of related triptan drugs, which activate 5HT_1D receptors, are important treatments for migraine headaches (Chapter 20). Clozapine, and many second-generation antipsychotic drugs, such as risperidone and olanzapine, are antagonists at 5HT_2A receptors in addition to their D₂ antagonist properties. As stated earlier, 5HT_2A receptor antagonism appears to counteract some of the risk of Parkinson-like side effects caused by D₂ receptor antagonism that is required for their therapeutic effects. Hopes that 5HT_2A receptor antagonism also contributes to the therapeutic effects of antipsychotic drugs have not been borne out. The idea that 5HT_2A receptor blockade might be antipsychotic derived partly from the observation that the hallucinogens, sometimes also described as “psychedelic drugs,” are partial agonists at 5HT_2A receptors 6–3. As was mentioned above, 5HT₃ antagonists, such as ondansetron and granisetron, are effective antiemetics that lack the Parkinson-like side effects of D₂ receptor antagonists. There is great interest in the therapeutic potential of drugs that activate or inhibit the other 5HT receptor subtypes, for example, 5HT_2C agonists as antiobesity drugs (Chapter 10) or 5HT₄ agonists as antidepressants (Chapter 15), but clinical experience with most of these molecules remains in early stages.

Alternative signaling cascades

Studies over the past decade have shown that the activation of GPCRs leads to the stimulation of so-called alternative signaling cascades mediated, not by G proteins, but by a family of proteins called arrestins (or β-arrestins based on their initial description in conjunction with the β-AR). Early studies showed that the phosphorylation of the β-AR, bound by ligand, by G protein receptor kinases (GRKs) triggers the recruitment of β-arrestin, which then mediates downregulation of the receptor (Chapter 4) via its internalization 6–15. More recently, however, such recruitment of β-arrestin has been shown to activate several additional signaling cascades, including MAP-kinase and Ca²⁺ pathways. Interestingly, receptor agonists can differ in their ability to activate “canonical” (G protein–mediated) versus “noncanonical” (β-arrestin–mediated) pathways—a phenomenon referred to as ligand-directed or biased signaling—and such diversity of signaling has been documented for many types of GPCRs. An example of biased signaling at DA receptors, which might be relevant to antipsychotic drug action, is discussed in Chapter 17.

Synthetic and Degradative Pathways

ACh is synthesized in a reversible reaction in which an acetyl group is transferred from acetyl coenzyme A (CoA) to choline by the enzyme choline acetyltransferase (ChAT) 6–16. Its synthesis is regulated by the rate-limiting availability of choline. Choline is transported into neuronal terminals, either in its free form or bound to membrane phospholipids, by distinct high-affinity and low-affinity transport mechanisms 6–17. Most ACh is synthesized in terminals, which are rich in ChAT, and also in mitochondria, which are the site of acetyl CoA synthesis.

The synaptic actions of ACh are terminated by the enzyme acetylcholinesterase (AChE), which hydrolyzes ACh into acetate and choline 6–17. AChE is an extraordinarily efficient enzyme that is capable of hydrolyzing 1000 ACh molecules per second per molecule of enzyme. This enzyme occurs in the cytoplasm and in the outer cell membrane; thus, it can metabolize ACh both intracellularly and extracellularly. Anticholinesterases, which inhibit AChE, cause released ACh to accumulate extracellularly, producing excessive stimulation of ACh receptors throughout the nervous system. Reversible inhibitors, such as physostigmine and neostigmine, inactivate AChE for as many as 4 hours and are used clinically to treat glaucoma, myasthenia gravis (Chapter 12), and smooth muscle dysfunction of the bladder and intestines. Unlike physostigmine, neostigmine cannot enter the brain because it is a quaternary ammonium compound and thus is too highly charged to cross the blood–brain barrier (Chapter 2). Centrally acting anticholinesterases, such as tacrine and donepezil, are used to increase central ACh concentrations in patients with Alzheimer disease (see below). Irreversible anticholinesterases completely inhibit ACh breakdown. Restoration of AChE activity requires new AChE synthesis. These irreversible inhibitors are used as insecticides, and are highly toxic if ingested by humans. These agents were a major class of nerve gases. Just before World War II, first German and then Allied scientists began to explore potential uses for organophosphate agents in chemical warfare. Their efforts resulted in the development of several lethal compounds, including sarin, soman, and tabun, which can cause death within 5 minutes of exposure. The primary cause of death is respiratory failure, which typically is preceded by cognitive impairment and severe autonomic symptoms. Treatment for exposure to these toxins involves combined administration of a muscarinic receptor antagonist, such as atropine, with the AChE antagonist, pralidoxime, which paradoxically restores AChE function.

Functional Anatomy

ACh is synthesized by widely projecting neurons with cell bodies in the basal forebrain and brainstem 6–18 and by interneurons in striatum.

Widely projecting cholinergic neurons arise from eight nuclei that are clustered in two areas, the basal forebrain and the upper brainstem. The basal forebrain cholinergic nuclei are composed of the medial septal nucleus, the diagonal band, and the nucleus basalis of Meynert. The dense innervation of the sensory and limbic cortices arising predominantly from the diagonal band and nucleus basalis is thought to influence arousal, cortical responsiveness to sensory input, and emotional states. Cholinergic neurons of the diagonal band and the nucleus basalis also innervate regions of the hippocampus and cerebral cortex involved in learning and memory. Because these basal forebrain cholinergic neurons tend to undergo early and severe degeneration in Alzheimer disease, likely contributing to cognitive impairment, they have been investigated as therapeutic targets. These neurons are dependent on nerve growth factor (NGF) for trophic support (Chapter 8); accordingly, NGF and drugs aimed at NGF signaling pathways have been tested as treatments but have not proven successful. It is because of the loss of ACh in the hippocampus and cerebral cortex that AChE inhibitors have been used in Alzheimer disease. Unfortunately, they are only modestly efficacious; at best they delay symptomatic deterioration for a few months, but do not slow neurodegeneration and do not produce dramatic clinical improvement (Chapter 18).

The pedunculopontine nucleus and the laterodorsal tegmental nucleus within the brainstem innervate dopaminergic neurons of the VTA and play a role in reward. They also innervate other brainstem nuclei as well as thalamocortical circuits to control sleep and arousal (Chapter 13). While these nuclei, as well as the nucleus basalis, innervate the basal ganglia, the most significant cholinergic innervation of striatum comes from large cholinergic interneurons intrinsic to this region. These interneurons are critical components of the complex circuitry that underlies motor learning and motor control. As mentioned above, loss of dopaminergic input to the striatum in Parkinson disease leads to a profound motor disorder that can be partly counteracted by muscarinic cholinergic antagonists such as trihexyphenidyl and benztropine. These can be used in the early stages of the disease, or to supplement other drugs, such as L-dopa. Antimuscarinic agents also are used to treat the extrapyramidal side effects of antipsychotic drugs. Muscarinic cholinergic antagonists are believed to act at muscarinic receptors on striatal medium spiny neurons, where they partially compensate for the decreased dopaminergic signaling in striatal circuits (Chapter 18).

Storage and Release

The cholinergic nerve–muscle synapse is the best studied synapse in neurobiology and has taught us an enormous amount about synaptic transmission as well as the mechanisms of synaptogenesis in both development and repair. Central cholinergic synapses differ in important regards, most significantly by being nerve–nerve synapses. In presynaptic terminals, ACh is concentrated in storage vesicles by a vesicular transporter 6–18 that can be inhibited by vesamicol, resulting in depletion of ACh from vesicles. Vesicles fuse with the presynaptic terminal membrane in response to depolarization, releasing ACh into the synapse. Several toxins interfere selectively with this vesicular release of ACh. Botulinum toxin A and tetanus toxin, which prevent ACh release from cholinergic motor neuron terminals, cause paralysis. Small injections of botulinum toxin (Botox) are used therapeutically to treat dystonias (by causing profound muscle relaxation) and also cosmetically to inhibit the movement of facial muscles that cause wrinkles. Black widow spider venom, which contains α-latrotoxin, promotes massive vesicular release of ACh and a subsequent overstimulation of postsynaptic neurons. It exerts its effects in part by uncoupling Ca²⁺ signals from the release process. α-Latrotoxin stimulates exocytosis by binding to neurexins and latrophilins, critical components of nerve terminals (Chapter 3).

Because ACh is not returned to the presynaptic terminal by a membrane transporter, it cannot be recycled like the monoamines. Instead, choline, released by the action of AChE on ACh, is returned to cholinergic terminals by a high-affinity choline transporter, where it is used to synthesize new transmitter. Hemicholinium is a high-affinity inhibitor of the choline transporter.

Acetylcholine Receptors

ACh acts on two major types of receptors named for naturally occurring agonists: (1) muscarinic receptors, which are members of the GPCR superfamily, and (2) nicotinic receptors, which are members of the ligand-gated ion channel superfamily. Other members of the latter family include 5HT₃ receptors, the ionotropic glutamate receptors, GABA_A receptors, and glycine receptors. Structural similarities among these ligand-gated channels are represented in 6–19.

Muscarinic receptors

Muscarinic receptors were named for their ability to bind muscarine, a compound derived from the poisonous mushroom Amanita muscaria. They are located peripherally in the autonomic nervous system (Chapter 9) and in the CNS. There are five subtypes, all expressed in brain, each with the canonical seven membrane-spanning domains that characterize GPCRs. Muscarinic receptors can be subgrouped based on their patterns of G protein coupling: M₁, M₃, and M₅ receptors couple to G_q, and M₂ and M₄ receptors couple to G_i 6–6. Electrophysiologic responses elicited by the activation of any given subtype can vary from one cell to another, depending on the second messenger systems and ion channels expressed in the cell. M₁, M₃, and M₅ receptors produce their effects by stimulating the phosphatidylinositol system. As with most other G_i-linked receptors, M₂ and M₄ receptors elicit mostly inhibitory responses; they act by activating inwardly rectifying K⁺ channels, inhibiting voltage-gated Ca²⁺ channels, and inhibiting adenylyl cyclase.

M₁, M₃, and M₄ receptors are primarily located in the cerebral cortex and hippocampus, where their activity may mediate some of the effects of ACh on learning and memory. In the striatum, M₁ and M₄ subtypes occur in abundance and mediate cholinergic signaling in extrapyramidal motor circuits and in responses to rewards. M₂ receptors are concentrated in the basal forebrain, the site of several cholinergic nuclei, where they act as autoreceptors to control ACh synthesis and release. The M₅ receptor is the least abundant of the muscarinic receptors and is expressed at low levels throughout brain.

General muscarinic agonists include several alkaloids, such as muscarine, pilocarpine, and arecoline, and many synthetic compounds, such as carbachol and oxotremorine. Centrally acting agonists produce arousal as well as peripheral effects such as excessive salivation and sweating. Arecoline is an ingredient of betel leaves, which are chewed in some Asian cultures to produce mild increases in arousal. Muscarinic cholinergic agonists are used clinically to treat glaucoma and urinary retention, and to ameliorate the dry mouth that characterizes Sjögren syndrome, a disorder of autoimmune degeneration of the salivary glands (Chapter 9). An increasing number of subtype-selective muscarinic receptor agonists and antagonists are being developed for clinical use; most target the autonomic nervous system and regulate peripheral organ function, although interest continues in the possible use of such agents to enhance cognitive function (Chapter 14).

Prototypical muscarinic antagonists include atropine and scopolamine. Neither of these is subtype-selective. Atropine is a derivative of belladonna, whose name (beautiful woman) reflects its historical cosmetic use as a pupillary dilator. Belladonna is produced by the deadly nightshade plant, named because ingestion of excessive amounts is dangerous. Indeed, any excess use of antimuscarinic drugs produces cognitive impairment, and at higher doses delirium (Chapter 14), along with tachycardia and other dangerous autonomic symptoms. As stated earlier, muscarinic antagonists, such as benztropine, are commonly used to treat Parkinsonian symptoms induced by first-generation antipsychotic drugs. Many psychotropic drugs, including TCAs and low-potency antipsychotic drugs (eg, chlorpromazine and thioridazine), have muscarinic antagonist properties, as do many first-generation histamine H₁ antagonists (antihistamines) used to treat allergies and as over-the-counter hypnotic agents. In addition to expected autonomic effects, such as dry mouth, constipation, and urinary retention, these drugs can cause delirium. The latter is seen most commonly in geriatric patients treated with high doses of these agents or when combinations of drugs that share anticholinergic properties are used.

Nicotinic receptors

Nicotinic ACh receptors (nAChRs) are located at the neuromuscular junction, autonomic ganglia, adrenal medulla, and CNS. These ligand-gated ion channels were first characterized based on their ability to bind nicotine isolated from the tobacco plant Nicotiana tabacum. Their activation by ACh leads to the rapid influx of Na⁺ and Ca⁺ and subsequent cellular depolarization. A prominent feature of nAChRs is their very rapid desensitization. Unlike desensitization of GPCRs, which requires the actions of other proteins (Chapter 4), desensitization of nAChRs is an intrinsic property of the receptors themselves. The same may hold true for other ionotropic receptors. The latter type of desensitization may be similar to inactivation of voltage-gated Na⁺ channels (Chapter 2).

nAChRs expressed in neurons and those expressed in muscle differ in their subunit composition. nAChRs are composed of five subunits organized around a central pore 6–20. Most receptors are composed of heterologous subunits, of which at least 12 have been identified to date. Eight are classified as α subunits (α₂–α₉) and three as β subunits (β₂–β₄); α₁ and β₁ subunits are expressed in muscle. Like other members of the ligand-gated ion channel family, each subunit has four membrane-spanning domains and disulfide loops 6–20. The second transmembrane (M2) region of each subunit in each pentameric receptor lines the pore of the channel to regulate its gating properties.

Most neuronal nAChRs contain both α and β subunits, and their stoichiometry is such that the ratio of these subunits typically is two to three. However, some nAChRs are homomeric; α₇ subunits, for example, are capable of forming functional channels in vitro without the presence of other subunits. Several major types of nAChR complexes have been identified based on their affinities for certain toxins; for example, one population of receptors (ie, α₇ homomeric receptors) binds α-bungarotoxin with high affinity and nicotine with low affinity, whereas most other populations bind nicotine with high affinity and are insensitive to bungarotoxin.

Only a small number of ligands bind at nAChRs. Curare blocks muscular and, to a lesser extent, neuronal nAChRs. Succinylcholine, a paralytic agent that is used clinically during anesthesia, acts as a weak partial agonist; it mildly activates nAChRs and subsequently induces their prolonged desensitization. Several blocking agents, including hexamethonium and mecamylamine, inhibit nAChRs that mediate neurotransmission in both sympathetic and parasympathetic ganglia (Chapter 9). Very few antagonists that are selective for subtypes of neuronal nAChRs have been developed. Perhaps the most selective antagonist available is methyllycaconitine, which preferentially antagonizes α₇ homomeric receptor complexes. Recently, varenicline, a partial agonist at α₄/β₂ nAChRs, has been introduced to promote smoking cessation (Chapter 16). The selective agonist epibatidine binds with high affinity at nicotinic receptors but has been used only for experimental purposes (eg, as an antinociceptive agent).

As their name implies, nAChRs bind nicotine, which is a highly addictive substance (Chapter 16). The normal role of nicotinic receptors, however, appears to lie in arousal, attention, and memory. The widespread use of tobacco by people with schizophrenia has raised the questions of whether nicotine is being used to self-medicate cognitive impairments. Some epidemiologic studies indicate that smoking reduces the likelihood of developing two neurodegenerative diseases, Alzheimer disease and Parkinson disease. Unfortunately, the use of nicotine or tobacco products is fraught with complications, most notably addiction and the diverse ill effects of inhaling or chewing tobacco (including cardiovascular disease, respiratory disease, and diverse cancers). Nevertheless, interest remains high in the pharmaceutical industry to develop more selective nicotinic agents for a range of medicinal purposes including the enhancement of cognition (Chapter 14).

Synthetic and Degradative Pathways

Histamine is produced in one step by decarboxylation of the amino acid histidine by histidine decarboxylase 6–21. Histidine also can be decarboxylated by AADC, which, as previously discussed, is involved in the synthesis of DA, NE, and 5HT. Histamine is metabolized into methyl histidine by histamine methyltransferase. Alternatively, diamine oxidase may convert the neurotransmitter into imidazole acetaldehyde.

Functional Anatomy

Within the brain, histamine is synthesized exclusively by neurons with their cell bodies in the tuberomammillary nucleus (TMN) that lies within the posterior hypothalamus (PH) 6–22. There are approximately 64,000 histaminergic neurons per side in humans. These cells project throughout the brain and spinal cord. Areas that receive especially dense projections include the cerebral cortex, hippocampus, neostriatum, nucleus accumbens, amygdala, and hypothalamus.

The TMN is innervated by other widely projecting systems including monoaminergic and orexinergic neurons. Histaminergic neurons project back, in turn, to monoamine and cholinergic neurons involved in arousal, attention, learning, and memory 6–23. While the best characterized function of the histamine system in the brain is regulation of sleep and arousal, histamine is also involved in learning and memory through its direct actions on the cerebral cortex, hippocampus, and amygdala, and indirectly by its effects on cholinergic and monoamine neurons. It also appears that histamine is involved in the regulation of feeding and energy balance. The firing rate of histaminergic neurons in the TMN is strongly correlated with states of arousal. Firing is fastest during periods of wakefulness and slower during sleep; indeed, these cells fall silent during slow-wave sleep (Chapter 13).

In the periphery, histamine is released from mast cells, where it plays a role in inflammatory, allergic, pruritic, and algesic responses. Histamine released in the stomach activates H₂ receptors to cause release of acid.

Storage, Release, and Reuptake

The mechanisms underlying the storage, release, and reuptake of histamine remain poorly defined 6–24. It is presumed that histamine is concentrated in synaptic vesicles, yet the vesicular transporter responsible for such localization has not been determined; it is speculated that VMAT2 may mediate this function. When histamine is released from nerve terminals in response to electrical stimulation, it acts on presynaptic and postsynaptic histamine receptors. Histamine is metabolized by the cytoplasmic enzyme, histamine N-methyltransferase. It is not clear how released histamine might be transported into neurons if this is, indeed, the major mechanism of inactivation. The OCT-2 and -3 can transport histamine when expressed in cell culture. Whether it functions as a significant histamine transporter in the brain is not yet established.

Histamine Receptors

Four histamine receptors (H₁–H₄) have been identified; all are members of the GPCR superfamily 6–7. H₁ receptors couple to G_q, and H₂ receptors couple to G_s. The H₃ receptor is thought to couple to G_i and to act both as an inhibitory autoreceptor and as a heteroreceptor that regulates the release of other neurotransmitters from nerve terminals. H₃ receptors have their highest distribution in the striatum, nucleus accumbens, and cerebral cortex. Antagonists and inverse agonists are being explored as potential therapeutic agents to increase alertness and enhance cognition, although clinical trials have thus far been discouraging. H₄ receptors appear to play a role in inflammation in peripheral tissues. They are expressed at low levels in the brain.

Many drugs block H₁ receptors, either as an unwanted side effect (eg, the TCA doxepin and the antipsychotic drug clozapine) or as a desired mechanism as in the case of many allergy medications such as diphenhydramine. CNS-penetrant H₁ receptor antagonists are sedating. Newer antihistamines used as allergy medications, such as loratadine and terfenadine, are nonsedating because they do not cross the blood–brain barrier. Centrally acting antihistamines are the active ingredient in many over-the-counter sleep aids. Most agents that are selective for the histamine H₂ receptor do not cross the blood–brain barrier. These agents such as cimetidine and ranitidine inhibit the secretion of gastric acid.

Unlike the other neurotransmitters released by widely projecting systems in the brain, the orexins (also known as hypocretins) are peptides. Orexin A and B (or hypocretin 1 and 2) are produced in the lateral hypothalamic area (LHA) and PH 6–25 and bind to two receptors that, like nearly all neuropeptide receptors, are G protein–linked: orexin receptors 1 and 2 (OX₁ and OX₂). OX₁ is coupled via G_q, whereas OX₂ is coupled via G_i/o and in some cases by G_q. Like all neuropeptides (Chapter 7), the orexins are synthesized by transcription and translation and packaged in dense core vesicles for release. Differences in vesicles and release mechanisms between small molecule and peptide neurotransmitters are discussed in Chapter 7.

Orexin neurons project to and activate monoaminergic and cholinergic neurons involved in the maintenance of a long “awake” period. Lack of orexin, possibly caused by degeneration of orexinergic neurons, produces narcolepsy (Chapter 13). Orexin neurons are regulated by peripheral mediators that carry information about energy balance, including glucose, leptin, and ghrelin. They also receive inputs from limbic structures. Orexin neurons are, therefore, in a position not only to regulate sleep–wake cycles but also to respond to significant environmental and metabolic signals. Accordingly, orexin plays a role in the regulation of energy homeostasis, reward, and perhaps more generally in emotion. There is a great deal of interest in generating orexin receptor antagonists, both OX_1/2 dual antagonists (eg, suvorexant) and selective OX₂ antagonists for the treatment of insomnia. Conversely, the possibility that orexin agonists might promote wakefulness awaits testing in clinical trials.