CHAPTER 7: Neuropeptides

• Neuropeptides are small proteins or polypeptides that serve as neurotransmitters in the nervous system generally acting via G protein–coupled receptors. Other signaling peptides such as growth factors and cytokines are considered to be distinct even though they may have some overlapping functions.

• Like the monoamines and acetylcholine, neuropeptide transmitters serve primarily modulatory roles in the nervous system.

• The synthesis of neuropeptides, like that of all proteins, requires the transcription of DNA and translation of the resulting messenger RNA (mRNA) into protein.

• Neuropeptides are synthesized as large precursor prepropeptides that undergo extensive posttranslational processing, which includes cleavages into smaller peptides and enzymatic modification. The “pre” refers to an N-terminal signal sequence that directs newly synthesized peptides into the regulated secretory pathway.

• As a result of alternative RNA splicing and differential cleavage of propeptides in different tissues, a single gene can give rise to diverse signaling peptides with distinct functions.

• Unlike small-molecule neurotransmitters, which are packaged in small synaptic vesicles, neuropeptides are generally packaged in large dense core vesicles; both types of vesicles may be found in the same neuron.

• Neuropeptides may diffuse for long distances within the extracellular space before binding to their specific receptors.

• Neuropeptide transmitters act almost exclusively via activation of G protein–coupled receptors.

• Neuropeptides and their receptors modulate many diverse functions of the central nervous system, including sleep, arousal, reward, feeding, pain, cognition, stress responses, and emotions.

Neuropeptides are short proteins or polypeptides that serve as neurotransmitters. They generally bind to G protein–linked receptors; there are rare exceptions in which peptides, such as insulin, have receptors that are enzymes (eg, protein tyrosine kinases) and act in a neurotransmitter-like fashion. Stimulation of G protein–coupled receptors produces slower responses than stimulation of ligand-gated channels (Chapter 4). Moreover, many of the actions mediated by G proteins and second messengers alter the response properties of neurons resulting in “modulation” rather than simple excitation or inhibition. More than 100 neuropeptide transmitters are known; they play diverse roles in the nervous system, including regulation of sleep and arousal, emotion, reward, feeding and energy balance, pain and analgesia, and learning and memory. However, much remains to be discovered about neuropeptide function because selective agonists and antagonists are still lacking for many neuropeptide receptors.

This chapter focuses on general aspects of neuropeptide synthesis, release, and action, and describes several neuropeptides in more detail to illustrate these concepts. Several neuropeptides are discussed at greater length in other chapters; for example, hypothalamic peptides are considered in conjunction with the regulation of neuroendocrine function, stress responses, and feeding behavior (Chapter 10), and opioid peptides are considered in connection with pain (Chapter 11). Orexin (hypocretin) peptides are described in Chapter 6 because of their widely projecting organization in the nervous system and in Chapter 13 in relation to sleep and arousal.

Peptides are small proteins or polypeptides and thus composed of amino acids covalently linked by peptide bonds 7–1. The term neuropeptide is reserved for small proteins or polypeptides that act as neurotransmitters in the nervous system. Other signaling peptides in the nervous system, such as growth factors and cytokines (Chapter 8), are generally distinguished from the neuropeptides even though they may have overlapping functions. Neuropeptides are found within the central nervous system and in the periphery, including both the sympathetic and parasympathetic nervous systems (Chapter 9). In addition to their neurotransmitter functions, some neuropeptides (eg, oxytocin and vasopressin) are released directly into the blood by neurons and act as hormones (Chapter 10); others (eg, luteinizing hormone) act as hormones secreted by endocrine glands; yet others (eg, cholecystokinin) act within peripheral organs such as the digestive system. A sampling of neuropeptides is listed in 7–1.

Neuropeptide Synthesis

In contrast to small-molecule neurotransmitters, the synthesis of neuropeptides, like that of all proteins, requires the transcription of DNA into messenger RNA (mRNA) and translation of mRNA into protein 7–2. A striking aspect of neuropeptide synthesis is that many steps can be regulated so that a single gene can give rise to diverse neuropeptides in a tissue-specific manner.

Diversity can be generated by alternative splicing of primary RNA transcripts to produce different mRNAs (Chapter 4). Thus, for example, calcitonin and calcitonin gene–related peptide (CGRP) are generated in a tissue-specific fashion by alternative splicing of a single primary transcript to yield peptides with very different biologic actions. Calcitonin is produced in the parafollicular “C” cells of the thyroid gland and plays a central role in Ca²⁺ and phosphorus metabolism. CGRP is produced in neurons and, among its other actions, is a potent vasodilator, which has made its receptor a potential target for treating migraine (Chapter 20). The tachykinin family (the members of which share a C-terminal sequence) provides another example of alternative splicing of neuropeptide-encoding mRNAs. There are two genes that encode tachykinins in humans, preprotachykinin (PPT) A and B. PPT-A is alternatively spliced and, as a result, encodes three distinct prepropeptides that give rise to multiple peptides, including substance P and neurokinin A (NKA) 7–3. PPT-B gives rise to neurokinin B (NKB).

Following splicing, the mature mRNA is exported to the cytoplasm for translation. The initial protein product translated on the ribosome is not an active signaling molecule, but a precursor polypeptide (a prepropeptide) that undergoes processing that converts it into one or more neuropeptides. Prepropeptides contain an N-terminal signal sequence (or “pre” sequence) that directs the newly synthesized protein into the lumen of the rough endoplasmic reticulum (ER) and thus into the proper, regulated secretory pathway 7–2. The signal sequence is cleaved by a signal peptidase even before translation is completed, described as cotranslational processing. The removal of the signal sequence results in a propeptide that is released from the ribosome after translation is complete, transferred to the Golgi complex, and subsequently packaged within large dense core vesicles (LDCVs). Within the Golgi complex and within LDCVs, the propeptide undergoes additional posttranslational processing, which involves additional cleavages and covalent modifications.

Propeptide Processing

Different patterns of tissue-specific cleavage are another step by which a diversity of signaling peptides can be generated. Posttranslational processing can be illustrated by the cleavage and modification of proopiomelanocortin (POMC; 7–4), the precursor of several peptides with distinct biologic actions, including adrenocorticotropic hormone (ACTH), α-melanocyte-stimulating hormone (α-MSH; also called melanocortin), and β-endorphin.

The endoproteases, prohormone convertases 1 and 2 (PC1 and PC2), which make the initial cleavages within propeptides, do so at dibasic amino acid pairs (Lys–Arg, Lys–Lys, Arg–Arg, or Arg–Lys). The resulting peptides are further modified by exopeptidases, which remove the free N- and C-terminal Lys or Arg 7–2. In addition to the action of proteases, many neuropeptides are further modified. N-terminal acetylation of α-MSH significantly enhances its biologic activity; conversely, acetylation of β-endorphin reduces its activity. Peptides with a C-terminal glycine, such as α-MSH, may also undergo α-amidation.

There is an interesting and successful example of a peptide-processing enzyme serving as a drug target: inhibitors of angiotensin-converting enzyme (ACE), also known as peptidyl dipeptidase A, such as captopril, enalapril, and lisinopril, are widely used to treat hypertension. ACE cleaves the decapeptide angiotensin I to yield the far more active octapeptide, angiotensin II. ACE is also involved in inactivation of an additional neuropeptide, bradykinin. Because angiotensin II is a potent vasoconstrictor and bradykinin is a vasodilator, ACE inhibitors decrease blood pressure. This example may not be generalizable to enzymes such as PC1 and PC2, because they act on a very large number of neuropeptides and would thus lack specificity.

Storage and Release

Neuropeptides are processed and stored in LDCVs, which are assembled in the Golgi apparatus and transported to the synapse. In contrast, glutamate, γ-aminobutyric acid (GABA), and other small-molecule neurotransmitters are stored in small clear synaptic vesicles (SSVs), which can be assembled in synaptic terminals. In central neurons, monoamines (eg, norepinephrine, dopamine, and serotonin) are largely stored in SSVs, although they are stored in LDCVs in adrenal chromaffin cells and various cultured cell lines. LDCVs containing peptides commonly coexist with SSVs containing small-molecule neurotransmitters. A few examples of neuropeptides and small-molecule neurotransmitters that are colocalized to the same terminals are given in 7–3. There are also examples of multiple peptides being colocalized within the same neurons. One striking example is some neurons of the supraoptic nucleus of the hypothalamus that may contain oxytocin, enkephalin, dynorphin, cholecystokinin, and cocaine- and amphetamine-regulated transcript (CART).

Although LDCVs and SSVs can be colocalized to the same terminals, their contents are released by different mechanisms, and indeed in response to different types of stimulation. SSVs are clustered in active zones abutting the synaptic cleft (Chapter 3). LDCVs appear to be excluded from these zones and are found at greater distances from the synapse 7–5. The exocytosis of SSVs occurs in response to large, transient increases in intracellular Ca²⁺, whereas the exocytosis of LDCVs requires increases in Ca²⁺ of lesser magnitude but longer duration, so that the Ca²⁺ can diffuse in adequate concentrations to reach these vesicles. Typically, a single action potential can cause SSVs to fuse with the cell membrane, but a rapid train of action potentials may be required to trigger release of neuropeptides from LDCVs. Thus, specific patterns of electrical activity in a neuron may lead to the preferential release of a neuropeptide or a small-molecule neurotransmitter, or may prompt the release of both. Because neuropeptides tend to be released under conditions of sustained activity, they may regulate strongly stimulated synapses by providing positive or negative feedback.

Long-Distance Signaling by Neuropeptides

Another difference between neuropeptide signaling and small-molecule neurotransmitters relates to the diffusion distances after release. When most small-molecule neurotransmitters are released into a synapse, molecules that do not bind a receptor are rapidly cleared by a transporter or, in the case of acetylcholine rapidly metabolized by a highly active enzyme, acetylcholinesterase. Neuropeptides are not rapidly cleared from the synapse. Their action is terminated by endopeptidases and exopeptidases (different from those involved in synthesis) that cleave peptide bonds. These peptidases reside on extracellular membranes, and, in contrast to acetylcholinesterase, their concentration and activity is such that peptides can diffuse relatively large distances in the nervous system. In fact, some neuropeptide receptors are found at significant distances from release sites.

As noted, most neuropeptide receptors belong to the superfamily of G protein–coupled receptors. Like small-molecule neurotransmitters, a given neuropeptide may have several receptor subtypes, as indicated in 7–4. However, in cases that have been studied, receptors for neuropeptides bind their ligands with greater affinities than do the receptors for small-molecule transmitters; for example, acetylcholine binds to its receptors in the 100 μM to 1 mM range, whereas some neuropeptides can bind with nanomolar affinity. This high affinity means that neuropeptides can act at low concentrations, which is what would be expected if they often diffuse for great distances.

Neuropeptide Receptor Types and Subtypes

The interactions between neuropeptides and their receptors are quite complex. Imagine a small molecule such as norepinephrine interacting with the binding pocket of its receptor. Only so many atoms of the norepinephrine molecule can possibly interact ionically or sterically with the receptor; thus, the molecular modeling of feasible interactions is relatively straightforward. Now envision a peptide such as neuropeptide Y (NPY), which is 36 amino acids in length. How does such a large molecule fit into the binding pocket of a G protein–coupled receptor? Which conformation has the highest affinity for the receptor, and which amino acid residues are critical for binding? Most importantly from a pharmacologic point of view, how might nonpeptide analogs be developed to mimic or antagonize NPY binding? All of these questions are difficult to answer and currently are the focus of investigation. Many, perhaps most, available ligands for neuropeptide receptors are modified peptide analogs, and peptides are far from ideal as potential drugs. Synthetic peptides are subject to proteolysis by ubiquitous peptidases, and, more importantly, they generally cannot cross the blood–brain barrier. Hence, nonpeptide agents are essential if neuropeptide systems are to be exploited for the treatment of central nervous system disorders.

Like small-molecule neurotransmitters, neuropeptides may have multiple receptor subtypes 7–4 with distinct patterns of expression. In rare cases, such as norepinephrine and epinephrine, small-molecule neurotransmitters can share receptors. This is more often the case for neuropeptides, such as the receptors of corticotropin-releasing factor (CRF; also known as corticotropin-releasing hormone [CRH]). The CRF₁ receptor shows high affinity for both CRF and a related peptide urocortin, whereas the CRF₂ receptor binds preferentially to urocortin. Each of the melanocortin receptor family members, termed MC_1–5, is activated by different peptides derived from POMC, with ACTH, α-MSH, and γ-MSH displaying varying degrees of potency. MC₄ receptors also can be antagonized by a distinct peptide called agouti-related peptide (ARP); indeed, MC₄ was the first receptor determined to have both endogenous agonists and an antagonist. These MC₄ ligands are involved in the regulation of feeding behavior (Chapter 10).

Although neuropeptide receptors tend to be localized in synapses, they also have been detected on the plasma membranes of axons, cell bodies, and dendrites. In fact, some of these receptor subtypes are hypothesized to reside primarily in extrasynaptic locations. This situation has been described for certain other G protein–coupled receptors, including those for some small-molecule neurotransmitters, such as dopamine. In contrast, the ligand-gated channels that subserve fast excitatory and inhibitory neurotransmission are expressed solely at synapses.

Neuropeptide receptors, like most G protein–coupled receptors, undergo internalization after sustained binding to a ligand; subsequently, the internalized receptors are either recycled to the plasma membrane or degraded (Chapters 4 and 6). For several neuropeptide receptors, such as neurotensin receptors, internalization may lead to transport from the synapse to the cell body. Interestingly, neuropeptide-receptor complexes have been detected near the cell nucleus.

Neuropeptide Functions

The functions of most small-molecule neurotransmitters have been discovered partly because of the availability of selective agonists and antagonists. Such agents have enabled investigators to ascertain the cellular and behavioral effects of these transmitters by mimicking or blocking their actions. In contrast, a lack of pharmacologic tools has been a major handicap in functional investigations of neuropeptide systems. Unfortunately, only a small number of peptide agonists and antagonists that cross the blood–brain barrier are known.

It has also been difficult to interpret measurements of neuropeptide concentration in nervous tissue. When neuronal activity is inhibited, for example, tissue levels of a neuropeptide may increase because synthesis may not be inhibited. This is in contrast, for example, to catecholamine biosynthesis where accumulation of end products inhibits the rate-limiting enzyme, tyrosine hydroxylase (Chapter 6). Conversely, sustained neuronal activation can lead to release and thus depletion of peptide stores. In vivo microdialysis has been used to overcome this challenge by measuring extracellular levels of a neuropeptide, for example, CRF and substance P, following some stimulation.

Because of the limited pharmacologic tools and the obstacles to neuropeptide measurement, studies of neuropeptide function have relied to a great degree on the construction of gene knockouts and other genetically engineered mice. While often difficult to interpret, especially, in mouse lines lacking the peptides or receptors of interest throughout development, such tools have significantly advanced our understanding of function, as will be illustrated in some of the cases described below.

Opioid Peptides

Primarily because of their potent analgesic properties, but also because of their antitussive and antidiarrheal effects, opiates have long ranked among the most important drugs in the pharmacopoeia. Indeed, of the drugs commonly used today, morphine is one of a small number that were available in the 19th century. The medical importance of opiate analgesics (Chapter 11), combined with their addictive liabilities (Chapter 16), has produced much research in an unsuccessful attempt to develop potent opiate analgesics that are also nonaddictive. Products of this research include the discovery of lipophilic, small-molecule opioid receptor antagonists, such as naloxone and naltrexone, which have been critical tools for investigating the physiology and behavioral actions of opiates. In addition, naloxone is effective in the treatment of opiate overdoses, and naltrexone, which is longer-acting, is used in the long-term treatment of opiate addiction (by blocking binding of opiates to their receptors) and of alcoholism (presumably by blocking endogenous opioid peptides that contribute to rewarding responses to alcohol). More significantly, this research motivated the discoveries of opioid receptors and endogenous opioid peptides in the nervous system 7–1.

Opioid peptides are encoded by three distinct genes 7–6. These precursors include POMC, from which the opioid peptide β-endorphin and several nonopioid peptides are derived, as discussed earlier; proenkephalin, from which met-enkephalin and leu-enkephalin are derived; and prodynorphin, which is the precursor of dynorphin and related peptides. Although they come from different precursors, opioid peptides share significant amino acid sequence identity. Specifically, all of the well-validated endogenous opioids contain the same four N-terminal amino acids (Tyr–Gly–Gly–Phe), followed by either Met or Leu (see 7–6).

There are three opioid receptors, μ, κ, and δ 7–4, and a fourth related receptor (ORL-1 that binds nociceptin; see below). However, there is growing evidence that heterodimers form (eg, μ–δ dimers), yielding a receptor with distinct properties. As well, several isoforms of the μ receptor, generated by alternative splicing, have been shown to mediate distinct functions. The σ receptor, a single transmembrane-spanning protein that binds phencyclidine,¹ was once considered to be a possible fourth opioid receptor because it binds benzomorphan opiate drugs such as pentazocine. However, it is not blocked by opioid receptor antagonists, and instead binds diverse drugs that are unrelated to opioids. The functional significance of the σ receptor remains poorly understood.

The μ, κ, and δ opioid receptors are all linked to the G_i/o family of G proteins (Chapter 4). Among endogenous opioid peptides, β-endorphin binds preferentially to μ receptors. Two other brain peptides, endomorphin-1 and -2, which lack the signature opioid peptide sequence shown in 7–6 (Tyr–Gly–Gly–Phe), bind selectively and with high affinity to μ receptors as well. Enkephalins bind with high affinity to δ receptors and dynorphin peptides to κ receptors. However, opioid peptides do not bind exclusively to the receptors for which they have highest affinity; in vivo binding is also likely to be influenced by relative locations of released peptides and opioid receptors. Moreover, the complex pharmacology of opiate drugs suggests that posttranslational modifications and the aforementioned heterodimerization of μ and δ receptors create a far richer possibility for opiate drug and peptide binding than the three cloned receptors would initially suggest.

Morphine-like opiate drugs preferentially bind to μ receptors, which are concentrated in regions associated with descending analgesic pathways, such as the periaqueductal gray matter, rostroventral medulla, medial thalamus, and dorsal horn of the spinal cord (Chapter 11). Significantly, these receptors also reside in reward-related regions including the ventral tegmental area (VTA) of the midbrain and the nucleus accumbens, where they are responsible for the addictive effects of opiates (Chapter 16) and may play a more general role in the hedonic effects of natural rewards, such as food. In addition, μ receptors are expressed in the dorsal striatum and in the locus ceruleus (LC). In the LC, μ receptors mediate important aspects of opiate physical dependence and withdrawal (Chapter 16).

Enkephalins, rather than any clinically available drugs, are the molecules with the greatest affinity for δ opioid receptors. δ receptors are expressed not only in the dorsal horn of the spinal cord where they play a role in analgesia but also in many brain regions.

Some κ receptor agonists such as nalbuphine and butorphanol exert clinically useful analgesic effects acting via κ receptors (Chapter 11), but are also potent μ receptor antagonists. Pentazocine, also used as an analgesic, is a κ receptor agonist and a partial μ receptor agonist or weak antagonist. As such, these drugs may precipitate withdrawal if used as analgesics in individuals who are dependent on morphine or heroin. κ receptors are found in the dorsal horn of the spinal cord, in the dorsal striatum and nucleus accumbens, in deep cortical layers, and in many other brain regions. Even though they are analgesic, κ receptor agonists and dynorphin peptides may produce dysphoria rather than euphoria because they are expressed on the presynaptic terminals of dopamine neurons that project from the VTA to the nucleus accumbens and other forebrain regions. Because κ receptors are coupled via G_i/o to a K⁺ conductance that hyperpolarizes dopamine terminals, κ agonists decrease dopamine release. In contrast, μ receptors are expressed on inhibitory interneurons in the VTA that suppress firing of dopamine neurons. Morphine-like opiates acting via μ receptors thereby disinhibit dopamine neurons and stimulate dopamine release, an effect opposite to κ agonists.

Another peptide, alternatively termed nociceptin or orphanin FQ, binds to a G protein–coupled receptor termed the nociceptin receptor, also known as ORL1. The terminology derives from ORL1 having been an orphan receptor because it did not have a known ligand (hence the term orphanin). Nociceptin/orphanin F/Q is a hectadecapeptide closely related to dynorphin A. It is derived from the pronociceptin/orphanin FQ gene. Nociceptin/orphanin F/Q has been reported to have antiopioid effects, and thus pronociceptive functions at least in some experimental paradigms, raising the possibility that ORL1 antagonists may be analgesic. However, possible antinociceptive effects of nociceptin/orphanin F/Q are also under investigation.

Corticotropin-Releasing Factor

CRF (also called CRH) is a 41-amino-acid peptide that was first isolated in the search for a hypothalamic releasing factor that causes ACTH secretion from the anterior lobe of the pituitary gland. CRF shares this capability with vasopressin in many species, including humans (see below). It is synthesized by a subset of neurons in the paraventricular nucleus (PVN) of the hypothalamus. A subset of these neurons project to the median eminence from which CRF is released into the portal hypophyseal circulation, from which it acts on pituitary corticotrophs (these neuroendocrine functions are described in Chapter 10). CRF is not only delivered into the portal circulation, however; PVN neurons and other neurons, located elsewhere in brain, synthesize CRF and release it synaptically in the brainstem, cerebral cortex, central nucleus of the amygdala, and the bed nucleus of the stria terminalis (BNST), among other regions. CRF localized within the amygdala and BNST appears to play a role in anxiety and fear-related behavior (Chapter 15). Amygdala CRF is also important in mediating the negative emotional symptoms of withdrawal from most, and possibly all, addictive drugs (Chapter 16).

Two CRF receptors have been identified and cloned. CRF₁ receptors are expressed widely in the brain, while CRF₂ receptors exhibit a much narrower distribution. They are concentrated in the lateral septal nuclei of the forebrain. The endogenous ligand for CRF₂ is not CRF, but a related 40-amino-acid peptide, urocortin. Urocortin can exert potent hypotensive and anorexigenic effects. Other members of the CRF family are urocortin II and III, urotensin I (isolated from certain fish), and sauvagine (isolated from frog skin).

CRF₁ receptor antagonists have been studied extensively for use as potential anxiolytic and antidepressant drugs (Chapter 15). Despite a great deal of research, no large clinical trial has yet demonstrated clear efficacy. One of the challenges is the difficulty in generating brain-penetrant CRF₁ antagonists, as noted earlier in this chapter. However, it is also possible that CRF, released in several brain areas in response to stress, actually serves an adaptive function such that antagonism of CRF might not be beneficial. The case may be stronger for CRF₁ antagonists being useful in the treatment of drug addiction withdrawal states (Chapter 16), but convincing clinical trials have yet to be performed. One might be concerned about the neuroendocrine side effects of CRF₁ antagonists—in producing a dangerous syndrome of cortisol deficiency (Chapter 10); however, administration of these drugs appears safe perhaps because vasopressin has an independent ability to release ACTH. Interest remains in the exploration of CRF₂ antagonists, although little clinical validation is currently available.

Oxytocin and Vasopressin

These closely related nonapeptides 7–7 have both neuroendocrine and more purely neural functions. Oxytocin and vasopressin are synthesized in the PVN and supraoptic nucleus of the hypothalamus and transported within the long axons of these neurons for storage and ultimately release into the blood as neurohormones. Axons of the magnocellular (large) neurons of these two hypothalamic nuclei project to the posterior pituitary (neurohypophysis) where the two peptides are stored in presynaptic terminals and released into the systemic circulation. Vasopressin acts in the distal tubules of the kidney to facilitate water reabsorption (it is also called antidiuretic hormone [ADH]), and oxytocin stimulates uterine contraction at parturition and milk letdown during nursing. Axons from the parvocellular (small) neurons of the PVN and supraoptic nucleus also project to the portal hypophyseal vessels. Vasopressin can act in the anterior pituitary, like CRF, to stimulate synthesis and release of ACTH, as stated above. The neuroendocrine functions of these peptides are described in Chapter 10; however, they also act within the brain.

Vasopressin has three receptors 7–4, V_1a and V_1b (once called V₃), and V₂. All are G protein–linked, V_1a and V_1b to G_q/11 and thus to the phosphatidylinositol second messenger system, and V₂ to G_s and to adenylyl cyclase (Chapter 4). A large body of research has implicated vasopressin and its V_1a receptor in the regulation of affiliative behavior 7–2. The V_1b receptor is located in the anterior pituitary where it stimulates ACTH secretion in concert with CRF. It is also expressed in the brain. V_1b knockout mice exhibit reduced aggression. Oxytocin has a single receptor that is linked to G_q/11.

Overall vasopressin and oxytocin play important roles in the ability of rodents to recognize previously encountered individuals of the same species and regulate social behavior through actions in diverse regions of the brain. Oxytocin appears to increase male–female bonding after mating and mother–infant bonding. In the amygdala, vasopressin acting via V_1a receptors may increase anxiety. Acting in a different region of the amygdala, oxytocin may decrease fear and anxiety and may promote prosocial behaviors by inhibiting avoidance and decreasing aggression. Is there any relevance for humans? Oxytocin can be delivered to humans via nasal spray, but resulting levels in CSF have not been adequately established. In a functional MRI experiment, oxytocin decreased amygdala activation in human volunteers in response to fearful faces or scenes 7–8. In several human experiments, oxytocin spray increased trusting behavior compared with a placebo spray. These results in humans are quite early, but suggest a role for oxytocin in regulating social interactions. Indeed, clinical trials are under way to evaluate the utility of oxytocin in treating autism (Chapter 14).

Tachykinins

Substance P, NKA (previously known as substance K), NKB, and neuropeptide K are members of the tachykinin family, all of which share the C-terminal sequence Phe–X–Gly–Leu–Met–NH₂. Substance P, NKA, and neuropeptide K are encoded by the PPT-A gene, and are produced by alternative splicing 7–3. The PPT-B gene encodes NKB. The three known tachykinin receptors, all of which are G_q/11-coupled, are designated NK₁, NK₂, and NK₃ 7–4. Substance P binds with highest affinity to the NK₁ receptor, NKA preferentially binds to the NK₂ receptor, and NKB binds somewhat selectively to the NK₃ receptor.

Of the tachykinins, substance P has gained the most attention. It was first discovered in 1931 in extracts of brain and gut. It is expressed in the dorsal horn of the spinal cord, amygdala, medulla, hypothalamus, substantia nigra, cerebral cortex, and striatum. In the latter region, it is colocalized with GABA and dynorphin in the striatonigral neurons that make up the “direct” striatothalamocortical pathway of great interest in Parkinson disease (Chapters 14 and 18).

Substance P was long thought to be a critically important neurotransmitter carrying pain signals. Primary afferent nociceptors that synapse in the dorsal horn of the spinal cord (Chapter 11) express a complex array of neuropeptides, of which the most abundant include substance P, NKA, and CGRP. Substance P is colocalized with glutamate in the synaptic terminals of a class of nociceptors called C fibers. NK₁ receptors are found in abundance in the dorsal horn. Accordingly, NK₁ receptors were considered an important target for the development of nonopioid analgesics. The first clues that this strategy might not succeed came from mice in which the PPT-A or NK₁ receptor gene had been knocked out; the phenotypes did not show substantial alterations in pain threshold. More definitively, potent and selective NK₁ antagonist drugs did not have significant effects in clinical tests on humans. Based on NK₁ receptor expression in the amygdala and on animal models of distress, NK₁ antagonists have also been studied as possible antidepressants. To date, most clinical trials have shown no efficacy. The main clinical use of NK₁ antagonists has been in blocking the nausea and vomiting caused by cancer chemotherapy. These drugs, such as aprepitant, are likely exerting their actions in the medullary vomiting centers.

Substance P is released not only in the dorsal horn but also in retrograde fashion from the free nerve endings of nociceptive neurons. This retrograde release contributes to the phenomenon of neurogenic inflammation; other peptides such as bradykinin also contribute (Chapter 11).

Neurotensin

Neurotensin is a 13-amino-acid peptide derived from a larger precursor that also contains neuromedin N, a related 6-amino-acid peptide. Neurotensin is expressed in the brain, adrenal gland, and gut in slightly different forms that illustrate tissue-specific posttranslational processing. Its C terminus contains one of three different Lys–Arg sequences, which are differentially cleaved depending on the tissue in which neurotensin is processed. In the brain, the precursor gives rise to both neurotensin and neuromedin N, whereas in the adrenal gland, neurotensin, a larger form of neuromedin N, and a larger form of neurotensin sequence are produced. In the gut, the most common products are neurotensin and large neuromedin N. Central administration of neurotensin produces hypothermia and analgesia.

There are three neurotensin receptors 7–4. Two of these, NTS₁ and NTS₂, are G protein–coupled receptors 7–4. NTS₃ is a single membrane-spanning receptor analogous to type I amino acid receptors, exceptional for neuropeptide signaling. Neurotensin mRNA is induced in striatopallidal neurons of the striatum by D₂ receptor antagonists, including antipsychotic drugs, and in striatonigral neurons by psychostimulant drugs such as cocaine and amphetamine. Therefore, it has been hypothesized that neurotensin influences dopamine signaling and perhaps contributes to the plasticity induced by drugs that act on dopamine systems in the brain. These observations, however, have not yet led to new treatments for psychotic disorders or drug addiction.

Orexins (Hypocretins)

Orexin A and B (also referred to as hypocretin 1 and 2), products of a single gene, were discovered by convergent approaches in rat brain. One group of investigators found a peptide that stimulated feeding (thus the name “orexin”), and the other group was searching for ligands for “orphan” hypothalamic G protein–linked receptors (thus the alternative name, hypocretin). The two known orexin receptors, OX₁ and OX₂, are coupled via G_q and G_i/o, respectively. There are reports of OX₂ coupling to G_q as well. Soon after the peptides were discovered, a loss-of-function mutation in the OX₂ receptor was found to be the cause of canine narcolepsy (Chapter 13), and orexin peptide knockout mice were shown to exhibit a narcolepsy-like syndrome. Subsequently, humans with narcolepsy were found to have presumed autoimmune destruction of orexin neurons and depletion of orexin from cerebrospinal fluid. These discoveries have stimulated great interest in the development of orexin receptor ligands to modulate sleep and alertness. The diverse functions of orexin peptides, which in addition to sleep and arousal include feeding and reward, result from an anatomic organization that is reminiscent of monoamine neurotransmitters (Chapter 6). Orexin peptides are synthesized solely by neurons in the lateral and posterior hypothalamus and project to and activate monoaminergic and cholinergic neurons, among many other neuronal types 6–25. Orexins are discussed further in Chapters 6 and 13.

Neuropeptide Y

NPY is the best known of a related peptide family that also includes peptide YY (PYY) and pancreatic polypeptide (PP). NPY gains its name for its N- and C-terminal tyrosines (“Y” is the single letter symbol for tyrosine). NPY is the most abundant neuropeptide in cerebral cortex. It also is concentrated in the dorsal horn of the spinal cord and arcute nucleus of hypothalamus. It is colocalized with norepinephrine in both the LC and sympathetic nervous system 7–3. NPY is a potent stimulator of feeding. Leptin, which inhibits feeding, acts partly by inhibiting the synthesis and release of hypothalamic NPY. Nonetheless, mice lacking NPY continue to feed normally, underscoring the complex interactions of hypothalamic systems that control feeding and energy balance (Chapter 10). In the periphery, NPY sensitizes smooth muscle to the effects of norepinephrine, resulting in a potent vasoconstrictor effect.

NPY, PYY, and PP bind to a group of six G protein–linked receptors, designated Y₁ to Y₆ 7–4 with varying selectivity; all couple to G_i/o. These receptors display marked region-specific distributions in brain and are located on both postsynaptic and presynaptic sites. Activation of the Y₁ receptor is thought to cause a decrease in anxiety-like behavior, acting, perhaps, in the amygdala. This hypothesis has generated interest in exploring the use of Y₁ agonists for the treatment of anxiety disorders. In contrast, activation of the Y₅ receptor has been proven to stimulate feeding, acting presumably within the hypothalamus; accordingly, Y₅ antagonists may be useful medications for the treatment of obesity. Applications of NPY pharmacology have not yet borne fruit in the clinic.

Although less is known about neuropeptides than about many small-molecule neurotransmitters, our understanding of the former is expanding rapidly based on the use of genetically engineered mice and the slowly growing catalog of selective agonists and antagonists. Given the modulatory influences of many peptides on functions such as sleep, arousal, feeding, anxiety, stress responses, reward, social interactions, and learning and memory, peptides and their receptors remain a potential storehouse of therapeutic targets that remain largely unexploited.