Synaptic physiology is a rapidly expanding, complex field that cannot be covered in detail in this book. However, it is appropriate to summarize information about the principal neurotransmitters and their receptors.
EXCITATORY & INHIBITORY AMINO ACIDS
Glutamate is the main excitatory transmitter in the brain and spinal cord and has been calculated to be responsible for 75% of the excitatory transmission in the CNS. There are two distinct pathways involved in the synthesis of glutamate (Figure 7–1). In one pathway, α-ketoglutarate produced by the Krebs cycle is converted to glutamate by the enzyme GABA transaminase (GABA-T). In the second pathway, glutamate is released from the nerve terminal into the synaptic cleft by Ca2+-dependent exocytosis and transported via a glutamate reuptake transporter into glia, where it is converted to glutamine by the enzyme glutamine synthetase (Figure 7–4). Glutamine then diffuses back into the nerve terminal where it is hydrolyzed back to glutamate by the enzyme glutaminase. In addition to uptake of released glutamate into glia, the membrane transporters also return glutamate directly into the nerve terminal. Within glutamatergic neurons, glutamate is highly concentrated in synaptic vesicles by a vesicular glutamate transporter.
Biochemical events at a glutamatergic synapse. Glutamate (Glu) released into the synaptic cleft by Ca2+-dependent exocytosis. Released Glu can act on ionotropic and G-protein–coupled receptors on the postsynaptic neuron. Synaptic transmission is terminated by the active transport of Glu via by a Na+-dependent glutamate transporters located on membranes of the presynaptic terminal [Gt(n)] and glia [Gt(g)]. In glia, Glu is converted to glutamine (Gln) by the enzyme glutamine synthetase; Gln then diffuses into the nerve terminal where it is hydrolyzed back to Glu by the enzyme glutaminase. In the nerve terminal, Glu is highly concentrated in synaptic vesicles by a vesicular glutamate transporter.
Glutamate acts on both ionotropic and metabotropic receptors in the CNS (Figure 7–4). There are three subtypes of ionotropic glutamate receptors, each named for its relatively specific agonist. These are the AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionate), kainate (kainate is an acid isolated from seaweed), and NMDA (N-methyl-D-aspartate) receptors. Table 7–2 summarizes some of the major properties of these receptors. Ionotropic glutamate receptors are tetramers composed of different subunits whose helical domains span the membranes three times and a short sequence that forms the channel pore. Four AMPA (GluR1–GluR4), five kainate (GluR5–GluR7, KA1, KA2), and six NMDA (NR1, NR2A–NR2D) subunits have been identified, and each is coded by a different gene.
The release of glutamate and its binding to AMPA or kainate receptors primarily permits the influx of Na+ and the efflux of K+, accounting for fast excitatory postsynaptic potential (EPSP). Most AMPA receptors have low Ca2+ permeability, but the absence of certain subunits in the receptor complex at some sites allows for the influx of Ca2+, which may contribute to the excitotoxic effect of glutamate (see Clinical Box 7–1).
Activation of the NMDA receptor permits the influx of relatively large amounts of Ca2+ along with Na+. When glutamate is in excess in the synaptic cleft, the NMDA receptor-induced influx of Ca2+ into neurons is the major basis for the excitotoxic actions of glutamate. The NMDA receptor is unique in several ways (Figure 7–5). First, glycine facilitates its function by binding to the receptor. In fact, glycine binding is essential for the receptor to respond to glutamate. Second, when glutamate binds to the NMDA receptor, it opens, but at normal membrane potentials, the channel is blocked by extracellular Mg2+. This block is removed only when the neuron containing the receptor is partially depolarized by the activation of adjacent AMPA and kainate receptors. Third, the excitatory postsynaptic potential induced by activation of NMDA receptors is slower than that elicited by activation of the AMPA and kainate receptors.
Diagrammatic representation of the NMDA receptor. When glycine and glutamate bind to the receptor, the closed ion channel (left) opens, but at the resting membrane potential, the channel is blocked by Mg2+ (right). This block is removed if partial depolarization is produced by other inputs to the neuron containing the receptor, and Ca2+ and Na+ enter the neuron. Blockade can also be produced by the drug dizocilpine maleate (MK-801).
CLINICAL BOX 7–1 Excitotoxins
Glutamate is usually cleared from the brain’s extracellular fluid by Na+-dependent uptake systems in neurons and glia, keeping only micromolar levels of the chemical in the extracellular fluid despite millimolar levels inside neurons. However, excessive levels of glutamate occur in response to ischemia, anoxia, hypoglycemia, or trauma. Glutamate and some of its synthetic agonists are unique in that when they act on neuronal cell bodies, they can produce so much Ca2+ influx that the neurons die. This is the reason why microinjection of these excitotoxins is used in research to produce discrete lesions that destroy neuronal cell bodies without affecting neighboring axons. Evidence is accumulating that excitotoxins play a significant role in the damage done to the brain by a stroke. When a cerebral artery is occluded, the cells in the severely ischemic area die. The surrounding partially ischemic cells may survive but lose their ability to maintain the transmembrane Na+ gradient. The elevated levels of intracellular Na+ prevent the ability of astrocytes to remove glutamate from the brain’s extracellular fluid. Therefore, glutamate accumulates to the point that excitotoxic damage and cell death occurs in the penumbra, the region around the completely infarcted area. In addition, excessive glutamate receptor activation may contribute to the pathophysiology of some neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson disease, and Alzheimer disease. THERAPEUTIC HIGHLIGHTS
Riluzole is a voltage-gated channel blocker that may antagonize NMDA receptors. It has been shown to slow the progression of impairment and modestly improve the life expectancy of patients with ALS. Another NMDA receptor antagonist memantine has been used to slow the progressive decline in patients with Alzheimer disease. A third NMDA receptor antagonist, amantadine, in conjunction with levodopa, has been shown to improve function in patients with Parkinson disease.
Essentially all neurons in the CNS have both AMPA and NMDA receptors. Kainate receptors are located presynaptically on GABA-secreting nerve endings and postsynaptically at various sites, most notably in the hippocampus, cerebellum, and spinal cord. Kainate and AMPA receptors are found in glia as well as neurons, but NMDA receptors occur only in neurons. The concentration of NMDA receptors in the hippocampus is high, and blockade of these receptors prevents long-term potentiation, a long-lasting facilitation of transmission in neural pathways following a brief period of high-frequency stimulation. Thus, these receptors may well be involved in memory and learning (see Chapter 15).
Activation of the metabotropic glutamate receptors (mGluR) leads to either an increase in intracellular inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) levels or a decrease in intracellular cAMP levels (Table 7–2). There are eight known subtypes of mGluR. These receptors are located at both presynaptic (mGluR2-4, 6-8) and postsynaptic sites (mGluR1, 5), and they are widely distributed in the brain. They appear to be involved in the production of synaptic plasticity, particularly in the hippocampus and the cerebellum. Activation of presynaptic mGluR autoreceptors on neurons in the hippocampus limits the release of glutamate from these neurons. Knockout of the gene mGluR1 causes severe motor incoordination and deficits in spatial learning.
A characteristic of an excitatory synapse is the presence of a thickened area called a postsynaptic density (PSD) on the membrane of the postsynaptic neuron. This is a complex structure containing ionotropic glutamate receptors and signaling, scaffolding, and cytoskeletal proteins. The mGluR are located adjacent to the PSD.
Pharmacology of Glutamate Synapses
Table 7–2 shows some of the pharmacologic properties of various types of glutamate receptors, examples of the agonists that bind to these receptors, and some of the antagonists that prevent activation of the receptors. The clinical applications of drugs that modulate glutamatergic transmission are still in their infancy. This is because the role of glutamate as a neurotransmitter was discovered much later than that of most other small-molecule transmitters. It was not identified as a neurotransmitter until the 1970s, more than 50 years after the discovery of chemical neurotransmission. One area of drug development is intraspinal or extradural administration of NMDA receptor antagonists for the treatment of chronic pain.
GABA is the major inhibitory mediator in the brain and mediates both presynaptic and postsynaptic inhibition. GABA, which exists as β-aminobutyrate in the body fluids, is formed by decarboxylation of glutamate (Figure 7–1) by the enzyme glutamate decarboxylase (GAD), which is present in nerve endings in many parts of the brain. GABA is metabolized primarily by transamination to succinic semialdehyde and then to succinate in the citric acid cycle. GABA-T is the enzyme that catalyzes the transamination. In addition, there is an active reuptake of GABA via the GABA transporter. A vesicular GABA transporter (VGAT) transports GABA and glycine into secretory vesicles.
Three subtypes of GABA receptors have been identified: GABAA, GABAB, and GABAC (Table 7–2). The GABAA and GABAB receptors (Figure 7–6) are widely distributed in the CNS, whereas in adult vertebrates the GABAC receptors are found almost exclusively in the retina. The GABAA and GABAC receptors are examples of ionotropic receptors, activation of which allows the entry of Cl– into neurons to mediate fast inhibitory postsynaptic potentials (IPSP). The GABAB receptors are examples of GPCR that are linked via G-proteins to alter the influx of K+ and Ca2+; Gi inhibits adenylyl cyclase to open a K+ channel, and Go inhibits or delays Ca2+ influx. Activation of GABAB receptors mediates both presynaptic and slow postsynaptic inhibition.
Diagram of GABAA and GABAB receptors, showing their principal actions. Two molecules of GABA (squares) bind to the GABAA receptor to allow an influx of Cl− to mediate fast inhibitory postsynaptic potentials. One molecule of GABA binds to the GABAB receptor, which couples to the α-subunit of a G-protein; Gi inhibits adenylyl cyclase to open a K+ channel and Go inhibits Ca2+ influx.
The GABAA receptors are pentamers made up of various combinations of six α subunits, four β, four γ, one δ, and one ε. This endows them with considerably different properties from one location to another. However, most synaptic GABAA receptors have two α, two β, and one γ subunit (Figure 7–6). GABAA receptors on dendrites, axons, or somas often contain δ and ε subunits in place of the γ subunit. The GABAC receptors are relatively simple in that they are pentamers of three ρ subunits in various combinations.
There is a chronic low-level stimulation of GABAA receptors in the CNS that is aided by GABA in the interstitial fluid. This background stimulation cuts down on the “noise” caused by incidental discharge of the billions of neural units and greatly improves the signal-to-noise ratio in the brain.
Pharmacology of GABA Synapses
Table 7–2 shows some of the pharmacologic properties of GABA receptors, including examples of agonists that bind to receptors and some of the antagonists that prevent activation of these receptors. The increase in Cl– conductance produced by GABAA receptors is potentiated by benzodiazepines (eg, diazepam). Thus, these are examples of neuromodulators. These drugs have marked antianxiety activity and are also effective muscle relaxants, anticonvulsants, and sedatives. Benzodiazepines bind to α subunits of GABAA receptors. Barbiturates such as phenobarbital are effective anticonvulsants because they enhance GABAA receptor—mediated inhibition as well as suppress AMPA receptor—mediated excitation. The anesthetic actions of barbiturates (thiopental, pentobarbital, and methoxital) result from their actions as agonists at GABAA receptors as well as by acting as neuromodulators of GABA transmission. Regional variation in anesthetic actions in the brain seems to parallel the variation in subtypes of GABAA receptors. Other inhaled anesthetics do not act by increasing GABA receptor activity; rather, they act by inhibiting NMDA and AMPA receptors.
Glycine has both excitatory and inhibitory effects in the CNS. When it binds to NMDA receptors, it makes them more sensitive to the actions of glutamate. Glycine may spill over from synaptic junctions into the interstitial fluid and in the spinal cord; for example, it may facilitate pain transmission by NMDA receptors in the dorsal horn. However, glycine is also responsible in part for direct inhibition, primarily in the brainstem and spinal cord. Like GABA, it acts by increasing Cl– conductance. Its action is antagonized by strychnine. The clinical picture of convulsions and muscular hyperactivity produced by strychnine emphasizes the importance of postsynaptic inhibition in normal neural function. The glycine receptor responsible for inhibition is a Cl– channel. It is a pentamer made up of two subunits: the ligand-binding α subunit and the structural β subunit. There are three kinds of neurons that are responsible for direct inhibition in the spinal cord: neurons that secrete glycine, neurons that secrete GABA, and neurons that secrete both. Neurons that secrete only glycine have the glycine transporter GLYT2, those that secrete only GABA have GAD, and those that secrete glycine and GABA have both. This third type of neuron is of special interest because the neurons seem to have glycine and GABA in the same vesicles.
Acetylcholine is the transmitter at the neuromuscular junction, in autonomic ganglia, and in postganglionic parasympathetic nerve-target organ junctions and some postganglionic sympathetic nerve-target junctions (see Chapter 13). In fact, acetylcholine is the transmitter released by all neurons that exit the CNS (cranial nerves, motor neurons, and preganglionic neurons). Acetylcholine is also found in the basal forebrain complex (septal nuclei and nucleus basalis), which projects to the hippocampus and neocortex, and the pontomesencephalic cholinergic complex, which projects to the dorsal thalamus and forebrain (Figure 7–2). These systems may be involved in regulation of sleep-wake states, learning, and memory (see Chapters 14 and 15).
Acetylcholine is largely enclosed in small, clear synaptic vesicles in high concentration in the terminals of cholinergic neurons. It is synthesized in the nerve terminal from choline and acetyl-CoA by the enzyme choline acetyltransferase (ChAT) (Figure 7–1 and Figure 7–7). Choline used in the synthesis of acetylcholine is transported from the extracellular space into the nerve terminal via a Na+-dependent choline transporter (CHT). Following its synthesis, acetylcholine is transported from the cytoplasm into vesicles by a vesicle-associated transporter (VAT). Acetylcholine is released when a nerve impulse triggers the influx of Ca2+ into the nerve terminal.
Biochemical events at a cholinergic synapse. Choline is transported into the presynaptic nerve terminal by a Na+-dependent choline transporter (CHT), which can be blocked by the drug hemicholinium. Acetylcholine (ACh) is synthesized from choline and acetyl Co-A (AcCoA) by the enzyme choline acetyltransferase (ChAT) in the cytoplasm. ACh is then transported from the cytoplasm into vesicles by the vesicle-associated transporter (VAT) along with peptides (P) and adenosine triphosphate (ATP). This step can be blocked by the drug vesamicol. ACh is released from the nerve terminal when voltage-sensitive Ca2+ channels open, allowing an influx of Ca2+, which leads to fusion of vesicles with the surface membrane and expulsion of ACh and co-transmitters into the synaptic cleft. This process involves synaptosome-associated proteins (SNAPs) and vesicle-associated membrane proteins (VAMPs) and can be prevented by the drug botulinum toxin. The released ACh can act on muscarinic G-protein—coupled receptors on the postsynaptic target (eg, smooth muscle) or on nicotinic ionotropic receptors in autonomic ganglia or the endplate of skeletal muscle (not shown). In the synaptic junction, ACh is readily metabolized by the enzyme acetylcholinesterase. Autoreceptors and heteroreceptors on the presynaptic nerve ending modulate neurotransmitter release.
Acetylcholine must be rapidly removed from the synapse if repolarization is to occur. The removal occurs by way of hydrolysis of acetylcholine to choline and acetate, a reaction catalyzed by the enzyme acetylcholinesterase in the synaptic cleft. This enzyme is also called true or specific cholinesterase. Its greatest affinity is for acetylcholine, but it also hydrolyzes other choline esters. Acetylcholinesterase molecules are clustered in the postsynaptic membrane of cholinergic synapses. Hydrolysis of acetylcholine by this enzyme is rapid enough to explain the observed changes in Na+ conductance and electrical activity during synaptic transmission. There are a variety of cholinesterases in the body that are not specific for acetylcholine. One found in plasma is capable of hydrolyzing acetylcholine but has different properties from acetylcholinesterase. It is called pseudocholinesterase. The plasma moiety is partly under endocrine control and is affected by variations in liver function.
Acetylcholine receptors are divided into two main types on the basis of their pharmacologic properties. Muscarine, the alkaloid responsible for the toxicity of toadstools, mimics the stimulatory action of acetylcholine on smooth muscle and glands. These actions of acetylcholine are called muscarinic actions, and the receptors involved are muscarinic cholinergic receptors. In sympathetic ganglia and skeletal muscle, nicotine mimics the stimulatory actions of acetylcholine. These actions of acetylcholine are called nicotinic actions, and the receptors involved are nicotinic cholinergic receptors. Nicotinic receptors are subdivided into those found in muscle at the neuromuscular junction (NM) and those found in the CNS and autonomic ganglia (NN). Both muscarinic and nicotinic acetylcholine receptors are also found within the brain.
The nicotinic acetylcholine receptors are members of a superfamily of ligand-gated ion channels (ionotropic receptors) that also includes the GABAA and glycine receptors and some of the glutamate receptors. Each nicotinic cholinergic receptor is made up of five subunits that form a central channel which, when the receptor is activated, permits the passage of Na+ and other cations. The five subunits come from several types designated as α, β, γ, δ, and ε that are each coded by different genes. The NM receptor is comprised of two α, one β, one δ, and either one γ or one ε subunit (Figure 7–8). The NN receptors are comprised of only α and β subunits. Each α subunit has a binding site for acetylcholine, and binding of an acetylcholine molecule to each of them induces a conformational change in the protein so that the channel opens. This increases the conductance of Na+, and the resulting influx of Na+ produces a depolarizing potential. A prominent feature of neuronal nicotinic cholinergic receptors is their high permeability to Ca2+. Many of the nicotinic cholinergic receptors in the brain are located presynaptically on glutamate-secreting axon terminals, and they facilitate the release of this transmitter.
Three-dimensional model of the nicotinic acetylcholine-gated ion channel. The receptor–channel complex consists of five subunits, all of which contribute to forming the pore. When two molecules of acetylcholine bind to portions of the α-subunits exposed to the membrane surface, the receptor—channel changes conformation. This opens the pore in the portion of the channel embedded in the lipid bilayer, and both K+ and Na+ flow through the open channel down their electrochemical gradient. (Reproduced with permission from Kandel ER, Schwartz JH, Jessell TM [editors]: Principles of Neural Science, 4th ed. New York, NY: McGraw-Hill; 2000.)
There are five types of muscarinic cholinergic receptors (M1–M5), which are encoded by five separate genes. These are metabotropic receptors that are coupled via G-proteins to adenylyl cyclase, K+ channels, and/or phospholipase C (Table 7–2). M1, M4, and M5 receptors are located in the CNS; M2 receptors are in the heart, M3 are on glands and smooth muscle. M1 receptors are also located on autonomic ganglia where they can modulate neurotransmission.
Pharmacology of Cholinergic Synapses
Table 7–2 shows some of the major agonists that bind to cholinergic receptors as well as some of the cholinergic receptor antagonists. Figure 7–7 also shows the site of action of various drugs that alter cholinergic transmission. For example, hemicholinium blocks the choline transporter that moves choline into the nerve terminal, and vesamicol blocks the VAT that moves acetylcholine into the synaptic vesicle. Also, botulinum toxin prevents the release of acetylcholine from the nerve terminal.
The chemical transmitter present at most sympathetic postganglionic endings is norepinephrine. It is stored in the synaptic knobs of the neurons that secrete it in characteristic small vesicles that have a dense core (granulated vesicles). Norepinephrine and its methyl derivative, epinephrine, are also secreted by the adrenal medulla (see Chapter 20), but epinephrine is not a mediator at postganglionic sympathetic endings. As discussed in Chapter 6, each sympathetic postganglionic neuron has multiple varicosities along its course, and each of these varicosities appears to be a site at which norepinephrine is secreted.
There are also norepinephrine-secreting and epinephrine-secreting neurons in the brain. Norepinephrine-secreting neurons are properly called noradrenergic neurons, although the term adrenergic neurons is also applied. However, the latter term should be reserved for epinephrine-secreting neurons. The cell bodies of the norepinephrine-containing neurons are located in the locus coeruleus and other medullary and pontine nuclei (Figure 7–2). From the locus coeruleus, the axons of the noradrenergic neurons descend into the spinal cord, enter the cerebellum, and ascend to innervate the paraventricular, supraoptic, and periventricular nuclei of the hypothalamus, the thalamus, the basal telencephalon, and the entire neocortex. The action of norepinephrine in these regions is primarily as a neuromodulator.
Biosynthesis & Release of Catecholamines
The principal catecholamines found in the body (norepinephrine, epinephrine, and dopamine) are formed by hydroxylation and decarboxylation of the amino acid tyrosine (Figure 7–1 and Figure 7–9). Some of the tyrosine is formed from phenylalanine, but most is of dietary origin. Phenylalanine hydroxylase is found primarily in the liver (see Clinical Box 7–2). Tyrosine is transported into catecholamine-secreting neurons via a Na+-dependent carrier. It is converted to dihydroxy-phenylalanine (dopa) and then to dopamine in the cytoplasm of the cells by tyrosine hydroxylase and dopa decarboxylase, respectively. The decarboxylase is also called amino acid decarboxylase. The rate-limiting step in synthesis of catecholamines is the conversion of tyrosine to dopa. Tyrosine hydroxylase is subject to feedback inhibition by dopamine and norepinephrine, thus providing internal control of the synthetic process.
Biochemical events at a noradrenergic synapse. Tyrosine (Tyr) is transported into the noradrenergic nerve terminal by a Na+-dependent carrier (A). The steps involved in the conversion of Tyr to dopamine and dopamine to norepinephrine (NE) are described in Figure 7–1. Metyrosine blocks the action of tyrosine hydroxylase (TH), the rate-limiting step in the production of catecholamines. Dopamine is transported from the cytoplasm into the vesicle by the vesicular monoamine transporter (VMAT), which can be blocked by the drug reserpine. NE and other amines can also be carried by VMAT. Dopamine is converted to NE in the vesicle. An action potential opens voltage-sensitive Ca2+ channels to allow an influx of Ca2+, and the vesicles then fuse with the surface membrane to trigger expulsion of NE along with peptides (P) and adenosine triphosphate (ATP). This process involves synaptosome-associated proteins (SNAPs) and vesicle-associated membrane proteins (VAMPs); it can be blocked by drugs such as guanethidine and bretylium. NE released into the nerve terminal can act on G-protein—coupled receptors on the postsynaptic neuron or neuroeffector organ (eg, blood vessels). NE can also diffuse out of the cleft or be transported back into the nerve terminal by the norepinephrine transporter (NET). NET can be blocked by cocaine and tricyclic antidepressants. Autoreceptors and heteroreceptors on the presynaptic nerve ending modulate neurotransmitter release.
CLINICAL BOX 7–2 Phenylketonuria
Phenylketonuria (PKU) is an example of an inborn error of metabolism. PKU is characterized by severe mental deficiency and the accumulation in the blood, tissues, and urine of large amounts of phenylalanine and its keto acid derivatives. It is usually due to decreased function resulting from mutation of the gene for phenylalanine hydroxylase. This gene is located on the long arm of chromosome 12. Catecholamines are still formed from tyrosine, and the cognitive impairment is largely due to accumulation of phenylalanine and its derivatives in the blood. The condition can also be caused by tetrahydrobiopterin (BH4) deficiency. Because BH4 is a cofactor for tyrosine hydroxylase and tryptophan hydroxylase, as well as phenylalanine hydroxylase, PKU cases due to BH4 deficiency have catecholamine and serotonin deficiencies in addition to hyperphenylalaninemia. These cause hypotonia, inactivity, and developmental problems. BH4 is also essential for the synthesis of nitric oxide (NO) by nitric oxide synthase. Severe BH4 deficiency can lead to impairment of NO formation, and the CNS may be subjected to increased oxidative stress. Blood phenylalanine levels are usually determined in newborns in North America, Australia, and Europe; if PKU is diagnosed, dietary interventions should be started before the age of 3 weeks to prevent the development of mental retardation. THERAPEUTIC HIGHLIGHTS
PKU can usually be treated successfully by markedly reducing the amount of phenylalanine in the diet. This means restricting the intake of high-protein foods such as milk, eggs, cheese, meats, and nuts. In individuals with a BH4 deficiency, treatment can include BH4, levodopa, and 5-hydroxytryptophan in addition to a low-phenylalanine diet. The US Food and Drug Administration approved the drug sapropterin, a synthetic BH4, for the treatment of some people with PKU.
Once dopamine is synthesized, it is transported into the vesicle by the VMAT. Here the dopamine is converted to norepinephrine by dopamine β-hydroxylase. Norepinephrine is the only small-molecule transmitter that is synthesized in synaptic vesicles instead of being transported into the vesicle after its synthesis.
Some neurons in the CNS and adrenal medullary cells also contain the cytoplasmic enzyme phenylethanolamine-N-methyltransferase, which catalyzes the conversion of norepinephrine to epinephrine. In these cells, norepinephrine leaves the vesicles, is converted to epinephrine in the cytoplasm, and then enters other vesicles for storage until it is released by exocytosis.
Catabolism of Catecholamines
Norepinephrine, like other amine and amino acid transmitters, is removed from the synaptic cleft by binding to postsynaptic receptors, binding to presynaptic receptors, reuptake into the presynaptic neurons, or catabolism. Reuptake via a NET is a major mechanism to terminate the actions of norepinephrine (Figure 7–3), and the hypersensitivity of sympathetically denervated structures is explained in part on this basis. After the noradrenergic neurons are cut, their endings degenerate with loss of NET to remove norepinephrine from the synaptic cleft. Consequently, more norepinephrine from other sources is available to stimulate the receptors on the autonomic effectors.
Epinephrine and norepinephrine are metabolized to biologically inactive products by oxidation and methylation. The former reaction is catalyzed by monoamine oxidase (MAO) and the latter by catechol-O-methyltransferase (COMT). MAO is located on the outer surface of the mitochondria. MAO is widely distributed, being particularly plentiful in the nerve endings at which catecholamines are secreted. COMT is also widely distributed, particularly in the liver, kidneys, and smooth muscles. In the brain, it is present in glial cells, and small amounts are found in postsynaptic neurons, but none is found in presynaptic noradrenergic neurons. Consequently, catecholamine metabolism has two different patterns.
Extracellular epinephrine and norepinephrine are for the most part O-methylated, and measurement of the concentrations of the O-methylated derivatives normetanephrine and metanephrine in the urine is a good index of the rate of secretion of norepinephrine and epinephrine. The O-methylated derivatives that are not excreted are largely oxidized, and vanillylmandelic acid is the most plentiful catecholamine metabolite in the urine.
In the noradrenergic nerve terminals, some of the norepinephrine is constantly being converted by intracellular MAO to the physiologically inactive deaminated derivatives, 3,4-dihydroxymandelic acid and its corresponding glycol. These are subsequently converted to their corresponding O-methyl derivatives, vanillylmandelic acid, and 3-methoxy-4-hydroxyphenylglycol.
Epinephrine and norepinephrine both act on α- and β-adrenergic receptors (adrenoceptors), with norepinephrine having a greater affinity for α-adrenoceptors and epinephrine for β-adrenoceptors. These receptors are metabotropic GPCR, and each has multiple subtypes (α1A, α1B, α1D, α2A, α2B, α2C, and β1β3). Most α1-adrenoceptors are coupled via Gq proteins to phospholipase C, leading to the formation of IP3 and DAG, which mobilizes intracellular Ca2+ stores and activates protein kinase C, respectively. Thus, at many synapses, activation of α1-adrenoceptors is excitatory to the postsynaptic target. In contrast, α2-adrenoceptors activate Gi inhibitory proteins to inhibit adenylyl cyclase and decrease cAMP. Other actions of α2-adrenoceptors are to activate G-protein–coupled inward rectifier K+ channels to cause membrane hyperpolarization and to inhibit neuronal Ca2+ channels. Thus, at many synapses, activation of α2-adrenoceptors inhibits the postsynaptic target. Presynaptic α2-adrenoceptors are autoreceptors which, when activated, inhibit further release of norepinephrine from postganglionic sympathetic nerve terminals. β-Adrenoceptors activate a stimulatory GS protein to activate adenylyl cyclase to increase cAMP.
α1-Adrenoceptors are located on smooth muscle and the heart, and α2-adrenoceptors are located in the CNS and on pancreatic islets cells and nerve terminals. β1-Adrenoceptors are located in the heart and renal juxtaglomerular cells. β2-Adrenoceptors are located in bronchial smooth muscle and skeletal muscle. β3-Adrenoceptors are located in adipose tissue.
Pharmacology of Noradrenergic Synapses
Table 7–2 shows some of the common agonists that bind to adrenoceptors as well as some of the common adrenoceptor antagonists. Figure 7–9 also shows the site of action of various drugs that alter noradrenergic transmission. For example, metyrosine blocks the action of tyrosine hydroxylase, the rate-limiting step in the synthetic pathway for catecholamine production in the nerve terminal. Reserpine blocks the VMAT that moves dopamine into the synaptic vesicle. Also, bretylium and guanethidine prevent the release of norepinephrine from the nerve terminal. Cocaine and tricyclic antidepressants block the NET. In addition to the agonists listed in Table 7–2, some drugs mimic the actions of norepinephrine by releasing stored transmitter from the noradrenergic endings. These are called sympathomimetics and include amphetamines and ephedrine.
In some parts of the brain, catecholamine synthesis stops at dopamine (Figure 7–1), which can then be secreted into the synaptic cleft. Active reuptake of dopamine occurs via a Na+- and Cl–-dependent dopamine transporter. Dopamine is metabolized to inactive compounds by MAO and COMT in a manner analogous to the inactivation of norepinephrine. 3,4-Dihydroxyphenylacetic acid and homovanillic acid are conjugated, primarily to sulfate.
Dopaminergic neurons are located in several brain regions (Figure 7–2). One region is the nigrostriatal system, which projects from the midbrain substantia nigra to the striatum in the basal ganglia and is involved in motor control. Another dopaminergic system is the mesocortical system; it arises primarily in the ventral tegmental area, which projects to the nucleus accumbens and limbic subcortical areas. The mesocortical system is involved in reward behavior and addiction and in psychiatric disorders such as schizophrenia (see Clinical Box 7–3). Studies using positive emission tomography (PET) scanning in healthy humans show that a steady loss of dopamine receptors occurs in the basal ganglia with age. The loss is greater in men than in women.
CLINICAL BOX 7–3 Schizophrenia
Schizophrenia is an illness involving deficits of multiple brain systems that alter an individual’s inner thoughts as well as their interactions with others. Individuals with schizophrenia suffer from hallucinations, delusions, and racing thoughts (positive symptoms); and they experience apathy, difficulty dealing with novel situations, and little spontaneity or motivation (negative symptoms). Worldwide, about 1–2% of the population lives with schizophrenia. A combination of genetic, biologic, cultural, and psychologic factors contributes to the illness. A large amount of evidence indicates that a defect in the mesocortical system is responsible for the development of at least some of the symptoms of schizophrenia. Attention was initially focused on overstimulation of limbic D2 dopamine receptors. Amphetamine, which causes release of dopamine as well as norepinephrine in the brain, causes a schizophrenia-like psychosis; brain levels of D2 receptors are said to be elevated in schizophrenics; and there is a clear positive correlation between the anti-schizophrenic activity of many drugs and their ability to block D2 receptors. However, several recently developed drugs are effective antipsychotic agents but bind to D2 receptors to a limited degree. Instead, they bind to D4 receptors, and there is active ongoing research into the possibility that these receptors are abnormal in individuals with schizophrenia. THERAPEUTIC HIGHLIGHTS
Since the mid-1950s numerous antipsychotic drugs (eg, chlorpromazine, haloperidol, perphenazine, and fluphenazine) have been used to treat schizophrenia. In the 1990s, new “atypical” antipsychotics were developed. These include clozapine, which reduces psychotic symptoms, hallucinations, suicidal behavior, and breaks with reality. However, a potential adverse side effect is agranulocytosis (a loss of the white blood cells), which impairs the ability to fight infections. Other atypical antipsychotics do not cause agranulocytosis, including risperidone, olanzapine, quetiapine, ziprasidone, aripiprazole, and paliperidone.
Five dopamine receptors have been cloned, but they fall into two major categories: D1-like (D1 and D5) and D2-like (D2, D3, and D4). All dopamine receptors are metabotropic GPCR. Activation of D1-type receptors leads to an increase in cAMP, whereas activation of D2-like receptors reduces cAMP levels. Overstimulation of D2 receptors may contribute to the pathophysiology of schizophrenia (Clinical Box 7–3). D3 receptors are highly localized, especially to the nucleus accumbens (Figure 7–2). D4 receptors have a greater affinity than the other dopamine receptors for the “atypical” antipsychotic drug clozapine, which is used primarily to treat schizophrenia in individuals who do not respond to other therapies.
Serotonin (5-hydroxytryptamine; 5-HT) is present in highest concentration in blood platelets and in the gastrointestinal tract, where it is found in the enterochromaffin cells and the myenteric plexus. It is also found within the brainstem in the midline raphe nuclei, which project to a wide portion of the CNS including the hypothalamus, limbic system, neocortex, cerebellum, and spinal cord (Figure 7–2).
Serotonin is synthesized from the essential amino acid tryptophan (Figure 7–1 and Figure 7–10). The rate-limiting step is the conversion of the amino acid to 5-hydroxytryptophan by tryptophan hydroxylase. This is then converted to serotonin by the aromatic L-amino acid decarboxylase. Serotonin is transported into the vesicles by the VMAT. After release from serotonergic neurons, much of the released serotonin is recaptured by the relatively selective serotonin transporter (SERT). Once serotonin is returned to the nerve terminal, it is either taken back into the vesicles or is inactivated by MAO to form 5-hydroxyindoleacetic acid (5-HIAA). This substance is the principal urinary metabolite of serotonin, and urinary output of 5-HIAA is used as an index of the rate of serotonin metabolism in the body.
Biochemical events at a serotonergic synapse. Tryptophan is transported into the serotonergic nerve terminal by a Na+-dependent aromatic L-amino acid transporter. The steps involved in the conversion of tryptophan to serotonin (5-hydroxytryptamine, 5-HT) are described in Figure 7–1. 5-HT is transported from the cytoplasm into vesicles by the vesicular monoamine transporter (VMAT). 5-HT release occurs when an action potential opens voltage-sensitive Ca2+ channels to allow an influx of Ca2+ and fusion of vesicles with the surface membrane. 5-HT released into the nerve terminal can act on G-protein–coupled receptors on the postsynaptic neuron (not shown). 5-HT can also diffuse out of the cleft or be transported back into the nerve terminal by the 5-HT transporter. 5-HT can act on presynaptic autoreceptors to inhibit further neurotransmitter release. Cytoplasmic 5-HT is either sequestered in vesicles as described or metabolized to 5-hydroxyindole acetaldehyde by mitochondrial monoamine oxidase (MAO).
Tryptophan hydroxylase in the CNS is slightly different from the tryptophan hydroxylase in peripheral tissues and is coded by a different gene. This is presumably why knockout of the TPH1 gene, which codes for tryptophan hydroxylase in peripheral tissues, has much less effect on brain serotonin production than on peripheral serotonin production.
There are seven classes of 5-HT receptors (from 5-HT1 through 5-HT7 receptors), and all except one (5-HT3) are GPCR and affect adenylyl cyclase or phospholipase C (Table 7–2). Within the 5-HT1 group are the 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, and 5-HT1F subtypes. Within the 5-HT2 group there are 5-HT2A, 5-HT2B, and 5-HT2C subtypes. There are two 5-HT5 subtypes: 5-HT5A and 5-HT5B. Some of the serotonin receptors are presynaptic and others are postsynaptic.
5-HT2A receptors mediate platelet aggregation and smooth muscle contraction. Mice in which the gene for 5-HT2C receptors has been knocked out are obese as a result of increased food intake despite normal responses to leptin (see Chapter 26), and they are prone to fatal seizures. 5-HT3 receptors are present in the gastrointestinal tract and the area postrema and are related to vomiting. 5-HT4 receptors are also present in the gastrointestinal tract, where they facilitate secretion and peristalsis, and in the brain. 5-HT6 and 5-HT7 receptors in the brain are distributed throughout the limbic system, and the 5-HT6 receptors have a high affinity for antidepressant drugs.
CLINICAL BOX 7–4 Major Depression
According to the National Institutes of Mental Health, nearly 21 million Americans over the age of 18 have a mood disorder that includes major depressive disorder, dysthymia, and bipolar disease. The largest group is those with major depression. Major depression has a median age of onset of 32 years and is more prevalent in women than men. Symptoms of major depression include depressed mood, anhedonia, loss of appetite, insomnia or hypersomnia, restlessness, fatigue, feelings of worthlessness, diminished ability to think or concentrate, and recurrent thoughts of suicide. Typical depression is characterized by feelings of sadness, early-morning awakenings, decreased appetite, restlessness, and anhedonia. Symptoms of atypical depression include pleasure-seeking behavior and hypersomnia.
The precise cause of depression is unknown, but genetic factors likely contribute. There is strong evidence for a role of central monoamines, including norepinephrine, serotonin, and dopamine. The hallucinogenic agent lysergic acid diethylamide (LSD) is a central 5-HT2 receptor agonist. The transient hallucinations produced by this drug were discovered when the chemist who synthesized it inhaled some by accident. Its discovery drew attention to the correlation between behavior and variations in brain serotonin content. Psilocin, a substance found in certain mushrooms, and N, N-dimethyltryptamine are also hallucinogenic and, like serotonin, are derivatives of tryptamine. 2,5-Dimethoxy-4-methyl-amphetamine and mescaline and other true hallucinogens are phenylethylamines. However, each of these may exert its effect by binding to 5-HT2 receptors. 3,4-Methylenedioxymethamphetamine (MDMA or Ecstasy) is a popular drug of abuse that produces euphoria followed by difficulty in concentrating and depression. The drug causes release of serotonin followed by serotonin depletion; the euphoria may be due to the release and the later symptoms to the depletion. THERAPEUTIC HIGHLIGHTS
In cases of typical depression, drugs such as fluoxetine (Prozac), which are selective serotonin reuptake inhibitors (SSRIs), are effective as antidepressants. SSRIs are also used to treat anxiety disorders. In atypical depression, SSRIs are often ineffective. Instead, monoamine oxidase inhibitors (MAOIs) such as phenelzine and selegiline have been shown to be effective as antidepressants. However, they have adverse consequences including hypertensive crisis if the patient ingests large quantities of products high in tyramine, which include aged cheese, processed meats, avocados, dried fruits, and red wines (especially Chianti). Based on evidence that atypical depression may result from a decrease in both serotonin and dopamine, drugs acting more generally on monoamines have been developed. These drugs, called atypical antidepressants, include bupropion, which resembles amphetamine and increases both serotonin and dopamine levels in the brain. Bupropion is also used as smoking cessation therapy.
Pharmacology of Serotonergic Synapses
Table 7–2 shows some of the common agonists that bind to 5-HT receptors as well as some of the common 5-HT receptor antagonists. In addition, tricyclic antidepressants inhibit the uptake of serotonin by block of SERT, similar to what was described for their actions at noradrenergic synapses. Selective serotonin uptake inhibitors (SSRIs) such as fluoxetine are widely used in the treatment of depression (see Clinical Box 7–4).
Histaminergic neurons have their cell bodies in the tuberomammillary nucleus of the posterior hypothalamus, and their axons project to all parts of the brain, including the cerebral cortex and the spinal cord. Histamine is also found in cells in the gastric mucosa and in heparin-containing cells called mast cells that are plentiful in the anterior and posterior lobes of the pituitary gland as well as at body surfaces. Histamine is formed by decarboxylation of the amino acid histidine. The three well-characterized types of histamine receptors (H1, H2, and H3) are all found in both peripheral tissues and the brain. Most, if not all, of the H3 receptors are presynaptic, and they mediate inhibition of the release of histamine and other transmitters via a G-protein. H1 receptors activate phospholipase C, and H2 receptors increase intracellular cAMP. The function of this diffuse histaminergic system is unknown, but evidence links brain histamine to arousal, sexual behavior, blood pressure, drinking, pain thresholds, and regulation of the secretion of several anterior pituitary hormones. In addition, a recently described histamine H4 receptor appears to play a role in regulating cells of the immune system.
ATP is an example of a small-molecule that is often co-localized and co-released from synaptic vesicles such as those in noradrenergic postganglionic sympathetic neurons (Figure 7–7), and it has recently been identified as a neurotransmitter. ATP has been shown to mediate rapid synaptic responses in the autonomic nervous system and a fast response in the habenula. ATP binds to P2X receptors, which are ligand-gated ion channel receptors. P2X receptors have widespread distributions throughout the body, including the dorsal horn, which implicates a role for ATP in sensory transmission. Antagonists of P2X receptors are under development for the treatment of chronic pain. ATP also binds to P2Y and P2U receptors, which are GPCR.
LARGE-MOLECULE TRANSMITTERS: NEUROPEPTIDES
Substance P is a polypeptide containing 11 amino acid residues that is found in the intestine, various peripheral nerves, and many parts of the CNS. It is one of a family of polypeptides called tachykinins that differ at the amino terminal end but have in common the carboxyl terminal sequence of Phe-X-Gly-LeuMet-NH2, where X is Val, His, Lys, or Phe. Other members of the family include neurokinin A and neurokinin B.
There are three neurokinin receptors (NK1–NK3), which are metabotropic GPCR. Substance P is the preferred ligand for NK1 receptors in the CNS, and activation of this receptor leads to increased formation of IP3 and DAG.
Substance P is found in high concentrations in the endings of primary afferent neurons in the spinal cord, and it is probably the mediator at the first synapse in the pathways for pain transmission in the dorsal horn. It is also found in high concentrations in the nigrostriatal system, where its concentration is proportional to that of dopamine, and in the hypothalamus, where it may play a role in neuroendocrine regulation. Upon injection into the skin, it causes redness and swelling, and it is probably the mediator released by nerve fibers that is responsible for the axon reflex. In the intestine, it is involved in peristalsis. Several recently developed centrally active NK-1 receptor antagonists have been shown to have antidepressant activity. They have also been used as antiemetics in patients undergoing chemotherapy.
The brain and the gastrointestinal tract contain receptors that bind morphine. The search for endogenous ligands for these receptors led to the discovery of two closely related pentapeptides (enkephalins) that bind to these opioid receptors: met-enkephalin and leu-enkephalin. These and other peptides that bind to opioid receptors are called opioid peptides. The enkephalins are found in nerve endings in the gastrointestinal tract and many different parts of the brain, and they appear to function as synaptic transmitters. They are found in the substantia gelatinosa and have analgesic activity when injected into the brainstem. They also decrease intestinal motility. Enkephalins are metabolized primarily by two peptidases: enkephalinase A, which splits the Gly-Phe bond, and enkephalinase B, which splits the Gly-Gly bond. Aminopeptidase, which splits the Tyr-Gly bond, also contributes to their metabolism.
Like other small peptides, the endogenous opioid peptides are synthesized as part of larger precursor molecules. More than 20 active opioid peptides have been identified. Unlike other peptides, however, the opioid peptides have a number of different precursors. Each has a prepro form and a pro form from which the signal peptide has been cleaved. Proenkephalin was first identified in the adrenal medulla, but it is also the precursor for met-enkephalin and leu-enkephalin in the brain. Each proenkephalin molecule contains four met-enkephalins, one leuenkephalin, one octapeptide, and one heptapeptide. Proopiomelanocortin, a large precursor molecule found in the anterior and intermediate lobes of the pituitary gland and the brain, contains β-endorphin, a polypeptide of 31 amino acid residues that has metenkephalin at its amino terminal. There are separate enkephalin-secreting and β-endorphin–secreting systems of neurons in the brain. β-Endorphin is also secreted into the bloodstream by the pituitary gland. A third precursor molecule is prodynorphin, a protein that contains three leuenkephalin residues associated with dynorphin and neoendorphin. Different types of dynorphins are found in the duodenum and the posterior pituitary and hypothalamus; β-neoendorphins are also found in the hypothalamus.
There are three classes of opioid receptors: μ, κ, and δ with various subtypes of each of these, but genes encoding only one subtype for each has been identified and characterized. As shown in Table 7–3, they differ in physiologic effects and affinity for various opioid peptides. All three are GPCR, and all inhibit adenylyl cyclase. Activation of μ receptors increases K+ conductance, hyperpolarizing central neurons and primary afferents. Activation of κ receptors and δ receptors closes Ca2+ channels.
TABLE 7–3Physiologic effects produced by stimulation of opioid receptors. ||Download (.pdf) TABLE 7–3 Physiologic effects produced by stimulation of opioid receptors.
|Receptor ||Endogenous Opioid Peptide Affinity ||Effect |
|μ ||Endorphins > Enkephalins > Dynorphins || |
Supraspinal and spinal analgesia
Increased secretion of growth hormone and prolactin
|κ ||Enkephalins > Endorphins and Dynorphins || |
Supraspinal and spinal analgesia
|γ ||Dynorphins > > Endorphins and Enkephalins ||Supraspinal and spinal analgesia |
Numerous other polypeptides are found in the brain. For example, somatostatin is found in various parts of the brain, where it may function as a neurotransmitter with effects on sensory input, locomotor activity, and cognitive function. In the hypothalamus, this growth hormone—inhibiting hormone is secreted into the portal hypophysial vessels; in the endocrine pancreas, it inhibits insulin secretion and the secretion of other pancreatic hormones; and in the gastrointestinal tract, it is an important inhibitory gastrointestinal regulator. A family of five different somatostatin receptors have been identified (SSTR1 through SSTR5). All are GPCR that inhibit adenylyl cyclase and exert various other effects on intracellular messenger systems. It appears that SSTR2 mediates cognitive effects and inhibition of growth hormone secretion, whereas SSTR5 mediates the inhibition of insulin secretion.
Vasopressin and oxytocin are not only secreted as hormones but also are present in neurons that project to the brainstem and spinal cord. The brain contains bradykinin, angiotensin II, and endothelin. The gastrointestinal hormones, including vasoactive intestinal polypeptide (VIP), cholecystokinin (CCK-4 and CCK-8), are also found in the brain. There are two kinds of CCK receptors in the brain, CCK-A and CCK-B. CCK-8 acts at both binding sites, whereas CCK-4 acts at the CCK-B sites. Gastrin, neurotensin, galanin, and gastrin-releasing peptide are also found in the gastrointestinal tract and brain. Neurotensin, VIP, and CCK receptors have been cloned and shown to be GPCR. The hypothalamus contains both gastrin 17 and gastrin 34. VIP produces vasodilation and is found in vasomotor nerve fibers. The functions of these peptides in the nervous system are unknown, although some of the peptides also expressed in the gastrointestinal system have been implicated in satiety (see Chapter 26).
Calcitonin gene-related peptide (CGRP) is present in the CNS and peripheral nervous system, gastrointestinal tract, cardiovascular system, and urogenital system. CGRP is co-localized with either substance P or acetylcholine. CGRP-like immunoreactivity is present in the circulation, and injection of CGRP causes vasodilation. CGRP and the calcium-lowering hormone calcitonin are both products of the calcitonin gene. In the thyroid gland, splicing produces the mRNA that codes for calcitonin, whereas in the brain, alternative splicing produces the mRNA that codes for CGRP. CGRP has little effect on Ca2+ metabolism, and calcitonin is only a weak vasodilator. Release of CGRP from trigeminal afferent fibers may contribute to the pathophysiology of migraine. Actions of this peptide are mediated by two types of metabotropic CGRP receptors.
Neuropeptide Y is a polypeptide that is very abundant throughout the brain and the autonomic nervous system. Neuropeptide Y acts on eight identified receptors: Y1–Y8; except for Y3, these are GPCR. Activation of these receptors mobilizes Ca2+ and inhibits adenylyl cyclase. It acts within the CNS to increase food intake, and Y1 and Y5 receptor antagonists may be used to treat obesity. It also acts in the periphery to cause vasoconstriction. It acts on heteroreceptors on postganglionic sympathetic nerve terminals to reduce release of norepinephrine.
OTHER CHEMICAL TRANSMITTERS
Nitric oxide (NO), a compound released by the endothelium of blood vessels as endothelium-derived relaxing factor, is also produced in the brain. It is synthesized from arginine, a reaction catalyzed in the brain by one of the three forms of NO synthase. It activates guanylyl cyclase and, unlike other transmitters, it is a gas, which crosses cell membranes with ease and binds directly to guanylyl cyclase. NO is not stored in vesicles like other classic transmitters; it is synthesized on demand at postsynaptic sites and diffuses to adjacent sites on the neuron. Synthesis may be triggered by activation of NMDA receptors, which leads to an influx of Ca2+ and activation of neuronal nitric oxide synthase. It may be the signal by which postsynaptic neurons communicate with presynaptic endings to enhance release of glutamate. It may also play a role in synaptic plasticity and thus in memory and learning.
Two types of endogenous cannabinoids have been identified as neurotransmitters: 2-arachidonyl glycerol and anandamide. These are also not stored in vesicles; they are rapidly synthesized in response to Ca2+ influx after a neuron is depolarized. Both act on a cannabinoid receptor (CB1) with a high affinity for Δ9-tetrahydrocannabinol, the psychoactive ingredient in marijuana. These receptors are primarily located on presynaptic nerve terminals. The CB1 receptor triggers a G-protein–mediated decrease in intracellular cAMP levels and is common in central pain pathways as well as in parts of the cerebellum, hippocampus, and cerebral cortex. In addition to inducing euphoria, CB1 receptor agonists have an anti-nociceptive effect, and CB1 receptor antagonists enhance nociception. Endogenous cannabinoids also act as retrograde synaptic messengers; they travel back across a synapse after release and bind to presynaptic CB1 receptors to inhibit further transmitter release. A CB2 receptor, which also couples to G-proteins, has also been cloned; it is located primarily in the periphery. Agonists of this class of receptor do not induce the euphoric effects of activation of CB1 receptors and they may have potential for use in the treatment of chronic pain.