SYNAPTIC PHYSIOLOGY OF THE AUTONOMIC NERVOUS SYSTEM

The Sympathetic and Parasympathetic Divisions Have Opposite Effects on Most Visceral Targets

All innervation of skeletal muscle in humans is excitatory. In contrast, many visceral targets receive both inhibitory and excitatory synapses. These antagonistic synapses arise from the two opposing divisions of the ANS, the sympathetic and the parasympathetic. In organs that are stimulated during physical activity, the sympathetic division is excitatory and the parasympathetic division is inhibitory. For example, sympathetic input increases the heart rate, whereas parasympathetic input decreases it. In organs whose activity increases while the body is at rest, the opposite is true. For example, the parasympathetic division stimulates peristalsis of the gut, whereas the sympathetic division inhibits it. Although antagonistic effects of the sympathetic and parasympathetic divisions of the ANS are the general rule for most end organs, exceptions exist. For example, the salivary glands are stimulated by both divisions, although stimulation by the sympathetic division has different characteristics than does parasympathetic stimulation (p. 929). In addition, some organs receive innervation from only one of these two divisions of the ANS. For example, sweat glands, piloerector muscles, and most peripheral blood vessels receive input from only the sympathetic division. Synapses of the ANS are specialized for their function. Rather than possessing synaptic terminals that are typical of somatic motor axons, many postganglionic autonomic neurons have bulbous expansions, or varicosities, that are distributed along their axons within their target organ (Fig. 15-7). It was once believed that these varicosities indicated that neurotransmitter release sites of the ANS did not form close contact with end organs and that neurotransmitters needed to diffuse long distances across the extracellular space to reach their targets. However, we now recognize that many varicosities form synapses with their targets, with a synaptic cleft extending approximately 50 nm across. At each varicosity, autonomic axons form an "en passant" synapse with their end-organ target. This arrangement results in an increase in the number of targets that a single axonal branch can influence, with wider distribution of autonomic output.
All Preganglionic Neurons-Both Sympathetic and Parasympathetic-Release Acetylcholine and Stimulate N2 Nicotinic Receptors on Postganglionic Neurons

Figure 15-7 Synapses of autonomic neurons with their target organs. Varicosities in the axon make multiple points of contact with their targets. Shown is a scanning electron micrograph of the axon of a postganglionic sympathetic neuron from a guinea pig, grown in tissue culture. (From Burnstock G: Autonomic neuromuscular junctions: Current developments and future directions. J. Anat 146: 1-30, 1986.)
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Table 15-1. PROPERTIES OF THE SYMPATHETIC AND PARASYMPATHETIC DIVISIONS SYMPATHETIC SYMPATHETIC PARASYMPATHETIC PARASYMPATHETIC PREGANGLIONIC POSTGANGLIONIC PREGANGLIONIC POSTGANGLIONIC Intermediolateral Prevertebral and Brain stem and sacral Terminal ganglia in or cell column in the paravertebral spinal cord (S2-S4) near target organ spinal cord ganglia (T1-L3) Yes No Norepinephrine Adrenergic Yes Acetylcholine Nicotinic No Acetylcholine Muscarinic

Location of neuron cell bodies Myelination

Primary Acetylcholine neurotransmitter Primary postsynaptic receptor Nicotinic

At their synapses between their postganglionic neurons and target cells, the two major divisions of the ANS use different neurotransmitters and receptors (Table 15-1). However, in both the sympathetic and parasympathetic divisions, synaptic transmission between preganglionic and postganglionic neurons

(termed "ganglionic transmission" because the synapse is located in a ganglion) is mediated by acetylcholine (ACh) acting on nicotinic receptors (Fig. 15-8). Nicotinic receptors are ligand-gated channels (i.e., ionotropic receptors) with a pentameric structure (p. 213). Table 15-2 summarizes some of the properties of nicotinic receptors. The nicotinic receptors on postganglionic autonomic neurons are a different molecular subtype (N2) than are those found at the neuro-muscular junction (N1). Both are ligand-gated ion channels that are activated by ACh or nicotine. However, whereas the N1 receptors at the neuromuscular junction (p. 212) are stimulated by decamethonium and preferentially blocked by D-tubocurarine, the autonomic N2 receptors are stimulated by tetramethylammonium, but resistant to D-tubocurarine. When activated, N1 and N2 receptors are both permeable to Na+ and K+, with entry of Na+ dominating. Thus, nicotinic transmission triggered by stimulating preganglionic neurons leads to rapid depolarization of postganglionic neurons.
All Postganglionic Parasympathetic Neurons Release Acetylcholine and Stimulate Muscarinic Receptors on Visceral Targets

Figure 15-8 Major neurotrans-mitters of the autonomic nervous system. In the case of the somatic neuron, the pathway between the central nervous system and effector cell is monosynaptic. The neuron releases acetylcholine (ACh), which binds to N 1-type nicotinic receptors on the postsynaptic membrane (i.e., skeletal muscle cell). In the case of both the parasympathetic and sympathetic divisions, the preganglionic neuron releases ACh, which acts at N2-type nicotinic receptors on the postsynaptic membrane of the postganglionic neuron. In the case of the postganglionic parasympathetic neuron, the neuro-transmitter is ACh, but the postsynaptic receptor is a muscarinic receptor, which is a metabotropic (i.e., G-protein-linked) receptor of one of five subtypes (M 1 -M5). In the case of most postganglionic sympathetic neurons, the neurotransmitter is norepinephrine. The postsynaptic receptor is a metabo-tropic (i.e., G-protein-linked) adrenergic receptor of one of two major subtypes ( and ).
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Table 15-2. SIGNALLING PATHWAYS FOR NICOTINIC, MUSCARINIC, AND ADRENERGIC RECEPTORS RECEPTOR TYPE N1 nicotinic ACh N2 nicotinic ACh AGONISTS* ACh (nicotine, decamethonium) ACh (nicotine, TMA) ANTAGONISTS D-Tubocurarine, -Bungarotoxin Hexamethonium Atropine, pirenzepine (M1) Atropine, methoctramine (M2) Phentolamine G LINKED 2ND PROTEIN ENZYME MESSENGER Gq Gi and Go Gq Gi Gs PLC Adenylyl cyclase PLC Adenylyl cylase Adenylyl cyclase Adenylyl cyclase Adenylyl cyclase IP3 and DAG [cAMP]i

M1/M3/M5 ACh (muscarine) muscarinic ACh M2/M4 ACh (muscarine) muscarinic ACh 1-Adrenergic 2-Adrenergic 1-Adrenergic NE Epi (phenylephrine)

IP3 and DAG [cAMP]i [cAMP]i

NE Epi (clonidine) Yohimbine Epi 3 NE (dobutamine, isoproterenol) Epi 3 NE (terbutaline, isoproterenol) Epi 3 NE (isoproterenol) Metoprolol

2-Adrenergic

Butoxamine

Gs

[cAMP]i

3-Adrenergic

SR-59230A

Gs

[cAMP]i

* Selective agonists are in parentheses. ACh, acetylcholine; cAMP, cyclic adenosine monophosphate; DAG, diacylglycerol; Epi, epinephrine; IP 3, inositol 1,4,5-triphosphate; NE, norepinephrine; PLC, phospholipase C; TMA, tetramethylammonium.

All postganglionic parasympathetic neurons act through muscarinic ACh receptors on the postsynaptic membrane of the target cell (see Fig. 15-8). Activation of this receptor can either stimulate or inhibit function of the target cell. Cellular responses induced by muscarinic receptor stimulation are more varied than are those induced via nicotinic receptors. Muscarinic receptors are metabotropic receptors that interact with heterotrimeric G proteins (p. 92) to (1) stimulate the hydrolysis of phosphoinositide and thus increase [Ca2+]i and activate protein kinase C, (2) inhibit adenylyl cyclase and thus decrease cyclic adenosine monophosphate (cAMP) levels, or (3) directly modulate K+ channels via the G-protein complex (see Table 15-2). Because they are mediated by second messengers, muscarinic responses, unlike the rapid responses evoked by nicotine receptors, are slow and prolonged. Muscarinic receptors exist in five different pharmacologic subtypes (M1 through M5) that are encoded by five different genes. All five subtypes are highly homologous to each other, but very different from the nicotinic receptors, which are ligand-gated ion channels. Subtypes M1 through M5 are each stimulated by ACh and muscarine and are blocked by atropine. Although a wide variety of antagonists inhibit the muscarinic receptors, none is completely selective for a specific subtype. However, it is possible to classify a receptor on the basis of its affinity profile for a battery of antagonists. Selective agonists for the different isoforms have not been available. A molecular characteristic of the muscarinic receptors is that the third cytoplasmic loop (i.e., between the fifth and sixth membrane-spanning segments) is different in M1, M3, and M5 on the one hand and M2 and M4 on the other. This loop appears to play a role in coupling of the receptor to the G protein downstream in the signal-trans-duction cascade. In general M1, M3, and M5 preferentially couple to Gq and then to phospholipase C, with release of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (p. 100). On the other hand M2 and M4 preferentially couple to Gi or Go to inhibit adenylyl cyclase and thus decrease [cAMP]i (p. 95). The five muscarinic subtypes have a heterogeneous distribution among tissues, and this heterogeneity varies among species. Muscarinic receptors are found both pre-synaptically and postsynaptically throughout the ANS. Many smooth muscles coexpress multiple muscarinic subtypes, each of which may play different roles in neuro-transmission. Thus, it is sometimes difficult to predict the effects of applying ACh to a particular tissue.
Most Postganglionic Sympathetic Neurons Release Norepinephrine onto Visceral Targets
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Figure 15-9 Muscarinic neurotransmission at ganglionic synapses. A, In this experiment, stimulation of the preganglionic sympathetic neuron of a frog releases acetylcholine, which triggers a fast EPSP (due to activation of nicotinic receptors on the postganglionic sympathetic neuron), followed by a slow EPSP (due to activation of muscarinic receptors on the postsynaptic neuron). EPSP, excitatory postsynaptic potential. (A, Data from Adams PR, Brown DA: Synaptic inhibition of the M-current: Slow excitatory post-synaptic potential mechanism in bullfrog sympathetic neurones. J Physiol 332: 263-272, 1982.) B, In this experiment on a rat sympathetic postganglionic neuron, the M current (mediated by a K+ channel) normally hyperpolarizes the neuron, thereby inhibiting the generation of action potentials. Thus, injecting current elicits only a single action potential. C, In the same experiment as that in B, adding muscarine stimulates a G-protein-linked muscarinic receptor and triggers a signal-transduction cascade that blocks the M current. One result is a steady-state depolarization of the cell. In addition, injecting current now elicits a train of action potentials. (B and C, Data from Brown DA, Constanti A: Intracellular observations on the

effects of muscarinic agonists on rat sympathetic neurones. Br J Pharmacol 70:593-608, 1980.)

Postganglionic sympathetic neurons release norepinephrine (see Fig. 15-8), which acts on target cells via adrenergic receptors. The sympathetic innervation of sweat glands is an exception to this rule. Sweat glands are innervated by sympathetic neurons that release ACh and act via muscarinic receptors (p. 573). The adrenergic receptors are all G-protein-coupled (i.e., metabotropic) receptors and are highly homologous to the muscarinic receptors. Two major types of adrenergic receptor are recognized, and , each of which exists in multiple subtypes (e.g., 1, 2, 1, 2, and 3). Additionally, there are heterogeneous 1 and 2 receptors, with three cloned subtypes of each. Table 15-2 lists the signalling pathways that are generally linked to these receptors. For example, 1 receptors in the heart activate the Gs heterotrimeric G protein and stimulate adenylyl cyclase, which antagonizes the effects of muscarinic receptors. Cholinergic Sympathetic Neurons Adrenergic receptor subtypes have a tissue-specific distribution. 1 Receptors predominate on blood vessels, 2 on presynaptic terminals, 1 in the heart, 2 in high concentration in the bronchial muscle of the lungs, and 3 in fat cells. This distribution has permitted the development of many clinically useful agents that are selective for different subtypes and tissues. For example, 1 agonists are effective as nasal decongestants, and 2 antagonists have been used to treat impotence. 1 Agonists increase cardiac output in congestive heart failure, whereas 1 antagonists are useful antihypertensive agents. 2 Agonists are used as bronchodilators in patients with asthma and chronic lung disease.
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The adrenal medulla (p. 1061) is a special adaptation of the sympathetic division, homologous to a sympathetic ganglion (see Fig. 15-8). It is innervated by preganglionic sympathetic neurons, and the postsynaptic target cells, which are called chromaffin cells, are analogous to postganglionic sympathetic neurons. Thus, chromaffin cells have nicotinic ACh receptors. However, rather than possessing axons that release norepinephrine onto a specific target organ, the chromaffin cells reside near blood vessels and release epinephrine into the bloodstream. This neuroendocrine component of sympathetic output enhances the ability of the sympathetic division to broadcast its output throughout the body. Norepinephrine and epinephrine both activate all five subtypes of adrenergic receptor, but with different affinities (see Table 15-2). In general, the receptors have a greater affinity for norepinephrine, whereas the receptors have a greater affinity for epinephrine.
Postganglionic Sympathetic and Parasympathetic Neurons Often Have Muscarinic As Well As Nicotinic Receptors

Two additional superimposed layers of complexity are also present in the ANS. First, some postganglionic neurons, both sympathetic and parasympathetic, have muscarinic in addition to nicotinic receptors. Second, at all levels of the ANS, certain neurotransmitters and post-synaptic receptors are neither cholinergic nor adrenergic. We will discuss the first exception in this section and the second in the following section. If we stimulate the release of ACh from preganglionic neurons or apply ACh to the ganglionic synapse, many postganglionic neurons exhibit both nicotinic and muscarinic responses. Because nicotinic receptors (N2) are lig-and-gated ion channels, nicotinic neurotransmission causes a fast, monophasic excitatory postsynaptic potential (EPSP). In contrast, because muscarinic receptors are G-protein linked, neurotransmission by this route leads to a slower electrical response. Thus, the result is a multiphasic postsynaptic response that, depending on the ganglion, can be a combination of a fast EPSP via a nicotinic receptor plus either a slow EPSP or a slow inhibitory postsynaptic potential (IPSP) via a muscarinic receptor. Figure 15- 9A is an example of a fast EPSP followed by a slow EPSP. A well-characterized effect of muscarinic neurotransmission in autonomic ganglia is inhibition of a specific K+ current called the M current. The M current is widely distributed in visceral end organs, autonomic ganglia, and the CNS. In the baseline state, the K+ channel that underlies the M current is active, thereby producing slight hyperpolarization. In the example shown in Figure 15-9B, with the stabilizing M current present, electrical stimulation of the neuron causes only a single spike. If we now add muscarine to the neuron, the stimulated muscarinic receptor turns off the hyperpolarizing M current and thus leads to a small depolarization. If we repeat the electrical stimulation in the continued presence of muscarine (Fig. 15-9C), repetitive spikes appear because the absence of the stabilizing influence of the M current increases the excitability of the neuron. Thus, muscarinic receptor activation modulates the repetitive firing properties of this neuron. The slow, modulatory effects of muscarinic responses greatly enhance the ability of the ANS to

control visceral activity.

Nonclassic Transmitters Can Be Released at Each Level of the ANS

In the 1930s, Sir Henry Dale first proposed that sympathetic nerves release a transmitter that is similar to epinephrine (now known to be norepinephrine) and parasympathetic nerves release ACh. For many years, attention was focused on these two neurotransmitters, primarily because they mediate large and fast postsynaptic responses that can be easily studied. In addition, a variety of antagonists are available to block cholinergic and adre-nergic receptors and thereby permit clear characterization of the roles of these receptors in the control of visceral function. More recently, it has become evident that some neurotransmission in the ANS involves neither adrenergic nor cholinergic pathways. Table 15-3 lists some of the multitude of neurotransmitters used in the ENS. Moreover, many neuronal synapses use more than a single neurotransmitter. Such cotransmission is now known to be common in the ANS. As many as eight different neurotransmitters may be found within some neurons, a phenomenon known as colocalization (see Table 12-1). Thus, ACh and norepinephrine play important but not exclusive roles in autonomic control. Sir Henry H. Dale Table 15-3. NEUROTRANSMITTERS AND NEUROMODULATORS PRESENT IN THE ENTERIC NERVOUS SYSTEM Acetylcholine (ACh) Norepinephrine (NE) Serotonin (5-HT) Dopamine Tachykinins (substance P, neurokinin A, neuropeptide K, neuropeptide ) Nitric oxide (NO) Adenosine triphosphate (ATP) Vasoactive intestinal polypeptide (VIP) -Aminobutyric acid (GABA) Cholecystokinin (CCK) Galanin Neuropeptide Y Somatostatin Gastrin-releasing peptide (GRP) Enkephalins Dynorphin Calcitonin gene-related peptide (CGRP) Neurotensin
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Table 15-4. NEUROTRANSMITTERS PRESENT WITHIN THE AUTONOMIC NERVOUS SYSTEM SYNAPSE Presynaptic CNS neurons Postsynaptic Preganglionic autonomic neurons TRANSMITTERS RELEASED Glutamate Glycine Substance P Serotonin Norepinephrine TRH

Enkephalins Neuropeptide Y Neurotensin Neurophysin II Oxytocin Somatostatin Preganglionic autonomic neurons Postganglionic autonomic neurons Acetylcholine Substance P CGRP Postganglionic autonomic neurons Target cell Norepinephrine Acetylcholine NO ATP Neuropeptide Y Galanin Somatostatin VIP Opioid peptides Visceral afferents Neurons in autonomic ganglia or spinal cord Peripheral autonomic ganglia and ENS Substance P CGRP Interneurons Dopamine Enkephalins
ATP, adenosine triphosphate; CGRP, calcitonin gene-related peptide; CNS, central nervous system; ENS, enteric nervous system; NO, nitric oxide; TRH, thyrotropin-releasing hormone; VIP, vasoactive intestinal peptide.

The distribution and function of nonadrenergic, non-cholinergic transmitters are only partially understood. However, these transmitters are found at every level of autonomic control (Table 15-4), where they can cause a wide range of postsynaptic responses. These nonclassic transmitters may cause slow synaptic potentials or may modify repetitive firing properties (as in the case of the M current) without having obvious direct effects. In other cases, nonclassic transmitters have no known effects and may be acting in ways that have not been determined. Although colocalization of neurotransmitters is recognized as a common property of neurons, it is not clear what controls the release of each of the many neurotransmitters. In some cases, the proportion of neurotransmitters released depends on the level of neuronal activity (p. 318). For example, medullary raphe neurons project to the intermediolateral cell column in the spinal cord, where they corelease serotonin, thyrotropin-releasing hormone (TRH), and substance P onto sympathetic pregan-glionic neurons. The proportions of released neurotrans-mitters are controlled by neuronal firing frequency; as the firing rate increases, TRH release makes up a higher percentage of the total release. Frequency-dependent modulation of synaptic transmission provides a mechanism for enhancing the versatility of the ANS.
Two of the Most Unusual "Nonclassic" Neurotransmitters, Adenosine Triphosphate and Nitric Oxide, Were First Identified in the Autonomic Nervous System

It was not until the 1970s that a nonadrenergic, nonchol-inergic class of sympathetic or parasympathetic neurons was first proposed by Geoffrey Burnstock and colleagues, who suggested that adenosine triphosphate (ATP) might act as the neurotransmitter. This idea, that a molecule used as an intracellular energy substrate could also be a synaptic transmitter, was initially difficult to prove. However, it is now clear that neurons use a variety of classes of molecules for intercellular communication (p. 304). Two of the most surprising examples of nonclassic transmitters, nitric oxide (NO) and ATP, were first identified and studied

as neurotransmitters in the ANS, but they are now known to be more widely used throughout the nervous system. ADENOSINE TRIPHOSPHATE.
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Figure 15-10 Cotransmission with adenosine triphosphate (ATP), norepinephrine, and neuropeptide Y (NPY) in the autonomic nervous system. In this example, stimulation of a postganglionic sympathetic neuron causes three phases of contraction of a vascular smooth muscle cell. Each phase corresponds to the release of a different neurotransmitter or group of transmitters. In phase 1, ATP binds to a P 2X purinoceptor (a ligand-gated cation channel) on the smooth muscle cell, leading to depolarization, activation of voltage-gated Ca 2+ channels, increased [Ca2+]i, and the rapid phase of contraction. In phase 2, norepinephrine binds to an 1-adrenergic receptor, which-via a Gq/PLC/IP3 cascade, leads to Ca2+ release from internal stores and the second phase of contraction. In phase 3, when present, NPY binds to a Y 1 receptor and somehow causes an increase in [Ca 2+]i and thus produces the slowest phase of contraction. ER, endoplasmic reticulum; IP 3 inositol 1,4,5-triphosphate; PLC, phospolipase C.

ATP is colocalized with norepinephrine in postganglionic sympathetic vasocon-strictor neurons. It is contained in synaptic vesicles, is released on electrical stimulation, and induces vascular constriction when applied directly to vascular smooth muscle. The effect of ATP results from activation of P 2 purinoceptors on smooth muscle, which include ligand-gated ion channels (P2X) and G-protein-coupled receptors (P2Y and P2U). P2X receptors are present on autonomic neurons and smooth-muscle cells of blood vessels, the urinary bladder, and other visceral targets. P2X receptor channels have a relatively high Ca2+ permeability (p. 317). In smooth muscle, depolarization can also activate voltage-gated Ca2+ channels (p. 189) and thus lead to an elevation in [Ca2+]i and a rapid phase of contraction (Fig.15-10). Norepinephrine, by binding to 1-adrenergic receptors, acts through a heterotrimeric G protein (p. 92) to facilitate the release of Ca2+ from intracellular stores and thereby produce a slower phase of contraction. Finally, the release of neuropeptide Y may, after prolonged and intense stimulation, elicit a third component of contraction. NITRIC OXIDE. In the 1970s, it was also discovered that the vascular endothelium produces a substance that induces relaxation of vascular smooth muscle. First called endothelium-derived relaxation factor (EDRF), in 1987 it was identified as the free radical NO. Nitric oxide is an unusual molecule for intercellular communication because it is a short-lived gas that is produced locally from L-arginine by the enzyme nitric oxide synthase (NOS; p. 110). Inhibition of NO signalling blocks long-term potentiation in hippocampal brain slices, which has led to speculation that NO acts as a retrograde messenger from postsynaptic neurons to presynaptic terminals during this cellular correlate of learning (p. 324). Similar effects of NO on synaptic strength have also been observed in autonomic neurons, even though learning has not typically been considered an important property of the ANS.

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Figure 15-11 Action of nitric oxide in the autonomic nervous system. Stimulation of a postganglionic parasympathetic neuron can cause more than one phase of relaxation of a vascular smooth muscle cell, corresponding to the release of a different neurotransmitter or group of transmitters. The first phase in this example is mediated by both nitric oxide (NO) and acetylcholine. The neuron releases NO, which diffuses to the smooth muscle cell. In addition, acetylcholine (ACh) binds to M 3 muscarinic receptors (G-protein linked) on endothelial cells, leading to production of NO, which also diffuses to the smooth muscle cell. Both sources of NO raise [cGMP] i in the smooth muscle cell and contribute to the first phase of relaxation. In the second phase, which tends to occur more with prolonged or intense stimulation, the neuropeptide VIP (or a related peptide) binds to receptors on the smooth muscle cell and causes a delayed relaxation via an increase in [cAMP] i or a decrease in [Ca2+ ]i. cGMP, cyclic guanosine monophosphate.

NOS is found in the preganglionic and postganglionic neurons of both the sympathetic and parasympathetic divisions, as well as in vascular endothelial cells. It is not specific for any type of neuron inasmuch as it is found in both norepinephrine- and ACh-containing cells, as well as neurons containing a variety of neuropeptides. Figure 15-11 shows how a parasympathetic neuron may simultaneously release NO, ACh, and vasoactive intestinal peptide, each acting in concert to lower [Ca 2+]i and relax vascular smooth muscle. Why NO is so ubiquitous and when its release is important are not known. However, evidence now indicates that abnormalities of the NO system are involved in the pathophysiology of adult respiratory distress syndrome, high-altitude pulmonary edema, stroke, and other diseases. Understanding its physiological and pathophysiological roles has led to the introduction of clinical treatments that modulate the NO system. Examples include the use of NO generators such as nitro-glycerin for treating angina, use of the cyclic guanosine monophosphate (cGMP) phosphodiesterase inhibitor sil-denafil (Viagra) for treating penile erectile dysfunction, and gaseous NO for treating pulmonary edema.
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