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Acetylcholine 

Acetylcholine
Chapter:
Acetylcholine
Author(s):

Leslie L. Iversen

, Susan D. Iversen

, Floyd E. Bloom

, and Robert H. Roth

DOI:
10.1093/med/9780195380538.003.0164
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The neurophysiological activity of acetylcholine (ACh) has been known since the early 19th century and its neurotransmitter role since the mid-1920s. With this long history, it is not surprising that students assume everything is already known about this transmitter. The main reason ACh assumed an early prominent role in guiding studies of neurotransmitters is the ease with which ACh can be studied. ACh is the transmitter at the neuromuscular junction, and thus both nerve terminal and its target can be readily accessed for experimental manipulations. Subsequent investigations also focused on the superior cervical ganglion, another peripheral site that was also easy to isolate and study. Lessons learned from experiments conducted on these peripheral tissues shaped our early approaches to defining the characteristics of neurotransmitters and neurotransmission. Unfortunately, the delay in developing sophisticated methods for determining where in the brain ACh was present in cholinergic tracts and terminals, which took decades to resolve, left this field far behind that of the biogenic amines. The following is the structural formula of ACh:

Acetylcholine

Synthesis

ACh is synthesized in a single step by a reaction catalyzed by choline acetyltransferase (ChAT):

Acetyl CoA + choline = ACh + CoA

Before entering into a discussion of ChAT, we should take note of Figure 6–1, which depicts the possible sources of acetyl coenzyme A (CoA) and choline. In brain slices, homogenates, acetone powder extracts, and preparations of nerve ending particles, glucose, or citrate are the best sources for ACh synthesis, with acetate rarely showing any activity. Regardless of its source, acetyl CoA is primarily synthesized in mitochondria. Since, as detailed later, ChAT appears to be in the synaptosomal cytoplasm, another still unsolved problem is how acetyl CoA is transported out of the mitochondria to participate in ACh synthesis. A probable carrier for acetyl CoA is citrate, which can diffuse into the cytosol and produce acetyl CoA via citrate lyase; a possible carrier is acetyl carnitine, and another possibility is Ca-induced leakage of acetyl CoA from mitochondria.

Figure 6–1. Acetylcholine (ACh) metabolism. CoA, coenzyme A; CTP, cytidine triphosphate.

Figure 6–1.
Acetylcholine (ACh) metabolism. CoA, coenzyme A; CTP, cytidine triphosphate.

Choline is transported to the brain both free and in phospholipid form (possibly as phosphatidylcholine) by the blood. Following the hydrolysis of ACh, approximately 35–50% of the liberated choline is transported back into the presynaptic terminal by a sodium-dependent, high-affinity active transport system to be reutilized in ACh synthesis. As outlined in Figure 6–1, the remaining choline may be catabolized or become incorporated into phos-pholipids, which can again serve as a source of choline. A curious observation is that when brain cortical slices are incubated for 2 hours in a Krebs–Ringer medium, choline accumulates to approximately 10 times its original concentration. Similarly, a rapid postmortem increase in choline has been observed. The precise source of this choline is unknown; a probable candidate is phosphatidylcholine.

Choline transport

Choline crosses cell membranes by two processes, referred to as high-affinity and low-affinity transport. High-affinity transport, with a Michaelis constant (Km) for choline of 1–5 μ‎M, is saturable, carrier mediated, dependent on sodium, and stimulated by chloride. It is also dependent on the membrane potential of the cell or organelle so that any agent (e.g., K+) that depolarizes the cell will concurrently inhibit high-affinity transport. Low-affinity choline transport, with a Km of 40–80 μ‎M, appears to operate by a passive diffusion process, to be linearly dependent on the concentration of choline, and to be virtually nonsaturable. In contrast to the other neurotransmitters, ACh is taken up in terminals only via low-affinity transport; it is only choline that exhibits high-affinity kinetics. Evidence suggests that the high-affinity transport of choline is specific for cholinergic terminals and is not present in aminergic nerve terminals. Furthermore, transport is kinetically (but not physically) coupled to ACh synthesis. Approximately 50–85% of the choline that is transported by the high-affinity process is utilized for ACh synthesis. Low-affinity transport, however, is found in cell bodies and in tissues such as the corneal epithelium, likely for the synthesis of choline-containing phospholipids. High-affinity choline transport that is coupled to phospholipid synthesis can also be found in tissues that do not synthesize ACh. Hemicholinium-3 is an extremely potent inhibitor of high-affinity transport (Km of 0.05–1 μ‎M) but a relatively weak inhibitor of low-affinity transport (Km of 10–120 μ‎M). There are three obvious mechanisms for regulating the level of ACh in cells: feedback inhibition by ACh on ChAT, mass action, and the availability of acetyl CoA and/or choline. Of these three possibilities, the major regulatory factor seems to be high-affinity choline transport. This view derives from early observations that choline is rate limiting in the synthesis of ACh coupled with findings in a number of laboratories. Using the septal–hippocampal pathway, a known cho-linergic tract, Kuhar and associates showed that changes in impulse flow induced via electrical stimulation or pentylenetetrazol administration (both of which increase impulse flow) or via lesioning or the administration of pento-barbital (both of which decrease neuronal traffic) will alter high-affinity transport of choline into hippocampal synaptosomes. In their studies, procedures that activated impulse flow increased the maximal velocity (Vmax) of choline transport, whereas agents that stopped neuronal activity decreased Vmax. In neither situation was the K changed, a result to be expected since the concentration of choline outside the neuron (5–10 μ‎M) normally exceeds the Km for transport (1–5 μ‎M). Recent evidence, however, suggests that this relationship between impulse traffic and choline transport does not occur in all brain areas (e.g., in the striatum, where cholinergic interneurons abound). In addition, the endogenous concentration of ACh is implicated in regulating the level of the transmitter in the brain. Thus, in several studies, an increase in choline uptake following depolarization of a preparation has been attributed to the release of endogenous ACh upon depolarization. Other studies, however, suggest that this increased choline uptake is not related to ACh release but, rather, to an increase in Na–K adenosine triphosphatase (ATPase) activity. The high-affinity choline transporter has been cloned by the Okuda group, who developed an antibody to map cholinergic neurons.

Choline acetyltransferase

The synthetic enzyme ChAT is the definitive marker for the presence of cho-linergic neurons in brain. Multiple mRNAs encode ChAT, resulting from differential use of three promoters and alternative splicing of the 5′ noncoding region. In the rat, the different transcripts encode the same protein, but in humans they give rise to multiple forms of the enzyme, including both active and inactive forms. The functional significance of these different transcripts is uncertain.

Although ChAT is the sole enzyme in ACh synthesis, it is not the rate-liming step in ACh synthesis. The full enzymatic activity of ChAT is not expressed in vivo. The activity of ChAT measured in vitro is much greater that would be expected on the basis of ACh synthesis in vivo. It has been suggested that the reason for this discrepancy might be related to the requirement to transport acetyl CoA from the mitochondria to the cytoplasm, which may be the rate-limiting step in ACh synthesis. Alternatively, intracellular choline concentrations and choline transport may ultimately determine the rate of ACh synthesis. This latter speculation has led to the administration of choline precursors in an attempt to boost ACh synthesis in the brain of Alzheimer’s patients in which there is a marked decrease in ACh in the cerebral cortex. Attempts to treat Alzheimer’s disease by administration of choline precursors such as lecithin have been unsuccessful in diminishing dementia.

With respect to the cellular localization of ChAT, the highest activity is found in the interpeduncular nucleus, caudate nucleus, retina, corneal epithe-lium, and central spinal roots (3000–4000 mg ACh synthesized/g/hour). In contrast, dorsal spinal roots contain only trace amounts of the enzyme, as does the cerebellum.

When highly purified from rat brain, ChAT has a molecular weight of 67–75 kDa: It has an apparent Km for choline of 7.5 × 10−4 M and for acetyl CoA of 1.0 × 10−5 M. Recent estimates suggest an equilibrium constant of 13. The enzyme is activated by chloride and inhibited by sulfhydryl reagents. A variety of studies on the substrate specificity of the enzyme indicate that various acyl derivatives of both CoA and ethanolamine can be utilized. The major gap in our knowledge of ChAT is that we do not know of any useful (i.e., potent and specific) direct inhibitor. Styrylpyridine derivatives inhibit it but suffer from the fact that they are light sensitive, somewhat insoluble, and possess varying degrees of anticholinesterase activity. Hemicholinium inhibits the synthesis of ACh indirectly by preventing the transport of choline across cell membranes.

Acetylcholinesterase

Everybody agrees that ACh is hydrolyzed by cholinesterases, but nobody is sure just how many cholinesterases exist in the body. All cholinesterases will hydrolyze not only ACh but also other esters. Conversely, hydrolytic enzymes such as arylesterases, trypsin, and chymotrypsin will not hydrolyze choline esters. The problem in determining the number of cholinesterases that exist is that different species and organs sometimes exhibit maximal activity with different substrates. For our purposes, we divide the enzymes into two rigidly defined classes: acetylcholinesterase (also called “true” or specific cholines-terase) and butyrylcholinesterase (also called “pseudo” or nonspecific cholin-esterase; the term propionylcholinesterase is sometimes used since in some tissues propionylcholine is hydrolyzed more rapidly than butyrylcholine). Although their molecular forms are similar, the two enzymes are distinct entities, encoded by specific genes. Evidence suggests that in lower forms butyrylcholinesterase predominates, gradually giving way to acetylcholinesterase with evolution. The type of cholinesterase found in a tissue is often a reflection of the tissue. This fact is used as a discriminating index between cholinesterases. In general, neural tissue contains acetylcholinesterase, whereas glial cells and nonneural tissue usually contain butyrylcholinesterase. However, this is a generalization, and some neural tissue (e.g., autonomic ganglia) contains both esterases, as do some extraneural organs (e.g., liver and lung). Because of its ubiquity, cholinesterase activity cannot be used as the sole indicator of a cholinergic system in the absence of additional supporting evidence. To generalize on this point, until neuron-specific, transmitter-degrading enzymes are discovered, it is a neurochemical commandment that, to delineate a neuronal tract, one should always assay with an enzyme involved in the synthesis of a neurotransmitter and not one concerned with catabolism.

Uptake, synthesis, and release of ach

Superior Cervical Ganglion, Brain, and Skeletal Muscle

The only major, thorough studies of ACh turnover in nervous tissue were done originally by MacIntosh and colleagues and subsequently by Collier using the superior cervical ganglion of the cat. Using one ganglion to assay the resting level of ACh and perfusing the contralateral organ, these investigators determined the amount of transmitter synthesized and released under a variety of experimental conditions, including electrical stimulation, addition of an anticholinesterase to the perfusion fluid, and perfusion media of varying ionic composition. Their results may be summarized as follows: During stimulation, ACh turns over at a rate of 8–10% of its resting content every minute (i.e., approximately 24–30 ng/minute). At rest, the turnover rate is approximately 0.5 ng/minute. Since there is no change in the ACh content of the ganglion during stimulation at physiological frequencies, it is evident that electrical stimulation not only releases the transmitter but also stimulates its synthesis. Choline is the rate-limiting factor in the synthesis of ACh. In the perfused ganglion, Na+ is necessary for optimum synthesis and storage, and Ca2+ is necessary for release of the neurotransmitter. Newly synthesized ACh appears to be more readily released upon nerve stimulation than depot or stored ACh. Approximately half of the choline produced by cholinesterase activity is reuti-lized to make new ACh.

At least three separate stores of ACh in the ganglion are inferred from these studies: surplus ACh, considered to be intracellular, which accumulates only in an eserine-treated ganglion and is not released by nerve stimulation but is released by K depolarization; depot ACh, which is released by nerve impulses and accounts for approximately 85% of the original store; and stationary ACh, which constitutes the remaining 15% that is nonreleasable.

Choline analogs, such as triethylcholine, homocholine, and pyrrolcholine, are released by nerve stimulation only after they are acetylated in the ganglia. Increasing the choline supply in the plasma during perfusion of the ganglion only transiently increases the amount of ACh that is releasable with electrical stimulation, despite accumulation of the transmitter in the ganglion. The compound AH5183 (vesamicol) inhibits ACh transport into synaptic vesicles and blocks release of ACh from the stimulated ganglia.

Most of these characteristics, which were established decades ago, are recapitulated in the central nervous system (CNS).

ACh is synthesized via ChAT and, once formed, is transported into vesicles by the vesicular cholinergic transporter (VAChT). This transporter is distinct from the plasma membrane transporter that accumulates choline. ACh is not taken up into cholinergic terminals by a high-affinity transport system. However, as first described by Parsons’ laboratory, ACh is transported into synaptic vesicles via a proton-pumping ATPase activity. A glycosylated ATPase pumps protons out of vesicles and drives ACh via a separate transporter into vesicles in exchange for the protons. VAChT was cloned on the basis of homology to a Caenorhabditis elegans gene(unc-17) that encodes a protein homologous with VMAT. VAChT is expressed in cholinergic neurons throughout the brain and is another useful marker in addition to ChAT for the presence of cholinergic neurons.

VAChT is present on chromosome 10, near the gene for ChAT, and is unique in that its entire coding region is contained in the first intron of the ChAT gene: Both genes are coordinately regulated.

Release

Following the discovery of presynaptically localized vesicles that contained ACh, the conclusion was almost unavoidable that these organelles are the source of the quantal release of transmitter as described in the neurophysiological experiments of Katz and collaborators. Thus, the obvious interpretation has been that as the nerve is depolarized, Ca2+ enters the terminal, vesicles in apposition to the terminal fuse with the presynaptic membrane, and ACh is released into the synaptic cleft to interact with receptors on the postsynaptic cell to change ion permeability. Synapsin I, a phosphoprotein that is localized in vesicles, may mediate the translocation of vesicles to the plasma membrane. It is of interest because botulinum toxin A binds to it and irreversibly degrades it, leading to a persistent blockade of neuromuscular transmission. The use of minute doses of locally injected botulinum toxin has found many medical uses (Chapter 17). Other vesicular proteins that have been implicated in the exocytotic process are the synaptotagmins, synaptophysins, and synaptobrevins (see Chapter 3). Synaptobrevin is the target for both tetanus toxin and botulinum toxin type B, which are zinc endopeptidases that inhibit ACh release by cleaving it. Synaptotagmin has been implicated as a Ca2+ sensor in the release process. The subsequent sequence of events is not clear, but in some manner the presynaptic membrane is pinocytotically recaptured and vesicles are resynthesized and simultaneously or subsequently repleted with ACh. This endocytotic event is apparently triggered by calcineurin, a Ca2+dependent protein phosphatase.

Cholinergic pathways

The identification of cholinergic synapses in the peripheral nervous system has been relatively easy, and we have known for a long time that ACh is the transmitter at autonomic ganglia, at parasympathetic postganglionic synapses, and at the neuromuscular junction. In the CNS, however, until the late 1970s, technical difficulties limited our knowledge of cholinergic tracts to the moto-neuron collaterals to Renshaw cells in the spinal cord. With respect to the aforementioned technical difficulties, the traditional approach has been to lesion a suspected tract and then assay for ACh, ChAT, or high-affinity choline uptake at the presumed terminal area. Problems with lesioning include making discrete, well-defined lesions and interrupting fibers of passage. This latter problem is illustrated by the discovery that a habenula–interpeduncular nucleus projection that, based on lesioning of the habenula, was always described as a cholinergic pathway is not: It turned out that what was lesioned were cholinergic fibers that passed through the habenula. Thus, although the interpeduncular nucleus has the highest choline uptake and ChAT activity of any area in the brain, the origin of this innervation remains largely unknown. A quantum leap in technology for tracing tracts in the CNS has occurred in the past several decades. Through the use of histochemical techniques (originally developed by Koelle and co-workers) that stain for regenerated acetylcholinesterase after DFP treatment (Butcher, Fibiger), autoradiography with muscarinic receptor antagonists (Rotter, Kuhar), immunohistochemical procedures with antibodies to ChAT (McGeer, Salvaterra, Cuello, Wainer), coupled with the employment of specific cholinotoxins, a clearer picture of cholinergic tracts in the CNS has emerged. The well-documented tracts are depicted in Figure 6–2. There is additional information that in the striatum and the nucleus accumbens septi, only cholinergic interneurons are found. Also, intrinsic cholinergic neurons have been reported to exist in the cerebral cortex, colocalized with vasoactive intestinal polypeptide and often in close proximity to blood vessels. Recent studies using electrophysiology, specific cholinotoxins, and molecular genetic techniques have enhanced our understanding of the roles played by acetylcholine in the CNS. Functional roles for CNS cholinergic neurons have been found in motivation and reward, sleep and arousal, and cognitive processes and stimulus processing. With respect to other neurotransmitter functions, ACh may participate in circuits involved with pain reception. Thus, the findings that nettles (Urtica dioica) contain ACh and histamine, that high concentrations of ACh injected into the brachial artery of humans result in intense pain, and that ACh applied to a blister produces a brief but severe pain indicate a relationship between ACh and pain. That ACh may act as a sensory transmitter in thermal receptors, taste fiber endings, and chemoreceptors has also been suggested, based on the excitatory activity of the compound on these sensory nerve endings.

Figure 6–2. Schematic representation of the major cholinergic systems in the mammalian brain. Central cholinergic neurons exhibit two basic organizational schemata: (1) local circuit cells (i.e., those that morphologically are arrayed wholly within the neural structure in which they are found), exemplified by the interneurons of the caudate putamen nucleus, nucleus accumbens, olfactory tubercle, and Islands of Calleja complex (ICj), and (2) projection neurons (i.e., those that connect two or more different regions). Of the cholinergic projection neurons that interconnect central structures, two major subconstellations have been identified: (1) the basal forebrain cholinergic complex composed of choline acetyl-transferase (ChAT)-positive neurons in the medial septal nucleus (ms), diagonal band nuclei (td), substantia innominata (si), magnocellular preoptic field (poma), and nucleus basalis (bas) and projecting to the entire nonstriatal telencephalon and (2) the pontomesen-cephalotegmental cholinergic complex composed of ChAT-immunoreactive cells in the pendunuclopontine (tpp) and laterodorsal (dltn) tegmental nuclei and ascending to the thalamus and other diencephalic loci and descending to the pontine and medullary reticular formations (Rt), deep cerebellar (DeC) and vestibular (Ve) nuclei, and cranial nerve nuclei. Not shown are the somatic and parasympathetic cholinergic neurons of cranial nerves III– VII and IX–XII and the cholinergic α‎ and γ‎ motor and autonomic neurons of the spinal cord. amyg, amygdale; ant cg, anterior cingulate cortex; CrN, dorsal cranial nerve nuclei; diencep, diencephalon; DR, dorsal raphe nucleus; ento, entorhinal cortex; frontal, frontal cortex; IP, interpeduncular nucleus; ins, insular cortex; LC, locus ceruleus; LR, lateral reticular nucleus; olfact, olfactory; pir, piriform cortex; PN, pontine nuclei; pr, perirhinal cortex; parietal, parietal cortex; post cg, posterior cingulate cortex; SN, substantia nigra; Sp5, spinal nucleus of cranial nerve V. (Modified from Butcher and Woolf [1986] and Woolf and Butcher [1989].)

Figure 6–2.
Schematic representation of the major cholinergic systems in the mammalian brain. Central cholinergic neurons exhibit two basic organizational schemata: (1) local circuit cells (i.e., those that morphologically are arrayed wholly within the neural structure in which they are found), exemplified by the interneurons of the caudate putamen nucleus, nucleus accumbens, olfactory tubercle, and Islands of Calleja complex (ICj), and (2) projection neurons (i.e., those that connect two or more different regions). Of the cholinergic projection neurons that interconnect central structures, two major subconstellations have been identified: (1) the basal forebrain cholinergic complex composed of choline acetyl-transferase (ChAT)-positive neurons in the medial septal nucleus (ms), diagonal band nuclei (td), substantia innominata (si), magnocellular preoptic field (poma), and nucleus basalis (bas) and projecting to the entire nonstriatal telencephalon and (2) the pontomesen-cephalotegmental cholinergic complex composed of ChAT-immunoreactive cells in the pendunuclopontine (tpp) and laterodorsal (dltn) tegmental nuclei and ascending to the thalamus and other diencephalic loci and descending to the pontine and medullary reticular formations (Rt), deep cerebellar (DeC) and vestibular (Ve) nuclei, and cranial nerve nuclei. Not shown are the somatic and parasympathetic cholinergic neurons of cranial nerves III– VII and IX–XII and the cholinergic α‎ and γ‎ motor and autonomic neurons of the spinal cord. amyg, amygdale; ant cg, anterior cingulate cortex; CrN, dorsal cranial nerve nuclei; diencep, diencephalon; DR, dorsal raphe nucleus; ento, entorhinal cortex; frontal, frontal cortex; IP, interpeduncular nucleus; ins, insular cortex; LC, locus ceruleus; LR, lateral reticular nucleus; olfact, olfactory; pir, piriform cortex; PN, pontine nuclei; pr, perirhinal cortex; parietal, parietal cortex; post cg, posterior cingulate cortex; SN, substantia nigra; Sp5, spinal nucleus of cranial nerve V. (Modified from Butcher and Woolf [1986] and Woolf and Butcher [1989].)

Cholinergic receptors

Cholinergic receptors fall into two classes, muscarinic (Table 6–1) and nicotinic (Table 6–2). At last count, five muscarinic receptors (M1–M5) had been cloned. All of them exhibit a slow response time (100–250 milliseconds), are coupled to G proteins, and either act directly on ion channels or are linked to a variety of second messenger systems. M1, M3, and M5 via Gq are coupled to phosphatidylinositol hydrolysis; M2 and M4 via Gi are coupled to cyclic ade-nosine monophosphate. When activated, the final effect can be to open or close K channels, Ca channels, or Cl channels, depending on the cell type. With this array of channel activity, therefore, stimulation of muscarinic receptors will lead to either depolarization or hyperpolarization. As noted in Table 6–1, second messenger systems have been described following activation of the muscarinic receptors. Knowing the messengers is fine, but it does not tell us anything about the message—that is, what the ultimate physiological effect is.

Table 6–1. Muscarinic Receptors

Currently Accepted Name

M1

M2

M3

M4

M5

Molecular biology classification

m1

m2

m3

m4

m5

Structural information

460 aa (human)

466 aa (human)

590 aa (human)

479 aa (human)

532 aa (human)

Subtype-selective agonists

McN-A343 (ganglion)

Pilocarpine (relative to M3 and M5)

l-689,660 Xanomcline

TBPB (allosteric agonist)

Bethanechol (relative to M4)

l-689,660

McN-A343 (relative to M2)

Xanomeline

Vu10010 (allosteric potentiator)

None known

Subtype-selective antagonists

Pirenzepine

Methoctramine

Hexahydrosila-difenidol

Tropicamide

None known

Telenzepine

AF-DX-116

Himbacine

AF-DX-384

p-Fluorohexahydrosila-difenidol

AF-DX-384

Gallamine (noncompetitive)

4-DAMP

Himbacine

Tripitramine

Receptor-selective agonists

Bethanechol

Metoclopramide

Muscarine

Pilocarpine

Oxotremorine M

Bethanechol

Metoclopramide

Muscarine

Pilocarpine

Oxotremorine M

Bethanechol

Metoclopramide

Muscarine

Pilocarpine

Oxotremorine M

Bethanechol

Metoclopramide

Muscarine

Pilocarpine

Oxotremorine M

Bethanechol

Metoclopramide

Muscarine

Pilocarpine

Oxotremorine M

Receptor-selective antagonists

Scopolamine

QNB, (±)

QNB, R(−)

Atropine

Scopolamine

QNB, (±)

QNB, R(−)

Atropine

Scopolamine

QNB, (±)

QNB, R(−)

Atropine

Scopolamine

QNB, (±)

QNB, R(−)

Atropine

Scopolamine

QNB, (±)

QNB, R(−)

Atropine

Signal transduction mechanisms

Gq/11 (increase IP3/DAG)

NO

Gi(cAMP modulation)

↑ K+ (G)

Gq/11 (increase IP3/DAG)

NO

Gi (cAMP modulation)

↑ K+ (G)

Gq/11 (increase IP3/DAG)

NO

Radioligands of choice

[3H]-Pirenzepine

[3H]-Telenzepine

[3H]-QNB

[3H]-AF-DX-384

[3H]-QNB

[3H]-4-DAMP

[3H]-QNB

[3H]-AF-DX-384

[3H]-QNB

[3H]-QNB

[3H]-NMS

AF-DX-116, 11-([2-[(Diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6-pyridol[2,3-b][1,4]benzodiazepin-6-one; AF-DX-384, 5,11-dihydro-11-[2-[2-[N, N-dipropylaminomethyl)piperidin-1-yl]ethylamino]-carbony]6II-pyridol[2,3-b][1,4] benzodiazepin-6-one; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide; l-689,660, 1-azabicyclo [2,2,2] octane, 3-(6-chloropyrazinyl)maleate; McN-A343, 4-(N-[3-chlorophenyl] carbamoyloxy)-2-butynyltrimethylammonium chloride; NMS, N-methylscopolamine; QNB, quinuclidinyl-α‎-hydroxydiphenylacetate (quinuclidinylbenzylate); NO, nitric oxide; IP3/DAG, inositol triphosphate/diacylglycerol; cAMP, cyclic adenosine monophosphate.

Source: Modified from Watling (2006).

Table 6–2. Nicotinic Receptors

Currently used name

Neuronal (CNS) (α‎-bungarotoxin insensitive)

Neuronal (CNS) (α‎-bungarotoxin sensitive)

Neuronal (autonomic ganglia)

Muscular

Subunits (arranged as pentamers)

α‎4β‎2 (major)

α‎3β‎4

α‎2?α‎3?

α‎7 homomers

α‎8?

α‎9?

α‎9/α‎10

α‎7 homomers

α‎3α‎5β‎4

α‎3α‎5β‎2β‎4

α‎1β‎1δ‎γ‎(ε‎)

Receptor selective agonists

Cytisine RJR-2403

Epibatidine

Anatoxin A

ABT-418

A-85380

Anatoxin A DMAC

GTS-21

AR-R-17779

Epibatidine

SIB-1553A

DMPP

Epibatidine

Anatoxin A

TMA

Receptor selective antagonists

Mecamylamine

Dihydro-β‎-erythroidine

Erysodine

α‎-Conotoxin AuIB (α‎3β‎4)

α‎-Conotoxin MII (α‎3β‎2)

Chlorisondamine

Methllycaconitine

α‎-Bungarotoxin

α‎-Conotoxin IMI

Hexamethonium Chlorisondamine?

Mecamylamine?

κ‎-Bungarotoxin

α‎-Bungarotoxin

Signal transduction mechanisms

Modulation of cation channel conductance permeability properties

Modulation of cation channel conductance permeability properties

Modulation of cation channel conductance permeability properties

Modulation of cation channel conductance permeability properties

Radioligands of choice

[3H]-Nicotine

[125I]-α‎-Bungarotoxin

[3H]-Epibatidine

[125I]-α‎-Bungarotoxin

[3H]-Epibatidine

[3H]-Cytisine

[3H]-Methyllycaconitine

[125I]-α‎-Bungarotoxin

[3H]-Methyllycaconitine

A-85380, 3-(2[S]-Azetidinylmethoxy)pyridine; ABT-418, (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole; AR-R-17779, (−)-spirol [1-azabicyclo[2.2.2]octane-3,5′-oxazolidin-2′ one (4a); DMAC, 3-(4)-dimethylaminocinnamylidine anabaseine; DMPP, N,N-dimethyl-N′-phenyl-piperazinium iodide: GTS-21, [3-(2,4-dimethoxybenzylidene)-anabaseine; RJR-2403, N-methyl-4- (3-pyridinyl]-3-buten-1-amine: SIB-1553 A, 4-[[2-(1-methyl-2-pyrrolidinyl)ethyl]thio]phenyl hydrochloride; TMA, tetramethylammonium.

Source: Modified from Watling (2006).

Cholinergic transmission in many of the most critical CNS circuits is mediated primarily by muscarinic acetylcholine receptors (mAChRs). Of the five identified mAChR subtypes (termed M1–M5), M1 and M4 are most heavily expressed in the CNS and are the most likely candidates for mediating the effects on cognition, attention, and sensory processing. In contrast, the most prominent adverse effects of muscarinic cholinergic agents (bradycardia, gas-trointestinal distress, salivation, and sweating) are mediated by activation of peripheral M1, M2, and M3 mAChRs.

The pharmacological antagonists that have been used to define three of the muscarinic subtypes are pirenzepine, which has a high affinity for M1; AFDX116 and methoctramine with a high affinity for M2; and 4-diphenylacetoxy-N-methylpiperidine methiodide, which exhibits the highest affinity for M3. Most antagonists do not show more than a fivefold selectivity for one subtype over all other subtypes. The two classic muscarinic antagonists atropine and quinu-clidinylbenzylate do not distinguish the subtypes but block all equally well.

The selectivity of classical muscarinic agonists among receptor subtypes is also very low due to the highly conserved nature of the orthosteric binding site among receptor subtypes. It would be a major advance if we knew whether each subtype subserved a specific function, such as bradycardia or smooth muscle contractibility. If this were the case (and it probably is), more specific drugs devoid of side effects could be developed. More selective muscarinic agonists that will define the various subtypes are clearly needed and several have recently emerged. A new, highly selective M4 allosteric potentiator, VU10010, has been developed that potentiates the M4 response to acetylcholine approximately 50-fold while having no activity at other mAChR subtypes. This compound and other positive allosteric modulators of M4 do not activate the receptor directly but bind to an allosteric site on the receptor and increase affinity for ACh and coupling to G proteins. The discovery of a highly selective allosteric modulator of M4 provides an unprecedented opportunity to selectively increase activity of this receptor and develop a more detailed understanding of the functional roles of M4 in brain circuits that are heavily modu-lated by cholinergic innervation. One of the most important roles of cholinergic systems in the CNS is modulation of transmission through the hippocampal for-mation, a limbic cortical structure that has a critical role in learning and memory and is thought to be important for cholinergic regulation of cognitive function.

In addition to allosteric potentiators, a novel allosteric agonist termed TBPB has been characterized that is selective for the M1 mAChR subtype. Unlike allosteric potentiators, allosteric agonists directly activate the receptor rather than potentiating the effects of ACh.

The development of novel ligands that are highly selective for individual mAChR subtypes promises to provide important information concerning the function these receptor subtypes subserve. The ultimate hope is that selective allosteric activation of M1 and/or M4 AChRs could lead to new treatments for psychiatric disorders associated with a component of cognitive dysfunction (i.e., schizophrenia, Alzheimer’s disease, and Parkinson’s disease).

Until relatively recently, the identification of nicotinic cholinergic receptors in the CNS was an enigma. Using labeled α‎-bungarotoxin, nicotine, meca-mylamine, or dihydro-β‎-erythroidine, each investigation yielded mystifying results in which the antagonist could not be easily displaced by ACh or by unlabeled ganglionic or neuromuscular antagonists but occasionally was displaced by muscarinic agonists and antagonists. A major problem has been the low density of nicotinic compared to muscarinic receptors in the brain.

Conversely, much is known about the properties of the nicotinic cholinergic receptor of Torpedo and Electrophorus electricus organs. This reflects the abundance of the receptor in this tissue and the availability of two snake toxins, α‎-bungarotoxin and Naja naja siamensis, that specifically bind to the receptor and have facilitated its isolation and purification. In the past 15 years, however, research with monoclonal antibodies and cDNA has yielded considerable information about the mammalian nicotinic receptors (see Table 6–2).

As we enter the second century since the discovery of the nicotinic acetyl-choline receptors (nAChRs) by Langley in 1905, we know a great deal about their subunit structure, channel function, and regulation. nAChRs are members of the Cys loop family of transmitter-gated ion channels that include the 5-HT3, GABAA, and strychnine-sensitive glycine receptors. All nicotine receptors are formed as pentamers of subunits. Genes encoding a total of 17 subunits (α‎1–10, β‎1–4, δ‎, ε‎, and γ‎) have been identified to date. All these subunits are of mammalian origin with the exception of α‎8, which is avian. The crucial physiological role of nicotine receptors in the autonomic nervous system is well established and their many potential roles in the CNS are becoming clearer. In autonomic and sensory ganglia that have been examined, α‎3β‎4 receptors predominate. In the CNS, there are potentially a very large number of AChRs based on possible subunit combinations. In most brain regions, however, a relatively small number of subtypes appear to be represented. These include the homomeric α‎7 receptor and several heteromeric receptors comprising the α‎4β‎2 and the α‎3β‎4 subtypes. Although all nAChRs share many pharmacological characteristics, especially the heteromeric subtypes, a number of ligands and a few drugs have emerged that can distinguish among the receptor subtypes. This is predominately seen between those containing β‎2 and those containing β‎4 subunits. The critical involvement of nAChRs in nicotine addiction makes them an important target for intervention in this disorder. In addition, because CNS nAChRs are strategically located to modulate the release of a number of important neu-rotransmitters, including glutamate, dopamine, GABA, norepinephrine, and ACh, they may influence a wide variety of CNS functions and pathways. Thus, it is not surprising that drug-targeting nAChR subtypes may have therapeutic potential in a number of conditions as diverse as neuropathic pain, Parkinson’s disease, schizophrenia, Tourette’s syndrome, attention deficit hyperactivity disorder (ADHD), as well as addiction to and dependence on nicotine.

In view of the previous discussion, it is no surprise that considerable attention is being devoted to nicotine despite the fact that use of nicotine or tobacco products is fraught with complications, including their highly addictive properties (see Chapter 26). This interest was sparked by the potential beneficial effect of nicotine in enhancing vigilance, improving memory and learning in animal models, as well as nicotine’s antinociceptive properties suggesting potential use in the treatment of pain. Epidemiological studies indicating that smoking is associated with a lower incidence of Parkinson’s disease further fueled the fire. In clinical studies, the finding that tobacco smoking reduces the incidence of cognitive dysfunction in Parkinson’s and Alzheimer’s disease encouraged testing of nicotine’s efficacy in these disorders. Nicotine patches have been shown to improve cognition in Alzheimer’s patients but to be ineffective in Parkinson’s patients due in part to side effects. Increasing evidence, both preclinical and clinical, has demonstrated that α‎7 nAChR agonists and partial agonists can lead to improvements in cognitive performance. Thus, the α‎7 subtype of the nAChR has become a target of considerable interest in CNS drug discovery for disorders such as schizophrenia, Alzheimer’s disease, and ADHD, which exhibit a component of cognitive dysfunction. In contrast to direct agonist activation, a novel approach to modulating α‎7 nAChR function is to enhance the effects of the endogenous neurotransmitter ACh through positive allosteric modulation. Positive allosteric modulators are a class of compounds that selectively modulate the activity of ACh at α‎7 nAChRs in a manner that avoids desensitization and may have significant advantages over indiscriminate and direct activation of nAChRs by nicotine/nicotinic agonists or by acetylcholinesterase inhibitors.

Another recent development is the discovery of a series of potent and selective α‎4β‎2 nAChR partial agonists that exhibit dual agonist and antagonist activity in preclinical models. One of these agents, varenicline (Chantix), is now marketed for smoking cessation pharmacotherapy (see Chapter 26). The α‎4β‎2 receptor has also been implicated in learning deficiencies. Finally, nico-tine antagonists such as dihydro-β‎-erythroidine impair working memory. Obviously, pharmaceutical chemists have more than a casual interest in pursuing this interesting and potentially lucrative drug development area.

Figure 6–3 is a schematic model of an ACh synapse illustrating the presynaptic and postsynaptic molecular entities involved in the synthesis, storage, release, reuptake, and signaling of ACh and depicting potential sites of drug action. Structures of several protypic drugs that affect the cholinergic nervous system are illustrated in Figure 6–4.

Figure 6–3. Model of an ACh synapse illustrating the presynaptic and postsynaptic molecular entities involved in the synthesis, storage, release, reuptake, and signaling of ACh. Choline is transported into the presynaptic terminal by an active uptake mechanism and converted to ACh by a single enzymatic step. The acetyl coenzyme A required for ACh synthesis is provided by presynaptic mitochondria. Muscarinic M2 autoreceptors in the presynaptic terminal modulate the release of ACh. In contrast to monoamine synapses, the plasma membrane transporter of an ACh synapse does not return the neurotransmitter to the presynaptic terminal but, rather, recycles precursor choline. ACh is metabolized by ACh esterase (AChE) present on both pre- and postsynaptic membranes, which serves to terminate its action. Both G protein-coupled muscarinic receptors and ligand-gated ion channels (nicotinic receptors) may be present as depicted on the postsynaptic cell. Sites of drug action:
Site 1: ACh synthesis can be blocked by styrylpyridine derivatives such as NVP.
Site 2: ACh transport into vesicles is blocked by vesamicol (AH5183).
Site 3: Release is promoted by β‎-bungarotoxin, black widow spider venom, and La3+. Release is blocked by botulinum toxin, cytochalasin B, collagenase pretreatment, and Mg2+.
Site 4: Acetylcholinesterase is inhibited reversibly by physostigmine (eserine) or irreversibly by DFP, or soman.
Site 5: Choline uptake competitive blockers include hemicholinium-3, troxypyrrolium tosylate, or AF64A (noncompetitive).
Site 6: Presynaptic muscarinic receptors may be blocked by AFDX-116 (an M2 antagonist), atropine, or quinuclidinyl benzilate. Muscarinic agonists (e.g., oxotremorine) will inhibit the evoked release of ACh by acting on these receptors.
Site 7: Postsynaptic receptors are activated by cholinomimetic drugs and anticholines-terases. Nicotinic receptors, at least in the peripheral nervous system, are blocked by rabies virus, curare, hexamethonium, or dihydro-β‎-erythroidine; n-methylcarbamylcho-line and dimethylphenyl piperazinium are nicotinic agonists. Muscarinic receptors are blocked by atropine, pirenzepine, and quinuclidinyl benzilate.
AC, adenylyl cyclase; ChAT, choline acetyltransferase; CT, plasma membrane choline transporter; DAG, diacylglycerol; IP3, inositol triphosphate; M, muscarinic receptors; nAChR, nicotinic acetylcholine receptors; PLC, phospholipase C; VAT, vesicular acetyl-choline transporter (not yet isolated). (Modified from Nestler et al., 2001)

Figure 6–3.
Model of an ACh synapse illustrating the presynaptic and postsynaptic molecular entities involved in the synthesis, storage, release, reuptake, and signaling of ACh. Choline is transported into the presynaptic terminal by an active uptake mechanism and converted to ACh by a single enzymatic step. The acetyl coenzyme A required for ACh synthesis is provided by presynaptic mitochondria. Muscarinic M2 autoreceptors in the presynaptic terminal modulate the release of ACh. In contrast to monoamine synapses, the plasma membrane transporter of an ACh synapse does not return the neurotransmitter to the presynaptic terminal but, rather, recycles precursor choline. ACh is metabolized by ACh esterase (AChE) present on both pre- and postsynaptic membranes, which serves to terminate its action. Both G protein-coupled muscarinic receptors and ligand-gated ion channels (nicotinic receptors) may be present as depicted on the postsynaptic cell. Sites of drug action:

Site 1: ACh synthesis can be blocked by styrylpyridine derivatives such as NVP.

Site 2: ACh transport into vesicles is blocked by vesamicol (AH5183).

Site 3: Release is promoted by β‎-bungarotoxin, black widow spider venom, and La3+. Release is blocked by botulinum toxin, cytochalasin B, collagenase pretreatment, and Mg2+.

Site 4: Acetylcholinesterase is inhibited reversibly by physostigmine (eserine) or irreversibly by DFP, or soman.

Site 5: Choline uptake competitive blockers include hemicholinium-3, troxypyrrolium tosylate, or AF64A (noncompetitive).

Site 6: Presynaptic muscarinic receptors may be blocked by AFDX-116 (an M2 antagonist), atropine, or quinuclidinyl benzilate. Muscarinic agonists (e.g., oxotremorine) will inhibit the evoked release of ACh by acting on these receptors.

Site 7: Postsynaptic receptors are activated by cholinomimetic drugs and anticholines-terases. Nicotinic receptors, at least in the peripheral nervous system, are blocked by rabies virus, curare, hexamethonium, or dihydro-β‎-erythroidine; n-methylcarbamylcho-line and dimethylphenyl piperazinium are nicotinic agonists. Muscarinic receptors are blocked by atropine, pirenzepine, and quinuclidinyl benzilate.

AC, adenylyl cyclase; ChAT, choline acetyltransferase; CT, plasma membrane choline transporter; DAG, diacylglycerol; IP3, inositol triphosphate; M, muscarinic receptors; nAChR, nicotinic acetylcholine receptors; PLC, phospholipase C; VAT, vesicular acetyl-choline transporter (not yet isolated). (Modified from Nestler et al., 2001)

Figure 6–4. Structures of some drugs that affect the cholinergic nervous system.

Figure 6–4.
Structures of some drugs that affect the cholinergic nervous system.

Acetylcholine in disease states

Aside from myasthenia gravis and other autoimmune diseases, such as the Lambert–Eaton myasthenic syndrome (a presynaptic problem involving diminished release of ACh), the role of ACh in nervous system dysfunction is unclear. Certainly, a strong case can be made for familial dysautonomia, an autosomal recessive condition affecting Ashkenazi Jews that is diagnosed by a supersensitivity of the iris to methacholine. Huntington’s disease, involving a degeneration of Golgi type 2 cholinergic interneurons in the striatum, is partially ameliorated by physostigmine, although this does not reverse the progressive disease. Administration of physostigmine to patients with tardive dyskinesia has produced mixed results.

Alzheimer’s disease is characterized behaviorally by a severe impairment in cognitive function and neuropathologically by the appearance of neuritic plaques and neurofibrillary tangles (see Chapter 16). In vivo single photon emission computed tomography imaging of vesicular ACh transporter using [123I]-IBVM in early Alzheimer’s disease strongly supports the belief that cholinergic degeneration occurs in the early stage of Alzheimer’s disease and most likely is involved in the impairment of cognitive functions observed at this stage of the disease. The brain-penetrant anticholinesterase inhibitors are the current mainstays of symptomatic treatment for patients with early Alzheimer’s disease (see Chapter 16).

Cholinergic drugs

The enzymatic inactivation of ACh has been a fertile ground for the development of a large number of pharmaceutical agents. Anticholinesterase inhibitors such as tabun, VX, and sarin, initially developed for military use, are potent neurotoxins that have been used as nerve gases since World War II. Other anticholinesterases include organophosphates (e.g., parathion), which are widely used as insecticides. Whether the target is a tomato hornworm or human, anticholinesterases function in the same manner: Instead of the released ACh leading to discrete single depolarizations of muscle fibers, the accumulation of ACh at the neuromuscular junction leads to muscle fibrillations and, ultimately, depolarization inactivation of the muscle (i.e., the muscle is so excited that it stops contracting, resulting in flaccid paralysis). Anticho-linesterases have a number of other less aggressive uses as well. Competitive neuromuscular blocking agents such as succinylcholine are occasionally used as an adjunct to anesthetics during surgery to increase muscle relaxation. Anticholinergic agents can be used to reverse muscle paralysis produced by succinylcholine. Cholinesterase inhibitors such as neostigmine and pyridostigmine are the mainstay in the treatment of myasthenia gravis, which as mentioned previously is a disorder of the neuromuscular junction that is usually marked by the presence of anti-nicotinic receptor antibodies. In terms of their useful CNS effects, the brain-penetrant cholinesterase inhibitors donepezil, galantamine, and rivastigmine are the current mainstays of symptomatic treatment for patients with early Alzheimer’s disease. The target in this case is cortical brain ACh; in vivo imaging studies have demonstrated a significant decrease in cortical ACh in Alzheimer’s patient. Unfortunately, the efficacy of these agents is limited and largely symptomatic.

Muscarinic cholinergic agonists are not currently used to treat CNS disorders but are used clinically to treat urinary retention and glaucoma. They are also effective in ameliorating the symptoms of Sjogren’s syndrome, an autoimmune disorder characterized by degeneration of the salivary glands. However, as mentioned previously, several subtype-selective M1 and M4 mAChR allosteric agonists have become available, show efficacy in animal models, and are being tested for therapeutic effects in schizophrenia. Muscarinic antagonists such as benztropine and trihexyphenidyl are used to treat Parkinson’s disease and the parkinsonian symptoms elicited by some antipsychotic drugs. Scopolamine, administered by a slow-release transdermal patch, is effective in preventing motion sickness.

In recent years, the most important therapeutic advance has been the development of nAChR-specific drugs. The clinical efficacy of the α‎4β‎2 nAChR partial agonist varenicline (Chantix) for smoking cessation pharmacotherapy has been established (see Chapter 26). Chantix exhibits dual action by sufficiently stimulating α‎4β‎2 nAChR-mediated dopamine release to reduce craving when quitting smoking and by inhibiting nicotine reinforcement when smoking. Since nAChRs in the CNS are strategically localized to modulate the function of numerous transmitters, there is no doubt that targeting nAChR subtypes will lead to the development of a number of useful CNS therapeutics in the near future.

Selected references

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