INTERNATIONAL REVIEW OF
Neurobiology VOLUME 30
Editorial Board W. Ross ADEY JULIUS
AXELROD
SEYMOUR KETY KEITHKILLAM
Ross BALDESSARINI
CONAN KORNETSKY
SIR ROGERBANNISTER
ABELLAJTHA
FLOYD BLOOM
BORISLEBEDEV
DANIEL BOVET
PAULMANDEL
PHILLIP BRADLEY
HUMPHRY OSMOND
YURI BUROV
RODOLFO PAOLETTI
JOSE
DELGADO
SOLOMON SNYDER
SIRJOHN ECCLES
STEPHEN SZARA
ELKES
SIRJOHN VANE
JOEL
H . J. EYSENCK
MARATVARTANIAN
KJELLFUXE
STEPHEN WAXMAN
Bo HOLMSTEDT
RICHARD WYATT
PAULJANSSEN
ZANGWILL OLIVER
INTERNATIONAL REVIEW OF
NeurobioIogy Edited by JOHN R. SMYTHIES RONALD J. BRADLEY Department of Psychiatry and The Neurapsychiatry Research Program The Medical Center The University of Alabama at Birmingham Birmingham, Alabama
VOLUME 30
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Biochemistry of Nicotinic Acetylcholine Receptors in the Vertebrate Brain JAKOB
I. I1 . 111. IV . V.
SCHMIDT.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Probes Employed and Characterization of Their Targets . . . . . . . . . . . Relationships of Receptor Candidates . . . . . . . . . . . . . . . . . . . . . . . . . . . Relationship of Individual Binding Proteins to Synaptic Transmission Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 3 16 20 30 32
The Neurobiology of N-Acetylaspartylglutamate
RANDYD . BLAKELY A N D JOSEPH T . COYLE I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Quantitative Biochemical Studies of NAAG in Neural Tissues . . . . . . 111. Cellular Localization of NAAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Metabolism of NAAG in Neural Tissues . . . . . . . . . . . . . . . . . . . . . . . . . V . The Search for NAAG Receptors and Their Mechanism of Action . . VI . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
39 46 51 72 81 88 93
Neuropeptide.Processing. .Converting. and -Inactivating Enzymes in Human Cerebrospinal Fluid
LARSTERENIUS AND FREDNYBERG I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Individual Enzymes in CSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .
101 105 118 119
Targeting Drugs and Toxins to the Brain: Magic Bullets
LANCEL . SIMPSON I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Protein Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Multicomponent Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V
123 124 128
CONTENTS
vi IV. V. VI. VII. VIII.
Toxins as Magic Bullets.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Broadening the Concept of Magic Bullets . . . . . . . . . . . . . . . . . . . . . . . Chimeric Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chimeric Neuroactive Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
134 135 138 139 144 145
Neuron-Glia Interrelations
ANTONIA VERNADAKIS I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Neuron-Glia Interactions during Ontogenesis 111. Neuron-Glia Interactions and Synaptic Events
IV. V. Neuron-Glia VI . References . . . . . . . . . . . . .
...................... s ..................... ...............................
149
150
177 194 198 208 210
Cerebral Activity and Behavior: Control by Central Cholinergic and Serotonergic Systems
C. H. VANDERWOLF I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Hippocampal Activity in Relation to Behavior 111. Neocortical Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Cholinergic and Serotonergic Control of Cortical Activation V. Sleep and Waking.. . . . . . . . . . . . . . . . . . . . . . . . VI . Interpretation and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
225
INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF RECENT VOLUMES.. .................................
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B I O CHEMISTRY 0F NI COTI NI C AC ETYLC H0L INE RECEPTORS IN THE VERTEBRATE BRAIN By Jakob Schmidt Department of Biochemistry State University of New York ot Stony Brook Stony Brook, New York 11794
I. Introduction 11. Probes Employed and Characterization of Their Targets
A. Cholinergic Ligands B. Probes Specific for the AChR in the Neuromuscular Junction 111. Relationships of Receptor Candidates A. Relationship of the Targets of Various Nicotinic Drugs-The Agonist Receptor B. Nonidentity of the Toxin and Agonist Receptors C. Immunoreactive Proteins D. Relationship of Binding Proteins and Muscle Receptor-Like Genes and Messages IV. Relationship of Individual Binding Proteins to Synaptic Transmission A. A Success Story: Th e Agonist-Binding Protein B. A Persistent Conundrum: The Neuronal a-Bungarotoxin-Binding Protein V. Concluding Remarks References Note Added in Proof
I. Introduction
Over the past 20 years investigations of the nicotinic acetylcholine receptor (AChR) in the neuromuscular junction and its evolutionary homolog, the neuroeffector junction in the electric tissue of electric fish, have provided a paradigm of successful receptor research. The rapid progress in the biochemistry, molecular biology, cell biology, and biophysics of this unique membrane protein has been made possible by a constellation of favorable circumstances: the availability of densely INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 30
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Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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innervated electric tissue as a receptor source, the occurrence of irreversibly binding 'receptor-specific protein ligands (the curarimimetic neurotoxins) in elapid and hydropheid snake venoms, a large body of knowledge of the physiology of the neuromuscular junction, and a high degree of conservation of this molecule throughout the vertebrates. The receptor's various functions and activities (binding of agonists and of several classes of antagonists, channel opening and closing, desensitization, interaction with intracellular and extracellular elements, covalent modification, etc.) are beginning to be understood at the atomic level of resolution (for recent reviews, see McCarthy et al., 1986; Karlin et al., 1986; Popot and Changeux, 1984). Nicotinic AChR also are present in interneuronal synapses both in the CNS and in the autonomic nervous system. It has long been known that these receptors differ in their drug-binding properties, and therefore structurally, from their neuromuscular counterparts and, consequently, that whatever has been learned about peripheral receptors would have limited validity for neuronal AChR. The lack of an electroplaque equivalent, the absence of a reliable probe, and the possibility of receptor diversity within a given organism, as well as across species boundaries, have all contributed to the relatively slow progress in this field. Until 1980, much of the biochemical characterization of putative neuronal AChR had been done using curarimimetic neurotoxins as ligands. This early work has been reviewed by several groups (Morley et al., 1979; Schmidt et al., 1980; Oswald and Freeman, 1981; Morley and Kemp, 1981). Developments since then have been assessed in brief summaries (e.g., Morley et al., 1983b; Clarke, 1987a) and rather specialized commentaries; thus, Chiappinelli (1985) discussed snake venom toxins suitable for the characterization of neuronal receptors, Lunt (1986) reviewed insect receptor research, Wonnacott (1987) dealt with protein and alkaloid probes for neuronal nicotinic receptors, and Schuetze and Role (1987) reviewed developmental regulation of nicotinic receptors in the peripheral and central nervous systems. Another review, written from a pharmacological perspective and covering the literature through 1983, focused on the behavioral and biochemical evidence for the existence of specific nicotine targets in the central nervous system (Martin, 1986). The field has expanded during the 1980s, due to the introduction of new receptor probes, and after a slow and painful start has begun to advance vigorously. As a result, a more complete coverage has become necessary, even as it has become difficult. In the following I will attempt to summarize recent developments; the major emphasis will be on nicotinic receptors of the vertebrate central nervous system. In general;
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developments since 1980 will be covered, with occasional references to earlier work, mostly to provide a historical perspective. It appears advisable at the outset to provide a brief glossary of the terms to be used. Receptor will be considered to be a synonym for ligandbinding protein, regardless of physiological function and signal transduction mechanism, acetylcholine receptor (AChR) to be an acetylcholine (ACh)-binding protein with a synaptic function, and nicotinic receptor denotes a binding protein with a preference for nicotinic compounds. CIO designates receptors that resemble peripheral nicotinic (i.e., muscletype) receptors in their pharmacology, whereas C6 refers to nicotinic receptors similar to those found in autonomic ganglia. Toxin will stand for a-bungarotoxin (aBuTx), the principal toxic component of the venom of Bungurus multicinctus, unless specified otherwise, and toxin receptor for the neuronal aBuTx-binding component. The neuronally active bungarotoxin (aliases: x-bungarotoxin, toxin F, Bgt 3.1) will be referred to as vBuTx. Agonist receptor is the term that will be used for the neuronal receptor recognized by nicotinic agonists but not by aBuTx.
II. Probes Employed and Characterization of Their Targets
The biochemical characterization of ion channels, such as the one contained within the nicotinic AChR of muscle and suspected as an integral constituent of the neuronal receptor, faces a unique dilemma, namely, the mutually exclusive requirements for solubilization (as a prerequisite for purification) and for maintenance of membrane-bounded compartments (for testing physiological function). This problem has traditionally been solved by redefining the receptor as a ligand-binding protein and by demonstrating channel properties after purification using suitable membrane reconstitution protocols. The biophysical reconstitution technique, involving insertion of the purified receptor into lipid bilayers or vesicles and analysis of ligand-induced ion fluxes, has over the past several years given way to a molecular biological one, whereby appropriate messages are expressed in Xenopus laevis oocytes to produce functional channels measureable by patch clamp techniques. Research on neuronal receptors is following the same route. Much of the work so far has dealt with ligand binding studies; a major obstacle in this research has been the absence of a universally approved and accepted probe. A number of different ligands, antibodies, and nucleic acid sequences have been championed and utilized as specific markers by different groups and will be summarized here. Fortunately,
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developments have been characterized by the convergence, even fusion, of many of these initially separate lines of research (see Section I11 for details).
A. CHOLINERGIC LIGANDS The use of radioactive cholinergic ligands for the identification of nicotinic AChR is not new. Waser employed radioactive curare and decamethonium to study AChR in diaphragm preparations as early as 1956 (Waser and Luethi, 1956). Probably the first application of a radioligand for research on neuronal receptors is that reported by De Robertis in 1967, who used 14C-labeledD-tubocurarine on rat brain synaptosomal preparations (De Robertis et al., 1967). The low specific radioactivity of these early markers restricted their successful application to tissues of high receptor concentrations such as the electric tissue of Torpedo and insect brain (O’Brien and Gilmour, 1969; Farrow and O’Brien, 1970; Eldefrawi et al., 1971). Small cholinergic compounds were eclipsed by the curarimimetic neurotoxins (see below) in the 1970s and made a comeback only when they became available at sufficiently high specific radioactivity and after the exclusive reliance on aBuTx and its congeners began to be rejected. These small drugs can be conveniently grouped as (1) nicotine, (2) acetylcholine (in the presence of acetylcholinesterase inhibitors as well as muscarinic blockers) and other agonists, and (3) antagonists. 1. Nicotine
An early attempt to identify nicotinic receptors by means of radioactive nicotine was reported in 1974 by Schleifer and Eldefrawi. Using tritiated nicotine with specific activity of 2.75 Wmmol, they were able to demonstrate specific binding to mouse brain synaptosomal membranes (Kd 7.3 nM; B,,, 3.1 pmol/g tissue). An improved assay procedure enabled Yoshida and Imura (1979) to employ nicotine of considerably lower specific activity (250 mCi/mmol) to measure nicotine binding to rat brain synaptosomes. Such binding studies were resumed with brain preparations from several species when nicotine of much higher specific radioactivity became available. In 1980, two groups using tritiated nicotine with an activity of greater than 22 Ci/mmol demonstrated stereoselective binding to rat brain membranes (Romano and Goldstein, 1980; Abood et al., 1980). The existence of specific nicotine-binding sites in rat brain was confirmed in a number of laboratories (Martin and Aceto, 1981; Romano et al.,
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1981; Ikushima et al., 1982; Balfour and Benwell, 1983; Costa and Murphy, 1983; Clarke et al., 1984; Sloan et al., 1984; Sugiyama et al., 1985; Nordberg et al., 1985; Whiting and Lindstrom, 198613; also see below). Similar measurements were performed in mouse brain preparations (Sershen et al., 1981; Marks and Collins, 1982; Larsson and Nordberg, 1985), as well as in chicks (Whiting and Lindstrom, 1986b; also see below). In much of this work multiple binding sites comprising a wide spectrum of affinities were observed (e.g., Sloan et al., 1984, who reported Kd values ranging from 20 pM to 50 pM and higher). High-affinity sites (Kd values in the vicinity of 10 nM) have been described by most investigators. Low-affinity binding sites were usually also observed. At least some of this pronounced heterogeneity may be attributable to different incubation and assay conditions used in different laboratories; thus the dependence on temperature and pH (Vincek et al., 1980) and the possibility of proteolytic damage of the binding proteins (Lippiello and Fernandes, 1986) have been noted. More recent research suggests that poor reproducibility was largely due to the use of racemic mixtures of tritiated nicotine in the earlier studies. McKennis and colleagues (McKennis et al., 1961) were the first to synthesize the radiolabeled ( - )-isomer by methylating ( - )-nornicotine with [l4C]forma1dehyde. The resulting ( -)- [14C]nicotine,however, had a specific activity of less than 0.1 Ci/mmol. In 1980, (-)-[SH]nicotine with a specific activity of over 5 Ci/mmol was produced; preliminary binding studies on rat brain revealed a Kd value of 4.6 X l o p 7 M (Vincek et al., 1980), later revised to 6.3 X l o p 8 M (Vincek et al., 1981). Apart from these early measurements, results obtained subsequently with the optically pure isomer have been fairly reproducible between laboratories. Abood et al. (1983a) found two binding sites, with K d values of 0.2 and 1.7 nM, respectively; the less abundant high-affinity site was later shown to exhibit positive cooperativity (Abood et al., 1985). Lippiello and Fernandes (1986) measured a K d of 2.0 nM for a single noncooperative site, Jenner et al. (1986) found one of 6.3 nM, and Martino-Barrowsand Kellar (1987) determined a Kd of 3.5 a,also for a single type of noninteracting binding sites. In human brain, two high-affinity binding sites (Kd values of 8.1 and 86 nM) were observed (Shimohama et al., 1985). The distribution of nicotine-binding sites in the brain has been investigated both biochemically, following microdissection, and neuroanatomically, using autoradiography. Marks and Collins (1982), using the biochemical approach, found high levels of nicotine-binding sites in the midbrain and the striatum, intermediate densities in cortex, hindbrain, hippocampus, and hypothalamus, and little activity in the
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cerebellum of rats, a distribution later basically confirmed by Marks et al. (1986) and Martino-Barrows and Kellar (1986). For mouse brains, findings were similar, except that levels in the striatum were relatively low (Marks et al., 1986). Autoradiographic analysis of nicotine binding, first attempted by Schmiterlow et al. in 1967 (the experiment inevitably failed because of the low specific activity of the ligand used) and successfully accomplished much later by Clarke et al. (1984), permitted a more detailed mapping of receptor distribution in the central nervous system. In confirmation and extension of the biochemical work on rat brain, high densities were found in the interpeduncular nucleus, many thalamic nuclei, medial habenula, superior colliculi, and the molecular layer of the dentate gyrus, and moderate levels in a large number of structures including cortex, substantia nigra, and tegmentum, whereas hypothalamus and hippocampus were largely devoid of binding activity (Clarke et al., 1985; London et al., 1985b). In human brains, binding site density varied about 4-fold between regions tested, with the nucleus basalis of Meynert and the thalamus containing the highest levels. High-affinity nicotine-binding proteins have been isolated from chick and rat brains by means of immunosorbent chromatography (Whiting and Lindstrom, 1986a; see below). In chicks, two subtypes of such a nicotine receptor were identified, one of which is comprised of subunits of 48 and 59 kDa, whereas the other is made up of a 48- and a 75-kDa peptide chain. The smaller subunit was found to interact with an antimain immunogenic region monoclonal antibody (Whiting et al., 198713) and with antisera to the Torpedo a subunit (Whiting and Lindstrom, 1986a); since the 48-kDa subunits from both receptor types yield indistinguishable peptide maps, they are likely to be identical (Whiting et al., 1987b). Using immobilized anti-chick brain receptor antibodies, Lindstrom’s group also has isolated a nicotine-binding protein from rat brain which consists of subunits of 51 and 79 kDa (Whiting and Lindstrom, 1987a). Similar receptors have meanwhile been identified in bovine and human brains (Whiting and Lindstrom, 1987~). The high affinity for nicotine has been exploited for the construction of affinity resins; using hydroxyethyl-nicotine linked to agarose, Abood et al. (198313) partially purified the nicotine-binding activity from rat brain. More recently, the same group has reported on the use of immobilized antiidiotypic antibodies (directed against the binding site of antinicotine monoclonal antibodies) as an affinity resin (Abood et al., 1987); both purification protocols yielded preparations of comparable specific activity (80-250 pmoles/mg protein) and comprising a 65-kDa polypeptide as their major constituent. In general, it is assumed that (+)-nicotine binds to the same site, albeit with lower affinity (see Martin, 1986). Direct binding studies with
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the tritiated (+)-isomer revealed an affinity about three times lower than that of the (-)-isomer (Vincek et al., 1981). Most investigators have noted that the (+)-isomer is less potent by over an order of magnitude either as an agonist in behavioral and physiological experiments or, biochemically, as a displacer of ( -)-[3H]nicotine. In one report, the suggestion was made that it may produce specific effects of its own (Ikushima et al., 1982). 2. Other Agonists
Tritiated acetylcholine (ACh) of rather low specific activity (which rendered it marginally useful for work in vertebrate brain) had originally been employed in work on tissues of high binding site density, such as electric organ or insect nervous tissue (see e.g., Eldefrawi et al., 1971). Tritiated ACh was first tried as a receptor ligand in mammalian brain by Schleifer and Eldefrawi (1974). These authors reported the presence in mouse brains of nicotinic ACh-binding sites at a tissue concentration of 5.6 pmol/g that bound [3H]acetylcholinewith a K d value of 12.3 nM, remarkably close to the values reported much later. In 1982, Kellar and his colleagues (Schwartz et al., 1982) introduced the use of ACh of high specific activity, which they synthesized by acetylation of commercially available [3H]choline. This label has subsequently been used for biochemical binding studies as well as for autoradiographic localization of both nicotinic (Rainbow et al., 1984) and muscarinic (Schwartz, 1986) binding sites. Recently, tritiated N-methyl-carbamoylcholinehas also been introduced as a more stable and more selective alternative to tritiated ACh (Boksa and Quirion, 1987). The most potent blocker of nicotinic ACh-binding sites is the plant alkaloid cytisine, which, however, has found only limited use as a receptor label. To my knowledge, only the group of Demushkin has prepared tritiated cytisine, using it as a probe for the nicotinic acetylcholine receptor in the nervous system of the squid (Demushkin and Kotelevtsev, 1982). Decamethonium in tritiated form has also been used, mostly in invertebrate preparations such as fly heads (Mansour et al., 1977) and squid optic ganglion (Kato and Tattrie, 1974). 3. Antagonists
In biochemical studies of neurotransmitter receptors, antagonists have frequently been preferred due to their superior affinity, stability and selectivity. Since nicotinic receptors in the vertebrate brain largely resemble those in autonomic ganglia (Curtis and Ryall, 1966; Brown, 1979), the ganglion blockers appear as promising biochemical labels. a. Ganglionic Blockers. The classical blocker hexamethonium (after which the ganglionic receptor was named “C6”) is of little use for the
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pharmacologist because of its inability to pass the blood-brain barrier, and for the receptor biochemist because of its low receptor affinity. Ganglionic blockers that do not share these deficiencies include mecamylamine, a secondary amine that in many studies has been documented as a selective blocker of nicotine’s central actions. Binding studies with these drugs seem to have been attempted (e.g., see the discussion section of Williams and Robinson, 1984), but to my knowledge no experiment involving radiolabeled mecamylamine (or of similar drugs such as pempidine, trimethaphan, chlorisondamine) has been published. b. Neosumgutoxin. Hayashi et al. (1984) found that neosurugatoxin, the toxic principle from the ivory shell Babyloniajuponica, blocks binding of radioactive nicotine to rat brain membranes with an IC50 value of 70 nM, better than that of any other antagonist; this binding is of a noncompetitive nature (Yamada et al., 1985). They later showed that neosurugatoxin exerts a powerful block on nicotine-induced antinociception: systemically applied, it proved to be far more potent than mecamylamine, strongly suggesting that it was acting centrally (Yamada et al., 1986). Neosurugatoxin has also been shown to block the nicotinic agonist-evokedrelease of dopamine from striatal nerve terminals (Rapier et al., 1985). To my knowledge, neosurugatoxin has not yet been employed in a radiolabeled form for direct binding studies. c. Lophotoxin. The same appears to hold for lophotoxin, a diterpene lactone from Pacific sea whips of the genus Lophogorgia that binds with high affinity to an acetylcholine-bindingsite on the skeletal muscle receptor and also produces a prolonged block of nicotinic transmission in autonomic ganglia (Langdon and Jacobs, 1985; Sorenson et al., 1987). d . Hzktnbnicotoxin. Histrionicotoxin, an alkaloid from the tree frog Dendrobates histrionicus that is a potent blocker of the acetylcholine receptor at the neuromuscular junction, also inhibits neuronal receptor function, but it does so only at very high concentrations ( -0.1 mM) (Stallcup and Patrick, 1980). Not surprisingly, when the compound was tested in tritiated form in the avian retina, it exhibited too much nonspecific binding (i.e., binding not displaceable by nicotinic cholinergic ligands) to be of much use as a receptor ligand (Betz, 1982). e. D-Tubocururzhe. For the same reason, many investigators in recent years seem to have avoided D-tubocurarine. Its binding to a nicotinic type of binding site in the squid optic ganglion has been reported (Demushkin and Kotelevtsev, 1980). Another report mentions the use of this classical nicotinic antagonist in a binding study with mouse brain; Larsson and Nordberg (1985), comparing the binding activities of aBuTk, nicotine, and D-tubocurarhe, found two types of high-affinity(Kdvalues
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of 2 and 14 nM) binding site for the latter. They also observed that in contrast to toxin and nicotine, D-tubocurarine binds rather uniformly to all brain regions tested. It is not obvious that D-tubocurarine should be labeling only nicotinic AChR in the central nervous system, in which it exhibits primarily excitatory activity and in which it has been reported to bind to noncholinergic receptors (Hill et al., 1972). f: Dihydro-P-eythroidine.Williams and Robinson (1984) investigated rat brain using radiolabeled dihydro-fl-erythroidine, a widely used blocker of neuronal nicotinic responses; they found two binding sites whose dissociation constants they estimated to be 4 and 22 nM. Binding to the high-affinity site was potently inhibited by cholinergic agonists (cytisine > (-)-nicotine, lobeline > acetylcholine, (+)-nicotine > anabasine) and was sensitive to the muscarinic agents arecoline, atropine, and oxotremorine, whereas the nicotinic antagonists mecamylamine, hexamethonium, and pempidine were inactive in displacing the compound. The highest site density was found in the thalamus, followed by hypothalamus, hippocampus, and other brain regions. In both regional distribution and drug potency series, the site labeled by dihydro-fl-erythroidineclosely resembles the nicotinic binding site identified in agonist binding studies (see Schwartz et al., 1982; Marks et al., 1986; Martino-Barrows and Kellar, 1986). g . Substance r! Several lines of evidence suggest that substance P exerts a blocking action on nicotinic responses. At the Mauthner cellgiant fiber synapse of the hatchetfish (Steinacher and Highstein, 1976), in cat Renshaw cells (Belcher and Ryall, 1977; Krnjevic and kkic, 1977), and in bovine chromaffin cells (Livett et al., 1979), nicotinic activation is inhibited by this neuropeptide. In the rat interpeduncular nucleus, a proximity of substance P immunoreactivity and toxin-binding sites has been noted (Rotter and Jacobowitz, 1984). Based on experiments with the clonal cell line PC12, Stallcup and Patrick (1980) have speculated that substance P may act by binding to the nicotinic receptor at a regulatory site other than the agonist-binding site or the ion channel, causing enhancement of receptor desensitization. However, autoradiographic maps of radiolabeled substance P do not resemble the distribution of agonist receptors (Rothman et a l . , 1984). Furthermore, in embryonic chick sympathetic ganglia, the physical separation of the substance P target from the nicotinic receptor has been demonstrated using patch clamp techniques (Simmons et al., 1987). Galanin, a 29-residue neuropeptide, has been reported to inhibit excitatory postsynaptic potential (EPSP)-like responses to ACh in the guinea pig myenteric plexus (Tamura et al., 1987), but like substance P, it may accomplish this through action at a distance.
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B. PROBES SPECIFIC FOR THE ACHR IN THE NEUROMUSCULAR JUNCTION 1. Curarimimetzc Protein Toxins
Curarimimetic toxins are small proteins in the venoms of elapid and hydropheid snakes; they have played a crucial role in the purification and characterization of muscle-typenicotinic AChR and were used extensively in attempts to identify neuronal nicotinic receptors as well. Although the physiological significance of the toxin-binding sites in the nervous system has remained unresolved (for further discussion, see below), there have been several important developments in the use of these toxins since 1980. a. a-Bungarotoxin. By 1980, neuronal toxin receptors in many vertebrates from fishes to primates and even in invertebrates were already fairly well characterized with respect to drug-binding properties and neuroanatomical distribution; here it may suffice to mention that they preferentially bind nicotinic cholinergic drugs, with affinities for agonists in general somewhat higher than for antagonists, and that their distribution across tissues, cells, and subcellular compartments generally agrees with what is expected of a nicotinic receptor (see Morley et a l . , 1979; Schmidt et al., 1980; Oswald and Freeman, 1981; Morley and Kemp, 1981). A more detailed molecular description of these receptors, however, had to await advances in protein analysis such as gel-stainingprocedures of increased sensitivity, Western blotting, and microsequencing techniques. Much of this biochemical work was done with preparations selected either for their high receptor content or their general neurobiological interest. i. Punyication and biochemical characterization of toxin receptors. The optic lobe of newly hatched chicks has been known as a rich source of toxin receptors for over a decade; tissue concentrations of over 30 fmol/mg tissue have been reported (Kouvelas and Greene, 1976; Wang and Schmidt, 1976). An early attempt at structural characterization using a cross-linking approach yielded an estimate of a molecular mass of 52 kDa for the toxin-binding subunit (Marchand et al., 1977). Barnard and his group (Norman et a l . , 1982) observed a single peptide of 54 kDa in affinity-purified receptor, which could be labeled with bromoacetylcholine; the labeling could be blocked by toxin. Betz et al. (1982) identified several polypeptides (approximate molecular masses of 57, 35, and 25 kDa) associated with the toxin receptor. Monoclonal antibodies raised against this brain protein were found to cross-react to some extent with the toxin receptor from PC12; no cross-reactivitywas observed with peripheral nicotinic receptors (Betz and Pfeiffer, 1984). In 1985 three
NICOTINIC ACETYLCHOLINE RECEPTORS
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subunits of 48, 56, and 69 kDa were identified in the purified receptor that were shown to be receptor related by several criteria: (1) Sequencing revealed that the amino-terminal portion of the 48-kDa peptide resembles the corresponding segment of the muscle type AChR a subunit (the larger subunits turned out to have blocked termini). (2) All three subunits could be precipitated with a monoclonal antibody against chick muscle receptor. (3) The 56-kDa peptide could be affinity labeled with bromoacetylcholine (Conti-Tronconi et al., 1985). The density of toxin-binding sites in rat brain, roughly 5 fmol/mg, is considerably lower than that in the chick optic lobe; however, much of the early work on toxin receptor identification, pharmacology, and neuroanatomy had been accomplished with this preparation, and as a result considerable effort went into the isolation of this particular receptor. Kemp et al. (1985) purified the toxin receptor by means of aBuTxSepharose and found subunit peptides of 55, 53.5, and 49 kDa. Occasionally, especially upon storage, a band of 46 kDa was noted but was attributed to proteolysis of higher molecular weight peptides. The affinity label maleimido-benzyltrimethylammoniumwas found to bind to the 55-kDa subunit. Using a mixed ligand approach (i.e., simultaneous interaction of the binding protein with toxin-Sepharose and 1251-labeled toxin) they could show that there are two toxin-binding sites per receptor molecule. Whiting and Lindstrom (1987a) subsequently found four subunits, of 44.7, 52.3, 56.6, and 65.2 kDa, noting their striking similarity with the C10 or muscle type AChR. Seto et al. (1981) investigated the subunit composition of the a-bungarotoxin-binding component in mouse brain, and interpreted their findings as supporting a homooligomeric structure made up of several 52-kDa peptides. Analysis of purified toxin receptor by SDS-PAGE and Western blots has revealed that high-molecular-weight components (Mr of 200,000 and 120,000)from both bird and fish and even from invertebrate (ApZysiu calijornica), but not mammalian, brain are labeled with 1251-labeled aBuTx. Smaller peptides that are detected in purified receptor preparations lack binding activity and do not seem to oligomerize to make up the high-molecular-weight binding components, since reduction and alkylation of receptor samples does not abolish the binding activity (Hawrot et al., 1986; McLaughlin and Hawrot, 1987). At present, the relationship of these seemingly distinct toxin receptors to the oligomeric type is unclear. ii. Utilization of toxins in work on invertebrate receptors. Soon after curarimimetic snake toxins became available and their highly specific association with peripheral nicotinic receptors had been demonstrated,
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their usefulness for the identification and analysis of invertebrate receptors began to be explored. A considerable fraction of this early work was devoted to insects, especially the fruit fly (reviewed by Dudai, 1979). Emphasis since then has been on the purification and biophysical characterization of these putative receptors. March et al. (1982) reported on the purification of a toxin-binding component from Musca domestica that had nicotinic binding properties and comprised subunits of 42 and 26 kDa. Hall and her collaborators, working with Drosophila melanogaster, observed a more complex subunit structure, with polypeptide sizes of 100, 79, 65, 57, 42, and 24 kDa, in purified toxin receptors (March et al., 1982). In 1983 Filbin et al. reported on the biochemical characterization of an AChR with nicotinic properties from the supraesophageal ganglion of the locust Schistocerca gregari'a. In partially purified preparations they saw three major bands of 60,41, and 25 kDa, of which the largest could be specifically labeled with [SH]maleimido-benzyltrimethylammonium. Another locust, Locusta migraton'a, provided Breer and his colleagues with the starting material for receptor isolation (Breer et al., 1985). These workers found only one subunit of 65 kDa, prompting them to propose a homotetramer structure for the toxin receptor. They also observed that the receptor was recognized by several monoclonal antibodies against the Torpedo receptor. Using basically the same approach, Sattelle and Breer (1985) purified a nicotinic AChR from the central nervous system of the cockroach Perz'planeta americana and determined that it too contained a single polypeptide of 65 m a . The protein isolated from Locusta migratoriu was incorporated into planar lipid bilayers and shown to impart nicotinic cholinoceptive properties to the membrane (Hanke and Breer, 1986). A general comment on receptor subunit analysis may be in order. It is now generally accepted that all muscle-type AChR are composed of four types of peptide chains. However, this has not been easy to establish because of the proteolytic lability of one or more of the peptide chains. In addition, staining intensities of individual subunits may not reflect actual recoveries. Thus, it took special precautions during the extraction of Electrophorus electricus tissue to preserve the integrity of the 6 subunit (Lindstrom et al., 1980); in the case of the Torpedo electroplaque receptor, a structure composed of only two subunit types was championed as late as 1980. It is therefore likely that the homooligomeric structure proposed for mouse brain receptor (Seto et al., 1981) is due to a proteolytic artifact, especially since the corresponding protein from rat brains is composed of several different subunits. Eventually, the subunit composition of the toxin receptors from Locusta and Per@laneta may have to be revised also.
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b. Identzfcation and Use of Neuronally Active Snake Venom Toxins. As early as 1972, the presence of at least two proteins with curarimimetic properties in the venom of Bungarus multicinctus had been reported (Lee et al., 1972); Bennett later obtained indirect evidence for a whole family of a-toxins in this venom (Eterovic et al., 1975). Since curarimimetic snake poisoning is frequently accompanied by autonomic manifestations (Lee and Lee, 1979; Campbell, 1979), one might expect the presence in the venom of toxins active at ganglionic synapses, perhaps directed against the ganglionic nicotinic AChR. Such toxins were indeed discovered as contaminants in commercial a-bungarotoxin preparations, some of which inhibited nicotinic transmission in the chick ciliary ganglion, although others did not (Chiappinelli and Zigmond, 1978). Zigmond and his collaborators found that the activity was due to the presence of a small quantity of a toxic protein different from the musclespecific a-toxin (Chiappinelli et al., 1981). Earlier, Ravdin and Berg (1979) had isolated a toxic peptide from Bungarus multicinctus venom that was capable of blocking nicotinic responses in the chick ciliary ganglion and that the authors referred to as Bgt 3.1 (as compared to the major muscle-specific toxin, designated Bgt 2.2). Chiappinelli coined the term x-bungarotoxin (Chiappinelli, 1983) for his neuronally active toxin, and Loring et al. (1984) named their protein toxin F. Complete sequence analysis of toxin F and x-toxin has revealed that they are the same protein (Grant and Chiappinelli, 1985). This also is likely to hold for toxin Bgt 3.1; it has the same molecular weight, and an N-terminal portion has been sequenced and found to be identical to that of x-toxin and toxin F (Loring et al., 1986). For reasons of simplicity and impartiality (and in the spirit of alliterative philhellenism) the term vBuTx (for a new, neuron-specific bungarotoxin) will be used to refer to this toxin in the remainder of this review. The neuronally active toxin, vBuTx, has been used extensively in chick ciliary ganglion (Ravdin and Berg, 1979; Ravdin et al., 1981; Chiappinelli, 1983; Dryer and Chiappinelli, 1983; Loring et al., 1984; Halvorsen and Berg, 1986), in which it was recently shown to bind specifically to synaptic membranes (Loring and Zigmond, 1987). It has also been found to display blocking activity in several other autonomic preparations such as chick sympathetic ganglia (Chiappinelii and Dryer, 1984) and the rat superior cervical ganglion, in which its affinity for nicotinic receptors is lower (Loring et al., 1983; Chiappinelli and Dryer, 1984). Toxins of similar specificity have been found in the venoms of Bungarus multicinctus (a-bungarotoxin 3.3) (Ravdin and Berg, 1979) and other snakes (x-flavitoxin from Bungarus flaziceps) (Chiappinelli et al., 1987). Skeletal muscle receptors also interact with vBuTx, but with low affinity ( K d 1.3 x 10-7 M for chick muscle receptor) (Chiappinelli,
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1985), as do aBuTx receptors present on chick ciliary ganglia (Halvorsen and Berg, 1986) and chick and rat retina (Loring et al., 1987; Aizenman et al., 1987). The biochemistry of specific vBuTx sites can be studied in the presence of sufficient aBuTx to saturate aBuTx sites. This research has recently been reviewed in considerable detail by Chiappinelli (1985). Based on their inhibitory activity and the predominantly synaptic localization of their specific targets, there is little doubt that these toxins label the nicotinic receptor in several autonomic ganglia. It is surprising that they have found little use in studies of the central nervous system. a-Bungarotoxin 3.3 has been used to identify nicotinic receptors in the rat hypothalamus; targets for this toxin and the major toxin active at the neuromuscular junction (Bgt 2.2) were found to be coextensive in autoradiographic analysis (Miller et al., 1982), suggesting that both toxins there may bind to a bona fide nicotinic AChR. Recent reports have described receptors for toxin F in chick retina (Loring et al., 1987), in rat retina (Aizenman et al., 1987), and in rat brain (Schulz and Zigmond, 1987) but have also sounded a note of caution: only half of the central binding sites have a nicotinic pharmacology and, in contrast to receptors recognized by other ligands, binding site density appears quite uniform throughout the brain. It cannot, therefore, be taken for granted that vBuTx reliably labels nicotinic receptors in the brain, as it obviously does in autonomic ganglia. 2. Antibodies as Probes f o r Neuronal AChR As shown below, antimuscle (or electroplax) receptor antibodies, both polyclonal and monoclonal, have been used to establish or refute structural relationships between AChR in muscle and toxin or agonist receptors on neurons. Since they had been shown to block ganglionic nicotinic receptors (Patrick and Stallcup, 1977), they have also been employed as ligands in their own right for the identification of neuronal nicotinic receptors. This research led, in Lindstrom’slaboratory, to the identification of a macromolecule in the chick brain that is recognized by a monoclonal antibody (mAb 35) directed against the main immunogenic region on the peripheral receptor (Lindstrom et al., 1983). Within the optic lobe of the chick brain, it was found to be localized to the spiriform nucleus (Swanson et al., 1983). Anti-main immunogenic region antibodies also labeled a synaptic membrane component in the chick ciliary ganglion (Jacob et al., 1984) that was later shown to be an integral membrane glycoprotein separable from the toxin receptor and displaying a tissue distribution expected for a nicotinic receptor (Smith et al., 1985). When the ACh sensitivity of the ganglion changes in response
NICOTINIC ACETYLCHOLINE RECEPTORS
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to cholinergic agonists or vBuTx, the antibody-binding sites in most cases are comodulated (Smith et al., 1986). Using the immobilized antibody, Lindstrom and his colleagues purified the agonist receptor from chick optic lobe and, performing an immunological cross-reactivity “walk’ or “bootstrap” operation, from rat brain as well (Whiting and Lindstrom, 1986a, 1987a). Antibodies to purified rat brain receptor in turn exhibited cross-reactivityto bovine and human agonist receptors (Whiting and Lindstrom, 1987~).In addition, these antibodies have been used to analyze subunit stoichiometry and to study receptor subtypes (for the remainder of the story see Section 111,A). 3 . cDNAs as Probes f o r AChR Genes and Their Transcrz$ts
The introduction of molecular cloning techniques has greatly facilitated the structural and functional analysis of the muscle type receptor and promises to be of similar importance in the study of neuronal receptors. The first steps toward the “molecular electrophysiology” of the neuronal nicotinic receptors have already been taken. Initially, Ballivet and his colleagues, on screening a chicken genomic library, discovered a variant receptor gene (Mauron et al., 1985). Since it exhibited more sequence similarity to known a genes than to other subunits, in particular the same arrangement of four cysteine residues in the presumptive extracellular region, they named it a-2 and speculated that it might encode a subunit of either an embryonic muscle receptor or a neuronal nicotinic receptor. Since then, Heinemann and Patrick and their collaborators have characterized two additional a-like and a p-like (or non-a) receptor sequences of neuronal origin. Screening a PC12 cDNA library with a mouse muscle receptor a-subunit probe they identified a sequence with unmistakable a-subunit features (Boulter et al., 1986). This gene, named a-3, was found, using in situ hybridization experiments, to be strongly expressed in the medial habenula and moderately in various other regions of the rat brain, including tegmentum, substantia nigra, thalamus, medial geniculate nucleus, and neocortex (Goldman et al., 1986). In cDNA libraries prepared from rat brain, two additional a-like messages were found that presumably arise by alternate splicing from an additional gene, termed a-4(Goldman et al., 1987); they are expressed in many parts of the rat brain. Recently, the equivalent of a - 2 has also been identified in rat brain and localized primarily to the hippocampus and the interpeduncular nucleus (Wada et al., 1987b). A receptor-like gene without cystine 192-193 has been identified and given the designation p-2 (Deneris et al., 1987). Two types of evidence suggest that it is
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functionally linked with a-type subunits: in the Xenopus laems oocyte expression system its message complements both a - 3 and a - 4 mRNA in the expression of functional nicotinic receptors (Boulter et al., 1987), and its distribution in the brain as measured by in situ hybridization resembles that of a - 3 and a - 4 combined. 111. Relationships of Receptor Candidates
A. RELATIONSHIP OF THE TARGETS OF VARIOUS -THE AGONISTRECEPTOR NICOTINIC DRUGS An excellent case can be made for the identity of the sites recognized by (-)-nicotine and by acetylcholine (if used in the presence of muscarinic blockers). The regional distribution of binding sites for the two ligands completely overlap when examined at the light microscopic level (Clarke et a l., 1985). Biochemical studies reveal that the two sites are similar in concentration in individual regions of the rat brain and undergo the same changes in density in response to experimental manipulations, such as prolonged nicotine exposure, or as a result of pathological processes, such as those occurring in Alzheimer’s disease. Binding of both drugs is equally sensitive to the presence of cholinergic ligands, to treatment with dithiothreitol and subsequent reoxidation with dithionitrobenzoate, to heat, and to proteases (Marks et al., 1986; Martino-Barrows and Kellar, 1987). At this point it seems advisable to introduce a common term for this neuronal membrane component; for the remainder of this review it will be referred to as the “agonist receptor.” The striking differences seen in agonist receptor preparations isolated from rat brain by the groups of Lindstrom and Abood remain to be clarified. Since the specific activity of the material purified by Abood et al. (1987) was fairly low (one ligand-binding site per 4000-12,500 kDa) (Abood et a l . , 1987), the possibility that the major polypeptide constituent of 65 kDa represents a contaminant cannot be excluded. Among the antagonists, dihydro-0-erythroidine seemingly binds to the agonist receptor with high affinity, whereas D-tubocurarine is likely to bind to other sites as well. Other antagonists, especially the classical ganglionic blockers, do not block agonist binding, but whether this is because they interact with entirely different receptors or because they bind to the ion channel, away from the ACh-binding site on the same receptor molecule, remains to be established. The most potent of them, mecamylamine and neosurugatoxin, have not been available in radioactive form, and their direct interaction with purified agonist receptor has not been tested.
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B. NONIDENTITY OF THE TOXIN AND AGONISTRECEETORS Several papers have specifically dealt with the relationship of agonistand toxin-binding sites. Drug-binding studies alone could not definitively distinguish between two separate binding sites on the same receptor and two separate receptors. Several lines of evidence have, however, convincingly documented that toxin receptors and agonist receptors are distinct entities. It was noted early on that the regional distribution of toxin- and agonist-binding sites did not correlate well (Schwartz et al., 1982; Marks and Collins, 1982); this observation has been repeatedly confirmed (e.g., Whiting and Lindstrom, 198613). Subsequently, light microscopic autoradiographic analyses of binding sites firmly established that the two binding sites had distinct and near-complementary regional distributions in the rat brain and could not possibly reside on the same macromolecule (Clarke et al., 1985). Clark et al. (1986), in a study of the rat interpeduncular nucleus, noted the unique and nonoverlapping subnuclear localization of agonist- and toxin-binding sites. In the chick ciliary ganglion, the toxin receptor resides in extrasynaptic regions (Jacob and Berg, 1983; but see Cocchia and Fumagalli, 1981), whereas the monoclonal antibody mAb35, which was subsequently shown to recognize the agonist receptor (Whiting and Lindstrom, 1986b), interacts with a synaptic constituent (Jacob et al., 1984), in addition to exhibiting low affinity for extrasynaptic toxin receptor. Separation of the two binding activities has also been accomplished by biochemical means (Schneider et al., 1985; Whiting and Lindstrom, 1987c; Wonnacott, 1986). The temporal segregation of binding activities similarly argues for two separate receptor molecules. Different time courses have been observed for the agonist and toxin receptors during development (Fiedler et al., 1987) and in response to nicotine treatment (Marks et al., 1983, 1985, 1986). C. IMMUNOREACTIVE PROTEINS As described above, a small fraction of antibodies raised against muscle-type receptors interact with the neuronal toxin receptor, whereas others recognize the agonist receptor. These antigenic similarities strongly suggest a common origin for this group of membrane proteins.
1. Antibodies and the Toxin Receptor Ever since Patrick and Stallcup (1977) reported that antibodies to peripheral AChR block the function of nAChR on sympathetic neurons,
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the expectation has been that antibodies would be useful tools for the characterization and, eventually, isolation of neuronal AChR. Early work in this field attempted to establish whether muscle-specific antibodies were capable of recognizing aBuTx receptors in neuronal preparations. Patrick’s initial observations suggested that antibodies to AChR from the electric organ of Electrophorus electricus, which block synaptic transmission in sympathetic ganglia, did not recognize the toxin receptors present on the same cells. In a more detailed study of several antisera directed against peripheral AChR, Morley and colleagues (Morley et al., 1983a)came to the conclusion that there was indeed no cross-reactivity. Conversely, Betz and Pfeiffer (1984) found that antibodies raised against the aBuTx receptor from chick optic lobe were unable to recognize skeletal muscle AChR. The majority of cross-reactivitystudies has yielded a somewhat different picture, suggesting that there is a subtle but detectable structural similarity. As early as 1978, Fulpius and his collaborators (Fontana et al., 1978) described antisera exhibiting activity against both muscle AChR and central nervous system toxin-binding sites. Block and Billiar reported in 1979 that 60% of rat brain toxin receptors cross-react with anti-Torpedo receptor antibodies. Similarly, Betz (1981) found that anti-Torpedoreceptor antiserum recognizes the toxin-binding protein from chick retina. A monoclonal antibody directed against the Torpedo receptor toxinbinding site was also reported to interact with mouse brain membranes (Mochly-Rosen and Fuchs, 1981). Antiserum against cat muscle receptor was found to cross-react with toxin receptor from chick optic lobe (Norman et al., 1982), whereas the toxin receptor from rat brain was recognized by an anti-rat muscle receptor antiserum (Wonnacott et al., 1982; Mills and Wonnacott, 1984). In a surprising turnaround, anti-eel electroplaque receptor antisera provided by Patrick, who had so persuasively demonstrated the nonidentity of toxin receptors and functional AChR in rat sympathetic ganglia, have been shown by Lukas to inhibit toxin binding not only in PC12, a rat pheochromocytoma line with neuronal characteristics (Lukas, 1986b), but also in rat brain (Lukas, 1986~);the extent of cross-reactivity, however, was only 3-5%, much lower than the 60% reported earlier by Block and Billiar (1979). In retrospect, it is not so surprising that the limited cross-reactivity between peripheral and central toxin-binding proteins was missed. Immunoprecipitatioas, as a rule, were carried out with the radiolabeled toxin-receptor complex as antigen, thus excluding antibodies that interact with the toxin-binding site, clearly a region of structural similarity, from the analysis. Patrick and Stallcup in their original work had considered this possibility; since neuronal cell extracts in the presence
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of excess aBuTx were found by them to inhibit immunoprecipitation of toxin-labeled muscle receptor to over 9574, they had concluded that “the failure to observe precipitation of [1251-aBuTx] -binding component complexes from PC12 is not due to occlusion of the necessary determinants by the toxin molecule.” However, the experiment only showed that some of the antibodies recognize determinants away from the toxin site of either receptor. At any rate, the presence of a certain fraction of cross-reactive toxin site-specific antibodies in anti-muscle receptor antisera is clearly compatible with the old as well as the more recent results. 2. Antibodies and the Agonist Receptor Patrick’s original suggestion that antiperipheral receptor antibodies may be used to identify neuronal receptors has been taken up by Lindstrom and his collaborators. Testing a panel of monoclonal antibodies against the fish electric organ receptor, they observed that several of the antibodies recognized a chick brain constituent with solubility and sedimentation properties similar to the muscle receptor (Lindstrom et al., 1983). A neuroanatomical analysis revealed that the antigen was localized to the spiriform nucleus (Swanson et a l . , 1983). Immobilization of the monoclonal antibody mAb35 afforded an immunosorbent that permitted purification of a protein from chick brain that contained subunits of 48 and 59 kDa (Whiting and Lindstrom, 1986a). When the drug-binding properties of this putative receptor were analyzed (Whiting and Lindstrom, 1986b), they turned out to be virtually indistinguishable from that of the chick optic lobe agonist receptor described by Schneider et al. (1985). Antibodies against this chick brain protein were subsequently found to recognize a similar protein in rat brain that consisted of a 51- and a 79-kDa subunit (Whiting and Lindstrom, 1987a). One of the antichick agonist receptor monoclonal antibodies, mAb270, proved particularly useful, as it not only recognized the rat brain agonist receptor but also enabled Lindstrom’s group to detect a second chick brain receptor with a subunit composition similar to the one in the rat brain protein (49 and 75 kDa). The two types of receptors account for essentially all the nicotine-binding activity in chick brain preparations. Although the ACh-binding subunits differ in size, they may share primary structure elements, as suggested by peptide mapping, and their drug-binding properties are indistinguishable (Whiting et al., 198713). The distribution of mAb270-binding sites in rat brain was subsequently shown by Swanson et al. (1987) to be very nearly identical to that of nicotine-binding sites (Clarke et al., 1985). That the agonist receptor interacts with antisera
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raised against Torpedo and narcine, has also been shown by Sugiyama and Yamashita (1986).
OF BINDINGPROTEINS AND MUSCLE D. RELATIONSHIP RECEPTOR-LIKE GENES AND MESSAGES
Primary structure elucidation of candidate receptor peptides and comparative analysis of peptide and mRNA distribution in the nervous system are rapidly providing answers to the question of how sequences and binding activities are related. Partial sequence analysis revealed that the large rat brain receptor subunit corresponds to Heinemann’s a - 4 mRNA (Goldman et al., 1987; Whiting et al., 1987a). Due to limited protein data, it is still unclear if a-2 or a - 3 mRNA codes for the 59-kDa subunit seen in chicken brains and likely to be present, albeit at low levels, in rat brains as well. Although sequence data have not been published yet, it appears likely, from the distribution and complementation studies mentioned aiove, that Lindstrom’s a-subunit corresponds to Heinemann’s 0-2 mRNA. [In the case of neuronally active toxins a triple terminology has persisted to this day. It is to be hoped that the confusing present nomenclature for the agonist receptor subunits and the genes and gene transcripts coding for them will soon be corrected. Whiting et al. (1987a) have recently suggested the use of the terms “AChbinding” and “structural”.] Research on the toxin receptor has also benefitted from the advent of molecular biology. Availability of a chick muscle a-subunit-specific probe and nucleotides coding for the 48-kDa subunit of the chick brain toxin receptor (Conti-Tronconi et al., 1985) has enabled Barnard and his colleagues to screen a cDNA library derived from optic lobes of l-dayold chicks and to identify and sequence a toxin receptor clone (Barnard et al., 1987).
IV. Relationship of Individual Binding Proteins to Synaptic Transmission
The binding of nicotinic drugs only suggests that a protein may have a synaptic function; its neurotransmitter receptor role must be established by additional types of evidence. In the following sections, this evidence is considered for the two candidates.
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A. A SUCCESS STORY:THEAGONIST-BINDING PROTEIN 1. The Agonist Receptor as a Nicotine Target That the agonist receptor (i.e., the high-affinity agonist-binding site) may represent a nicotine target is suggested by its affinity for that drug as well as for a number of other nicotinic activating agents. The virtual identity of the central nervous system binding sites for nicotine and ACh (in the presence of atropine) strongly suggests that physiological nicotine targets are AChR (Marks et al., 1986; Martino-Barrowsand Kellar, 1987), although additional low-affinity nicotine-binding sites may exist (such as those represented by the toxin receptor, which itself, however, may be an AChR, see Section IV,B). Among nicotinic agonists, cytisine, a widely distributed plant alkaloid, has been identified as the most potent ligand in several investigations (Schwartz et al., 1982; Schneider et al., 1985; Whiting and Lindstrom, 1986b; Martino-Barrows and Kellar, 1987). Dissociation or inhibition constants for agonists, as a rule, are several orders of magnitude lower than those for antagonists (Marks and Collins, 1982; Schwartz et a l . , 1982; Marks et al., 1986; Whiting and Lindstrom, 1986b), a conspicuous exception being dihydro-0-erythroidine (Williams and Robinson, 1984; Schneider et al., 1985; Martino-Barrows and Kellar, 1987). Chronic exposure to nicotine has been found to increase the number of agonist receptors, probably through protracted desensitization at the recognition site (Schwartz and Kellar, 1983a, 1985; Marks et al., 1983, 1985; Ksir et al., 1985); this does not necessarily support a synaptic role for these receptors but suggests that they represent sites at which nicotine acts in vivo. In support of this notion, London et al. (1985a) observed that the areas of nicotine-induced cerebral activity as detected by the 2-deoxyglucose technique coincide with the autoradiographic distribution of ( -)-[3H]nicotine. The more recent results of Gruenwald et al. (1987) confirm a dose-dependent increase in glucose mobilization in a number of regions (including the substantia nigra pars compacta, superior colliculus, interpeduncular nucleus, and lateral geniculate body), most of which also have high agonist receptor densities. 2 . The Agonist Receptor as a Synaptic Constituent
The tissue and subcellular locations of the agonist receptor strongly suggest a synaptic role. Several authors have noted the enrichment of agonist receptors in crude mitochondria1 and synaptosomal preparations (Schwartz et al., 1982; Benwell and Balfour, 1985; Schneider et al., 1985).
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Evidence for an association with neuronal cholinergic systems has been obtained by biochemical as well as neuroanatomical tissue distribution studies (Schwartz et al., 1982; Clarke et al., 1985; London et al., 1985b; Swanson et al., 1987). Although this type of analysis is far from complete (primarily because cholinergic components such as ACh, choline acetyltransferase, and hemicholinium binding sites have not been mapped systematically or at high enough resolution, and because acetylcholinesterase cannot be considered a reliable marker), it is safe to say that there is a high correlation between agonist receptor density and the presence of other cholinergic markers. It will be especially important to carry this analysis to the ultrastructural level. Although this is not feasible with small ligands such as nicotine itself, it can be done with protein labels. Receptor-specific antibodies have been shown to localize to synaptic regions in the chick ciliary ganglion (Jacob et al., 1984, 1986) and should prove equally useful in the central nervous system. What is unclear at the present time is to what extent these receptors are located pre- and postsynaptically. Nicotine is known to facilitate the release of several neurotransmitters in the brain including norepinephrin, dopamine, and serotonin, in addition to regulating its own release (Starke, 1981; Chesselet, 1984). Some of the nicotinic receptors in the central nervous system, therefore, should have a presynaptic localization. Lesion studies indeed strongly suggest the presence of agonist receptors on terminals of the habenulo-interpeduncularpathway (Clarke et al., 1986), on catecholamine- and serotonin-containingneurons in the striatum and hypothalamus (Schwartz et al., 1984; Clarke and Pert, 1985), and in all major projections of the optic nerve of the rat (Swanson et al., 1987). Chronic treatment with acetylcholinesteraseinhibitors leads to downregulation (Costa and Murphy, 1983; Schwartz and Kellar, 1983a, 1985); since acetylcholine is released at specific presynaptic sites, one may conclude that the agonist receptor has a synaptic location as well. 3. The Agonist Receptor as a Member of a Receptor Gene Family The agonist receptor is a multisubunit membrane protein that exhibits some remarkable structural similarities with the nicotinic receptor from skeletal muscle. It has a sedimentation coefficient of approximately 10 s, somewhat greater than that for the muscle type receptor and corresponding to a molecular mass of 250-300 kDa (Schneider et al., 1985; Whiting and Lindstrom, 1986a, 1987a). It contains both regulatory (i.e, ACh site-carrying) and structural subunits for which, in the case of the rat brain receptor, a 2 : 3 stoichiometry has been proposed (Whiting et al., 1987a). Some antibodies directed against epitopes on muscle type receptors (the so-called main immunogenic region on the
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a-subunit) recognize the smaller subunit of the agonist receptor. The larger subunits carry the ACh-binding sites whose reactivity toward agonists is affected by the reductive cleavage of a nearby disulfide bridge (Schwartz and Kellar, 1983b). As in the case of the muscle receptor, this has made possible the use of the sulfhydryl-reactive affinity labels bromoacetylcholine (Whiting and Lindstrom, 1986b) and maleimidobenzyltrimethylammonium (Whiting and Lindstrom, 1987b) for the covalent labeling of receptor subunits. In the C10 receptor, the two AChbinding sites differ strikingly in their reactivity toward D-tubocurarine and affinity labels, presumably as a consequence of the unique subunit environment of each a-subunit in the a2 pentamer. No such differences are seen in the agonist receptor (Whiting and Lindstrom, 1987b) possibly because the smaller number of subunit types leads to multimeric assemblies of greater symmetry. 4. The Agonist Receptor as a Functioning AChR a. Indirect Evidence. The functional significance of this neuronal receptor was investigated along several lines of inquiry. By 1986 it had become obvious that the agonist receptor in chick brains is recognized by the antimuscle type receptor antibody mAb35 (Whiting and Lindstrom, 198613). A receptor-like antigen had also been detected in chick ciliary ganglia by means of the same antibody, but attempts to block cholinergic transmission with mAb35 were unsuccessful (Smith et al., 1985). However, antisera raised in rats against the chick brain agonist receptor were found to inhibit nicotinic cholinergic responses of ciliary ganglion neurons (Stollberg et al., 1986). In addition, the ACh sensitivity of the chick ciliary ganglion was found to be comodulated with the agonist receptor-like antigen (Smith et al., 1986), whereas the toxin receptor had previously been shown to be regulated independently (Smith et al., 1983). In PCl2 cells, a monoclonal antibody (mAb270) to the same receptor “modulates” nicotinic acetylcholine sensitivity, suggesting antibodyreceptor interaction, and the antibody target is coregulated with the physiological nicotinic receptor on these cells upon treatment with nerve growth factor (NGF) (Whiting et al., 1987b). Taken together, these results strongly suggest that the immunoreactive component is the physiological AChR. b. Direct Evidence. The most convincing argument for the nicotinic receptor nature of the agonist receptor comes from the demonstration that messages coding for several subtypes of the agonist receptor, a - 2 , a-3, and a-4(the latter having been shown to code for the larger subunit of the rat brain receptor) (Whiting et al., 1987a), each combined with /3-2, upon injection into Xenopus laeuzi oocytes, impart nicotinic
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cholinoceptive properties to the host cell (Wada et a l . , 1987b; Boulter et al., 1987). Significantly, none of these receptors is blocked by aBuTx. Differences in pharmacological properties do exist, however, thus the a-2-0-2 combination is refractory to vBuTx (Wada et al., 1987b),whereas the others are inhibited by it (Boulter et al., 1987).
5 . Remaining Problems Some observations seem less readily reconcilable with a synaptic cholinoceptive role for the agonist receptor. In particular, its pharmacology requires a comment. With respect to agonist binding, tritiated nicotine and ACh bind to brain membranes with dissociation constants in the nanomolar range, yet exert pharmacological actions at micromolar concentrations (Russell, 1976; Martin et a l . , 1983). Perhaps binding in Witro prompts the receptor to undergo a conformational change to a desensitized, agonist-selective state (Romano and Goldstein, 1980; Schwartz et al., 1982), as is the case with peripheral muscle-type receptors. Another problem arises due to the inability of ganglionic nicotinic antagonists to inhibit agonist binding. One possible explanation is that these drugs interfere with receptor function by combining with a site different from the agonist recognition site; Ascher et al. (1979) have shown that in mammalian brain, certain ganglion blockers interact with the ion channel rather than the acetylcholine-binding site. Chick ciliary ganglion cells (Halvorsen and Berg, 1986), which clearly possess nicotinic receptors that are not identical with the toxin receptors found there, lack high-affinity agonist-binding sites. The same observation has been made for PC12 cells (Kemp and Morley, 1986; Whiting et a l . , 1987b). Perhaps receptors in PC12 cells and in autonomic ganglia in general are special and different from those prevailing in the CNS. At any rate, caution must be exercised when attempting to extrapolate from this cell type to any other neuron. Finally, at least one of the neuronal receptor peptides found in PC12 cells is not coordinately expressed with the physiological receptor (Smith et al., 1986). Such discrepancies may eventually be accounted for by the presence of multiple receptor-related proteins in this and other neuronal cell types.
B. A PERSISTENT CONUNDRUM: THENEURONAL a-BUNGAROTOXIN-BINDING PROTEIN 1. Phylogenetically Old Toxin Receptors
In lower vertebrates, aBuTx and other curarimimetic snake neurotoxins have been shown to block nicotinic cholinergic transmission, and consequently, the toxin-binding proteins have been accepted
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as bona fide AChR. Freeman and his colleagues (Freeman et al., 1980) observed in dendrites of goldfish tectum neurons upon administration of a-bungarotoxin a rapid irreversible block of the EPSP generated by optic nerve activation. At the same time, Schwartz et al. (1980) found that binding sites for aBuTx and for antibodies specific to AChR colocalize in the goldfish optic nerve and tectum. Kato et al. (1980) reported that erabutoxins a and b block the effect of iontophoretically applied acetylcholine on bullfrog sympathetic ganglia. Subsequently, aBuTx was shown to be active at the same preparation and to localize to synaptic membrane regions (Marshall, 1981). Later, Day et al. (1983), in a study of the Mauthner cell-giant fiber synapse of hatchetfish, observed the synaptic localization of 1251-labeledaBuTx and presumed that the toxin was labeling the nicotinic receptor. Oswald and his collaborators (Henley et al., 1986a) determined that the goldfish toxin receptor is recognized by monoclonal antibodies to the main immunogenic region in muscle receptors and have proceeded to use such antibodies to analyze AChR synthesis and transport in the goldfish visual system (Henley et al., 1986b);more recently they showed that the aBuTxbinding protein is one of two receptors with high affinity for tritiated nicotine (Henley and Oswald, 1987). The situation is similar in invertebrates. Early findings indicated that in molluscs (Shain et al., 1974) and insects (Harrow et al., 1979; Carr and Fourtner, 1980) curarimimetic toxins block nicotinic receptors. In addition toxin-insensitive receptors have been observed. An example is the cockroach metathoracic ganglion, in which aBuTx blocks the ACh response of the fast coxal depressor motoneuron (David and Sattelle, 1984), while the cholinoceptive dorsal unpaired median (DUM) neurons are rather insensitive to the toxin; differential sensitivity was shown not to arise from different target accessibility, “indicating that the possible existence of multiple [AChR] in insects should be explored further.” Evidence for multiple AChR with differing toxin sensitivity has also been reported for molluscs (Kehoe et al., 1976; Martinez-Soler et al., 1984). 2 . The Neuronal Toxin Receptor in Higher Vertebrates
The functional significance of aBuTx receptors in higher vertebrates has remained problematical. Careful analysis has shown repeatedly that toxin has no effect on cholinergic transmission in autonomic ganglia as well as in specific locations of the central nervous system (such as Renshaw cells in the spinal cord), at concentrations at which it saturates specific sites in these preparations (see the earlier reviews). This has not ruled out the possibility that it may play a neurotransmitter receptor role but clearly has put the burden of proof on the shoulders of the proponents of such a function. During the past several years the list
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of credentials of the neuronal toxin receptor was extended, but not, unfortunately, in a decisive way. a. The Toxin Receptor as a Member of the Cholinergic Receptor Family. In overall structure the toxin receptor resembles the muscle-type AChR as well as the agonist receptor: it is a membrane glycoprotein of approximately 300 kDa that consists of several subunits in the 40to 60-kDa range (see Section II,B,l,a,i). It displays the drug-binding properties and chemical reactivity expected of a nicotinic AChR (Lukas, 1984). In chick brain, the smallest of these polypeptides has an aminoterminal amino acid sequence exhibiting high homology with the muscletype a-subunit (Conti-Tronconi et al., 1985); preliminary data have appeared in the literature indicating that this subunit also resembles the muscle receptor in the sequence and disposition of putative transmembrane components (Barnard et al., 1987). In the rat brain receptor, whose subunit composition is very reminiscent of that of the muscle receptor (Whiting and Lindstrom, 1987a), Kemp et al. (1985) found evidence for the presence of two ligand-binding subunits, as is well established for the muscle AChR and has now also been demonstrated for the agonist receptor (Whiting et al., 1987a). Both brainderived receptors differ from the muscle receptor not only in their slightly bigger size (10 s versus 9 s), but also in their agonist preference, which is much more pronounced in the case of the agonist receptor and may be the consequence of tachyphylaxis, a rapid desensitization-likeprocess; an agonist-induced increase in agonist affinity has been demonstrated for the toxin receptor by Lukas and Bennett (1979), but not, to my knowledge, for the agonist receptor yet. Like the muscle type AChR and the agonist receptor, the toxin receptor has a disulfide bridge near the ligandbinding site (Lukas and Bennett, 1980), and, as in the case of the agonist receptor, it is one of the larger subunits that binds active site-directed affinity labels (Norman et al., 1982; Kemp et al., 1985). As described earlier, there is also some limited immunological crossreactivity between muscle AChR and brain toxin receptor, much of it confined to the toxin-binding site or its immediate surroundings. Since there must be a site complementary to aBuTx on both proteins, the significance of immunological cross-reactivity deduced by the use of antibodies whose antigen-combiningsites presumably resemble the toxin must remain limited. b. The Toxin Receptor as a Nicotine Target. The toxin receptor has M; see Morley et a l . , 1979; Schmidt an affinity for nicotine (& et al., 1980; Oswald and Freeman, 1981; Morley and Kemp, 1981) that is compatible with the concentrations of the drug thought to be present in the central nervous system when physiological effects are observed
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(Russell, 1976; Martin et al., 1983). Furthermore, chronic nicotine administration upregulates toxin-binding sites in the mouse brain, notably in the hippocampus (Marks et al., 1983, 1985). This would not necessarily imply a synaptic function for the toxin receptor but suggests that it (together with the agonist receptor, which is similarly regulated) is a target that nicotine reaches under physiological conditions. Correlation between toxin receptor levels and nicotine tolerance as measured by behavioral and physiological paradigms was poor, however. Increased uptake of dietary choline had earlier been shown to result in significant increases in toxin receptor levels in the rat brain (Morley et al., 1977). Miner et al. (1985, 1986) found that mice that were especially sensitive to nicotine-induced seizures exhibited higher toxin receptor concentrations in the hippocampus and argued that this correlation reflected a causal relationship. It is noteworthy that previously Caulfield and Higgins (1983) had implicated a ganglionic-type receptor in these seizures. c. The Toxin Receptor as a Synaptic and Extrasynaptic Protein. Several kinds of evidence have pointed to a synaptic localization and function of the toxin receptor. These included enrichment in synaptosoma1 fractions and a tissue localization at the light microscopic and ultrastructural levels that is largely compatible with other information concerning the anatomy of cholinergic pathways (see Schmidt et al., 1980, for details). In most cases, toxin receptors appear to be localized postsynaptically; their disappearance from the terminal fields of lesioned fiber tracts has suggested a presynaptic localization in several instances (Brecha et al., 1979; Clarke et al., 1986). In addition, toxin receptors occur extrasynaptically, either at sites without known cholinergic input (Hunt and Schmidt, 1978b) or at locations devoid of synapses, such as the dorsal root ganglion (Polz-Tejera et al., 1980). The latter situation can be explained by the elaboration of receptors in the perikaryon prior to their transport into the periphery; axonal transport of toxin receptors in the sciatic nerve (presumably in sensory fibers, as motoneuron terminals are free of toxin-binding activity) (Jones and Salpeter, 1983) has been demonstrated (Nincovic and Hunt, 1983). It is worth mentioning here that agonist receptors also have been observed in cell bodies and fiber tracts (Swanson et al., 1983, 1987; also seeJacob et al., 1986). The toxin receptor of the autonomic ganglion is located primarily in extrasynaptic areas such as chick ciliary ganglion (Jacob and Berg, 1983; Messing and Gonatas, 1983; Loring et al., 1985) and rat superior cervical ganglion (Fumagalli and De Renzis, 1984). These observations suggest the presehce of two types of nicotinic receptor: a toxin-insensitive receptor subserving synaptic transmission (and recognized by vBuTx), and a toxin-binding receptor in extrasynaptic regions. It has indeed been reported that the toxin blocks the membrane response to ACh
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iontophoretically applied to the surface of rat superior cervical ganglion cells (Dun and Karczmar, 1980). d . The Physiological Signijicance of the Toxin Receptor. There are a few reports describing blocking effects of aBuTx on neurons of higher vertebrates. Burne (1978) described an inhibitory, curarelike effect of erabutoxin b on some cholinergic responses of rat somatosensory cortical neurons; Fex and Adams (1978) noted that aBuTx reversibly blocks cholinergic inhibition in the cat cochlea. These older observations are suspect because of possible contamination of the toxin preparations used (not realized at the time) with neuroactive compounds. Nevertheless, toxin effects have also been reported more recently. A toxin-sensitive nicotinic receptor seems to be involved in the control of circadian rhythm in the suprachiasmatic nucleus of the hypothalamus (Zatz and Brownstein, 1981). In the inferior colliculus, aBuTx has been reported to block the effects of nicotinic agonists on single units (Farley et al., 1983). Toxin receptor levels in the mouse hippocampus are correlated with susceptibility to nicotine-induced seizures (Miner et al., 1986). If the neuronal toxin receptor is a nicotinic AChR, a reason must be found why the curarimimetic toxins are physiologically inert, and several explanations have been offered. Perhaps the toxin does not reach its target; Rehm and Betz (1981) observed that some treatments (phospholipase C, low pH, or detergent extraction) increase the number of toxinbinding sites in the chick retina and concluded that these sites are inaccessible to the toxin in the intact tissue, thus accounting for its ineffectiveness. That this argument is unlikely to hold for autonomic ganglia was demonstrated by Bursztajn and Gershon (1977), and for insect ganglia by Lane et al. (1982). Furthermore, inaccessibility is not a likely explanation for the ineffectiveness of the toxin at the Renshaw cell; Hunt and Schmidt (1978a) were able to label neurons in the spinal cord under conditions that leave nicotinic transmission unaffected (Duggan and Hall, 1976). Perhaps the toxin receptor is coupled to a signal transduction mechanism other than a cation channel and therefore escapes detection in experiments involving radioactive cations; Ono and Salvaterra (1981) and Fex and Adams (1978) have described toxin receptors coupled to, or containing, chloride channels. Perhaps agonists can bind in the presence of bound toxin; this was postulated, on the basis of kinetic data, by Wang et al. (1978). That toxin and agonist sites might reside on different subunits of the same receptor protein was considered by Morley et al. (1983b), whereas Lukas (1986b) more generally concluded that “structural heterogeneity of toxin binding sites (both in pharmacological and immunological terms) may account for the discrepancy--’at some sites’- between toxin binding activity and functional potency.” Perhaps
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only one of two agonist sites present on the receptor is able to bind the toxin, and signal transduction can be triggered by the activation of the toxin-insensitive site alone (Morley and Kemp, 1981). The observation that radiolabeled aBuTx interacts with a receptor immobilized on toxinagarose establishes the presence of two toxin-binding sites and strongly argues against the two agonist sites-one toxin site model (Kemp et a l . , 1985). e. Specijic Examples. A large number of structures are labeled by aBuTx in the mammalian brain (Hunt and Schmidt, 1978a; Arimatsu et al., 1981; Clark et al., 1985). In view of the possible heterogeneity of toxin receptors and their functions, it may be desirable to focus on a specific region. One of the most thoroughly analyzed areas is the rat hypothalamus, in which nicotine is known to have specific physiological effects. Although agonist receptor density is low there (Clarke et al., 1985; London et al., 1985), fairly high toxin receptor concentrations have been described in the older autoradiographic literature as well as more recently (Block and Billiar, 1981; Marks and Collins, 1982; Meeker et al., 1986; Miller and Billiar, 1986). The hypothalamic toxin receptor appears to resemble both the AChR in the neuromuscular junction and the agonist receptor: (1) it is recognized by antisera to Torpedo receptor (Block and Billiar, 1979), (2) the regional distributions of toxin receptors and binding sites for the neuronally active a-bungarotoxin 3.3 (Ravdin and Berg, 1979) are indistinguishable (Miller et al., 1982), and (3) in the supraoptic nucleus, toxin has been reported to colocalize with a peripheral receptor-specific monoclonal antibody, mAb35 (Mason, 1985). The last finding has been disputed recently by Swanson et al. (1987). Physiological effects of toxin administration have also been described. When applied locally to the suprachiasmatic nucleus, aBuTx appears to block the effect of light or cholinergic stimulation on serotoninN-acetyltransferase activity in the pineal gland (Zatz and Brownstein, 1983). Michels et al. (1987) observed that toxin potentiates the ACh-induced release of vasopressin from rat hypothalamus and they concluded that it did so by acting on a receptor that cannot easily be classified as either nicotinic or muscarinic. Toxin receptor levels in the hypothalamus are regulated by gonadal steroids (Morley et al., 1983c; Miller et al., 1984), supporting the notion that these receptors are essential links in the gonado-hypophysealfeedback loop. Altogether, it appears then that the nature and role of the toxin receptor remain difficult to pin down even within a specific region of the brain. The rat cerebellum might serve as another example. There the striking correlation of cholinergic marker enzymes and toxin receptors in folia I, IX, and X was noted a decade ago (Hunt and Schmidt, 1978a);
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it is noteworthy that the agonist receptor there has a similar distribution (Swanson et al., 1987). Recently, electrophysiological studies have revealed the presence of two types of nicotinic AChR, one with ganglionic properties that is found on Purkinje cells, the other residing on interneurons and resembling the neuromuscular type in its sensitivity to curare and aBuTx (De la Garza et al., 1987a,b). Perhaps it is no coincidence that the human medulloblastoma cell line TE671, which expresses the human skeletal muscle type nicotinic acetylcholine receptor (Lukas, 1986a; Luther et al., 1987), was derived from a cerebellar tumor (McAllister et al., 1977). The possibility that the muscle type receptor (consisting of 2 a-1, and one each of p-1, y, and 6 subunits) occurs on specific neurons of the central nervous system has not been ruled out yet. Weak signals in immunofluorescence and in situ hybridization experiments employing muscle receptor-specific probes have, however, usually been interpreted as due to cross-reactivity of different receptor molecules rather than to the presence of low levels of authentic muscle AChR (Swanson et al., 1983; Goldman et al., 1986). f: Other Roles for the Toxin Receptor? If the toxin receptor serves as a receiver for chemical signals other than ACh, it should be possible to identify such a hypothetical endogenous ligand by suitable competition experiments. An investigation of this kind, however, revealed only choline and, by implication, ACh as a potential ligand (Polz-Tejeraand Schmidt, 1983). This study could not rule out the presence of a rare and/or fragile ligand of a more exotic nature; but none has since been reported. Quik (1982) found evidence for an endogenous ligand of larger size (greater than 1 m a ) ; however, no further biochemical characterization of this inhibitor has appeared.
V. Concluding Remarks
After more than a decade of research the original belief that methods developed during the work on peripheral (i.e., neuromuscular) AChR should be adaptable to the analysis of neuronal receptors has been vindicated. The combined utilization of cross-reacting antibodies and nucleic acids probes has led to concrete results in the identification and biochemical characterization of nicotinic receptors in the central nervous system. What has been referred to as the agonist receptor in the preceding pages has turned out to be a closely related group of physiological AChR. Thus, in rodent brains three such nicotinic receptors have been identified and their regional distributions mapped. Less is known about either the structure or the function of the neuronal aBuTx receptor. Yet,
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additional evidence accumulated over the past years also points largely to a cholinoceptive role, although definitive clarification has to await reconstitution experiments involving the incorporation of the purified protein into artificial membranes or the expression of subunit-specific messages in the Xeno@w laeuzi oocyte system. The recent identification of a toxin receptor clone in a chick cDNA library (Barnard et al., 1987) is an important step toward this goal. The major finding has been that the neuronal nicotinic receptors, together with the muscle type receptor, belong to a larger gene family of ACh-gated cation channels. From considerations of subunit composition and affinity label reactivity as well as fragmentary sequence information, it appears likely that the toxin receptor likewise belongs to this diverging group of membrane proteins. Advances in the structural analysis of the GABA (Schofield et al., 1987) and glycine (Grenningloh et al., 1987) receptors suggest that chemically gated channel proteins in general may have arisen from a common ancestor. It is to be expected that in the near future the structural elucidation of other channel-containing neurotransmitter receptors and of nicotinic receptors in invertebrates will permit us to piece together the puzzle of AChR evolution. Research on nicotinic receptors in the central nervous system has also more practical implications. Since it is assumed that nicotine is the addictive (dependence-producing) component of tobacco smoke (Clarke, 1987b), a characterization of the nicotine targets involved is of great importance from a public health point of view. It is quite likely that the several agonist receptors as well as the toxin receptor serve as targets for nicotine reaching the brain, but an interaction with an additional site to which nicotine might bind at micromolar concentrations, with an affinity too low to be detectable by binding techniques, remains conceivable. The molecular mechanism of nicotine addiction, which may involve the phenomenon of upregulation touched upon in this review, and the pharmacology of smoking cessation remain topics for future research. This will require a greater emphasis on receptor metabolism and its regulation. Eventually this kind of research may contribute to an understanding of how neuronal excitability and the cholinoceptive phenotype are controlled.
Acknow ledgmentr
I would like to thank Drs. P. B. S. Clarke, R. J. Lukas, and S. Heinemann for reading this manuscript. Research in my laboratory was supported by NIH Grants NS 18839 and NS 20233.
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TH E NE UROBI0LOGY 0F N-ACETYLAS PARTYLGLUTAMATE By Randy D. Blakely Section of Molecular Neurobiology Howard Hughes Medical Institute Yale University School of Medicine New Haven, Connecticut 06510
and Joseph T. Coyle Departments of Neuroscience, Pharmacology, and Psychiatry The Johns Hopkins School of Medicine Baltimore, Maryland 21205
I. Introduction A. Acidic Amino Acid Neurotransmitters B. NAAG as a Neurotransmitter at Glutamatergic Synapses 11. Quantitative Biochemical Studies of NAAG in Neural Tissues A. Phylogenetic and Regional Distribution B. Developmental Patterns 111. Cellular Localization of NAAG A. Visualization of NAAG-Like Immunoreactivity in Vertebrate Neurons B. Excitotoxic, Mechanical, and Pathological Lesions of NAAG-Containing Pathways IV. Metabolism of NAAG in Neural Tissues A. Biosynthesis: Enzymatic or Ribosomal? B. Subcellular Compartmentation of Endogenous NAAG C. Degradation of NAAG by an N-Acetylated, a-Linked, Acidic Dipeptide-Preferring Peptidase (NAALADase) Activity: A Mechanism of NAAG Inactivation? V. T h e Search for NAAG Receptors and Their Mechanism of Action A. Electrophysiological Effects of NAAG B. In V i t ~ oBiochemical Approaches to NAAG Function VI. Conclusions References
1. Introduction
Neurons possess a rich vocabulary for the translation of electrical signals into the chemical vernacular of synaptic transmission. These chemical messages arise from compounds involved in many facets of INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 30
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Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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RANDY D. BLAKELY AND JOSEPH T. COYLE
intracellular metabolism, such as the amino acids glutamate (Glu), aspartate (Asp), and glycine, as well as molecules synthesized exclusively for neurotransmission, such as acetylcholine (ACh), the monoamines, and peptides (Snyder, 1980; Cooper et al., 1986). The neuropeptides range in size from the tripeptide thyrotropin-releasing hormone (TRH) to molecules of over 30 amino acids in length, with diversity in function enhanced by both pre- and post-translational structural modification (Mains et al., 1983; Amara, 1985). As well, neuronal chemical signaling is augmented by colocalization of peptides within neurons synthesizing the “classical” neurotransmitters (Bloom, 1984; Hokfelt et al., 1987). Because of diverse mechanisms of postsynaptic action, we are probably only beginning to understand the degree of functional complexity associated with the rich signaling repertoire possessed by both vertebrate and invertebrate neurons (Kaczmarek and Levitan, 1987; Iversen and Goodman, 1987). With our growing appreciation for the signaling complexity of neurons has come a renewed interest in delineating the set of chemical messengers secreted at central synapses. Many neurons once thought to use inhibitory amino acid neurotransmitters exclusively have been shown recently to synthesize at least one bioactive peptide (Hokfelt et al., 1987). However, the difficulties involved in unequivocally establishing the acidic amino acids Asp and Glu as neurotransmitters (Watkins and Evans, 1981; Fonnum, 1984; Robinson and Coyle, 1987) have prevented the identification of peptides potentially secreted at putative glutamatergic synapses, which make up a substantial fraction of excitatory brain synapses. In analogy to the extensive synthesis of peptides such as cholecystokinin (CCK) and somatostatin (Oertel et al., 1983; Hendry et al., 1984) within GABAergic neurons, which form a large proportion of brain inhibitory synapses, such a phenomenon seems hardly unexpected. Indeed, the brain dipeptide N-acetylaspartylglutamate (NAAG) has recently received attention as a neurotransmitter candidate for certain brain synapses biochemically and pharmacologically defined as glutamatergic (Zaczek et al., 1983; Coyle et a l . , 1986). NAAG is nonuniformly distributed within neural tissues (Miyake et al., 1981; Koller et al., 1984a), where its millimolar tissue levels (0.1-5.0 nmol/mg wet weight in rodent) probably represent one of the highest concentrations of any vertebrate brain peptide. Although the neurobiological significanceof NAAG is only beginning to be understood, a suggestion of a role as a neurotransmitter was virtually coincident with the peptide’s original isolation (Curatolo et al., 1967). Not until NAAG was isolated de novo from rodent brain as a selective, high-affinity inhibitor of membrane sites labeled by [3H]glutamate (Fig. l), however, did
NEUROBIOLOGY OF N-ACETY LASPARTY LGLUTAMATE
41
this poorly defined proposal of a neurotransmitter role take the form of an explicit hypothesis for NAAG as an endogenous ligand at a subpopulation of glutamate receptors (Zaczek et al., 1983; ffrench-Mullen et al., 1985; Koller and Coyle, 1985; Coyle et al., 1986). Zaczek et al. (1983) also demonstrated that NAAG injected into the rodent hippocampus caused cortical epileptiform activity with a potency and latency similar to that of other excitatory amino acid agonists, and markedly different from that obtained with the hydrolyzed peptide or Glu alone. Subsequent electrophysiological (ffrench-Mullen et al., 1985; Bernstein et al., 1985), lesion (Koller et al., 1984a; French-Mullen et al., 1985), ligand-binding (Koller and Coyle, 1984b; Koller and Coyle, 1985), and immunocytochemical (Anderson et al., 1986; Blakely et al., 1987b) studies, reviewed in detail below, have provided results consistent with a role of NAAG in glutamatergic systems, although this proposal has not been without its critics (Luini et al., 1984; Riveros and Orrego, 1984; Westbrook et al., 1986; Ory-Lavollke et al., 1987; Forloni et al., 1987). Given the recent focus upon NAAG as an endogenous ligand for excitatory amino acid receptors, we begin with a brief discussion of the neurotransmitter candidacy of Asp and Glu. In the subsequent sections, we have organized information relevant to the hypothesis of NAAG as a neurotransmitter at synapses, glutamatergic or other, and draw attention to certain of the more promising directions for future research into its neurobiological function(s). Although many of the metabolic and anatomical pathways described are, indeed, consistent with a role in a restricted set of glutamatergic pathways, other findings demand alternative concepts. This has become particularly apparent in studies on spinal cord, in which NAAG levels have been found to be enriched in the ventral horn and ventral roots and in which intense NAAG-like immunoreactivity has been visualized in cholinergic motoneurons (Forloni et al., 1987; Ory-LavollCeet a l . , 1987). Other findings demonstrate the presence of NAAG-like peptides in functionally related sets of neurons. Although these findings do not invalidate the hypothesis of a role at glutamatergic synapses, they argue against its generality and may herald a new perspective on the function of acidic brain peptides. A. ACIDICAMINOACIDNEUROTRANSMITTERS As early as 1954, the acidic acid L-glutamate (Glu) was shown to have potent excitatory effects when topically applied to motor cortex (Hayashi, 1954). Following the introduction of iontophoretic techniques, it became possible to demonstrate the excitatory effects of Glu and Asp
42
RANDY D. BLAKELY AND JOSEPH T. COYLE
A
2.5
5
-
2.0.
0
z-1.5. m
e
.-
10.
E
U
0 5-
NEUROBIOLOGY OF N-ACETYLASPARTYLGLUTAMATE
43
on single neurons throughout the vertebrate nervous system (Curtis et al., 1960;Johnson, 1978; Davies et al., 1980; Watkins and Evans, 1981), excitation that bears considerable electrophysiological resemblance to evoked synaptic potentials (Curtis et al., 1972; Watkins and Evans, 1981; Mayer and Westbrook, 1985). Yet, a definitive resolution of the synaptic actions of endogenous Glu and Asp has been hampered by their fundamental role in intermediary metabolism, as well as by their specific brain functions unrelated to excitatory neurotransmission (Letendre et al., 1980; Lajtha et al., 1981; McGeer and McGeer, 1981), which interfere with the unambiguous localization of Glu or Asp neurotransmitter pools (Fonnum, 1984). In additon, their pervasive excitation of neurons within the vertebrate nervous system immediately raised concerns as to their specific role in central neurotransmission (Curtis and Watkins, 1960). In the decades succeeding the original neurophysiological observations, however, the excitatory promiscuity of Glu and Asp has appeared less the result of nonspecific excitation and more a reflection of a widespread distribution of excitatory amino acid-utilizingpathways, including corticostriatal, corticospinal, corticocortico, hippocampal-septal, and intrahippocampal pathways, along with the projections of the lateral olfactory tract (LOT), cerebellar parallel and climbing fibers, and primary sensory afferents, to mention only several of the more prominent (Fagg and Foster, 1983; Fonnum, 1984). Most of these systems have been delineated on the basis of one or more of the following criteria, each with their own interpretive limitations: (1) selective decrements in the levels of Glu or Asp after viral or surgical lesions of neuronal pathways (Young et al., 1974; Hassler et al., 1982); (2) complementary lesioninduced reductions in sodium-dependent, high-affinity uptake of radiolabeled Glu, a reflection of a loss of presynaptic elements enriched in the major mechanism of transmitter Glu inactivation (Fonnum et al., 1981; Potashner and Tran, 1984); (3) autoradiographic localization of transported radiolabeled Glu/Asp in synaptic processes (Storm-Mathisen and Iversen, 1979; Ottersen and Storm-Mathisen, 1984); (4) calciumdependent evoked release of prelabeled or endogenous Glu-Asp (Fagg FIG. 1. HPLC separation of NAAG from forebrain extract. The peak of ~-[~H]glutamate displacement activity in the 2.25 M formate fraction of a Dowex AG-1 column was subjected to further purification by HPLC over a 10-pm anion exchange column. The column was eluted with a KCl gradient and eluant was monitored for (A) UV absorbance at 215 nm; (B) the ability of fractions to inhibit the specific binding of ~-[~H]glutamate to washed membranes prepared from rat cerebral cortex; and (C) the amino acid content (Aspartate -, Glutamate - - -) of the fractions after acid hydrolysis. (From Zaczek et al., 1983.)
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RANDY D. BLAKELY AND JOSEPH T. COYLE
and Lane, 1979;Malthe-Ssrenssen et al., 1980;Collins et , d . ,1981); or (5) antagonist inhibition of evoked synaptic responses (Davies et al., 1980; McClennan, 1983;Watkins, 1984). Most recently, immunocytochemical strategies, using antibodies directed against protein-coupled amino acid, have been introduced to identify putative Glu- or Asp- secreting neurons (Ottersen and Storm-Mathisen, 1984; Mad1 et al., 1986; Saito et al., 1986), although uncertain distinctions between potential neurotransmitter pools and those utilized for protein synthesis, ammonia detoxification, y-aminobutyric acid (GABA) synthesis, and many other intracellular facets of intermediary metabolism affect the interpretation from these techniques. Nonetheless, sufficient evidence from several independent strategies warrants the consideration of Glu and Asp as primary neurotransmitter candidates for a considerable proportion of central excitatory synapses (Emson, 1983). One major problem in the definitive assessment of the candidacy of Asp and Glu as neurotransmitters in specific excitatory pathways has been the dearth of selective and potent antagonists gohnson, 1978;Davies et aZ., 1980). More recently, the use of several relatively specific antagonists, including the glutamate analogs 2-amino-5-phosphonovaleric acid (APV), 2-amino-4-phosphonobutyric acid (APB), and their derivatives, has, however, permitted a finer analysis of receptors mediating synaptic excitation and has provoked speculation regarding potential endogenous ligands (Davies and Watkins, 1979;Koerner and Cotman, 1981;Hori et al., 1981; Crooks et al., 1985). Similar, if not identical, sensitivity to such antagonists for both the exogenously applied and endogenously released compound is a necessary, though not sufficient, criterion for neurotransmitter identification (Watkins and Evans, 1981; Cooper et al., 1986). Although these antagonists block excitatory postsynaptic potentials (EPSP) in a number of pathways, studies in several systems have failed to demonstrate pharmacological identity of the endogenous transmitter with either Asp or Glu (Ishida and Shinozaki, 1980;Hori et al., 1981;Shiells et al., 1981;Robinson, 1985). Although certain of these pharmacological observations may reflect the mixed agonist properties of these acidic amino acids (Watkins and Evans, 1981; Davies and Watkins, 1983;Mayer and Westbrook, 1984), the presence of extrasynaptic receptors (Davies and Watkins, 1979),or other difficulties arising from an unfaithful replication of endogenous transmitter delivery, it would appear plausible that a synaptic role of endogenous Asp- or Glu-like compounds, rather than the amino acids themselves, could explain problematic observations, Thus, several investigators have attempted to identify other potential endogenous ligands for glutamate receptor subtypes (Zaczek et al., 1983;Luini et al., 1984;Do et al., 1986).
NEUROBIOLOGY OF N-ACETYLASPARTYLGLUTAMATE
45
Notably, small acidic peptides, composed of Asp and/or Glu, may assume relatively few conformations in solution, especially in the presence of divalent cations (Dill et al., 1985; Lannom et al., 1986), and thereby be predisposed for recognition by distinct subclasses of excitatory amino acid receptors (Foster and Fagg, 1984). Interestingly, several competitive antagonists of excitatory amino acidinduced excitation are dipeptide derivatives of Asp or Glu, including y-D- glutamyl-glycine and p-D - aspartyl-p- alanine (Watkins, 1981), fi-D-aSpartyl-, and y-D-glutamyl-aminomethylphosphonate(Davies et al., 1984; Davies and Watkins, 1985). In addition, Goldberg and Teichberg (1985) have reported that peptide analogs of the potent excitatory amino acid agonist kainic acid represent a new class of specific antagonists for amino acid receptors. Although not ostensibly peptides, several of the other characterized antagonists of excitatory amino acid neurotransmission are long-chain w-phosphono analogs of mono- and dicarboxylic acids of up to seven carbons in length (2-amino-7phosphonoheptanoate) (Watkins, 1981; Perkins et al., 1982; Polc, 1985). Of potential relevance, virtually all of these derivatives have activity at either the N-methyl-D-aspartate (NMDA) or kainic acid (but not quisqualate) subclasses of excitatory amino acid receptors. Regardless, they reveal an ability of distinct excitatory amino acid receptors to recognize small, peptide-sized, organic acids in addition to their putative endogenous ligands, Asp and Glu. Moreover, these data suggest the possibility that small endogenous, acidic amino acid-rich peptides might behave as agonists at certain of these receptors.
B. NAAG
AS A
NEUROTRANSMITTER AT GLUTAMATERGIC SYNAPSES
Several endogenous peptides containing Glu and/or Asp have been described in neural tissues, although few have been the subject of quantitative studies. Glutathione (y-glutamyl-cystinyl-glycine), although present in high quantities in neural tissues (Lajtha et al., 19Sl), is found in virtually all cells, and is thought to function in amino acid transport (Meister, 1973) and as an important source of metabolic reducing equivalents (Hsia and Wolf, 1981; Perry et al., 1982). In contrast, NAAG and several peptides structurally similar to NAAG (Reichelt and Edminson, 1977; Kanazawa and Sutoo, 1981a,b; Marnela and Lahdesmaki, 1983; Marnela et al., 1984) may be involved in chemical neurotransmission. Notably, NAAG appears to be present in large quantities only in neural tissues (Miyamoto et al., 1966; Miyake et al., 1981; Koller et al., 1984a) and contains within its structure both of the putative excitatory
46
RANDY D. BLAKELY AND JOSEPH T. COYLE
amino acids, bearing a notable structural resemblance at its N-terminus to the nonendogenous excitatory amino acid agonist, NMDA. In view of the aforementioned problems associated with the unambiguous identification of the endogenous neurotransmitters of central excitatory pathways, the presence in brain of a N-blocked dipeptide composed of Asp and Glu has prompted speculation that NAAG or NAAG-like peptides, along with the traditional acidic amino acid candidates, might be neurotransmitters at excitatory amino acid synapses (Zaczek et al., 1983; Koller et al., 1984a; Coyle et al., 1986). If, indeed, NAAG is a neurotransmitter, several well-established criteria (Cooper et al., 1986) must be met. (1) NAAG should be synthesized in specific neuronal subpopulations, with a nonuniform regional distribution. (2) NAAG should be released from nerve terminals following the arrival of impulses into the synapse. (3) A mechanism should exist to inactivate the postsynaptic responses induced by the peptide, especially if rapid and transient responses follow synaptic release. (4) Receptors should be present on target cell membranes that transduce its effects. (5) A pharmacological identity should exist between NAAG and the endogenous neurotransmitter of identified pathways. As discussed below, certain of these criteria have been apparently satisfied, with findings that are highly suggestive of the extension of this general hypothesis to the specific function of neurotransmissionwithin putative glutamatergic neuronal pathways; other criteria either find only indirect support or remain unexamined; still other findings support neither a restriction of NAAG to glutamatergic systems nor a singular role as a mediator of fast chemical signaling in the nervous system. The latter results caution against simplistic models for the action of NAAG in the CNS and point toward neuromodulatory roles in chemically defined, nonglutamatergic pathways.
II. Quantitative Biochemical Studies of NAAG in Neural Tissues
A. PHYLOGENETIC AND REGIONAL DISTRIBUTION Nearly a decade after the discovery of the protected amino acid Nacetylaspartate (NAA) (Tallan et al., 1956), Curatolo and co-workers identified, with paper chromatography, a ninhydrin-negativecompound from a soluble extract of rabbit brain that upon acid hydrolysis, yielded equimolar concentrations of Glu and Asp (Curatolo, 1964; Curatolo et al., 1965). Using more quantitative liquid chromatographic pro-
NEUROBIOLOGY OF N-ACETYLASPARTYKLUTAMATE
47
cedures, Curatolo determined the distribution of this peptide, identified as NAAG, within the equine nervous system. The peptide was found to be present in relative abundance ( 1 nmol/mg wet weight), similar to the CNS levels of GABA (McGeer and McGeer, 1981), and to be distributed unevenly across brain regions, with lowest levels in cortex and highest levels in caudal brainstem structures. Shortly thereafter, Miyamoto and colleagues (1966) determined that the peptide bond present in endogenous bovine NAAG was formed through the a-carboxyl moiety of aspartate, rather than by 0-linkage, that the acyl group protecting the N-terminus was, indeed, an acetyl group, and that the peptide was a major constituent of rat, guinea pig, and chicken brains. With modern high-perform ance liquid and gas chromatographic procedures for the quantification of acidic brain peptides (Lenda, 1981; Miyake et al., 1981; Koller et al., 1984a), high concentrations of NAAG in neural tissues have been subsequently confirmed in a wide variety of vertebrates including frogs (Miyake et al., 1981), rodents (Reichelt and Kvamme, 1967; Miyake et al., 1981; Koller et al., 1984a), rabbits (Miyake et al., 1981), and primates (Reichelt and Kvamme, 1967; Koller et al., 1984b). Considerable differences in absolute levels, however, have been documented across taxa, with a greater than 10-foldreduction apparent when moving from frog and chick brain to fish (carp, goldfish) neural tissues, in which little, if any, material isochromatographic with NAAG is detectable (Miyake et al., 1981; Koller et al., 1984b). Thus, NAAG appears to be widely distributed, with peak levels found in lower vertebrates. In addition, Koller et al. (1984b) have also recently reported the presence of NAAG in several invertebrate groups including sea anemones and planarians (whole animal), as well as in cockroaches (head). Of note for later discussions of pathways involved in NAAG metabolism, NAA and NAAG levels exhibit a general lack of phylogenetic covariance. For example, NAAG levels exceed NAA levels by nearly 10-fold in frogs (Miyake et al., 1981; Koller et al., 1984b), whereas the reverse is true of mammalian brains. Studies with vertebrate tissues show both NAAG and NAA to be selectively enriched in neural tissues. In early studies, NAAG was undetectable in heart, liver, spleen, kidney, intestine, ovary, testis, lung, or muscle; only meager ( < 1% of brain levels) quantities of NAA were present in the same tissues (Miyamoto et al., 1966; Miyake et al., 1981; Koller et al., 1984a). On the basis of a lack of brain and spinal cord penetration by peripherally administered radiolabeled NAA, Berlinguet and Laliberte (1966) concluded that the high neural levels of this compound were unlikely to result from uptake of peripherally synthesized material. Similar definitive experiments for NAAG have, as yet, not been
-
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RANDY D. BLAKELY AND JOSEPH T. COYLE
conducted. Consistent with the previously cited equine studies, NAAG levels in both rabbit (Miyake et al., 1981) and rat (Koller et al., 1984a) brains display a marked enrichment in caudal regions of the neuraxis, reaching levels in brainstem and spinal cord nearly an order of magnitude above those occurring in telencephalic structures. Little regional variations is evident for NAA levels (Table I). In addition, spinal cord NAAG levels in cervical, thoracic, and lumbar segments are enriched in the ventral half of hemidissected cords and in ventral roots, relative to their dorsal homologs (Ory-Lavollte et al., 1987). These latter findings point toward the presence of NAAG in efferent motor units, consistent with recent immunocytochemical findings of intense staining of NAAG-like immunoreactivity in spinal and brainstem motoneurons (see below) (Blakely et al., 1986a; Ory-Lavollte et al., 1987; Forloni et al., 1987). Recently, an extremely sensitive radioimmunoassay for free NAAG has been developed (Guarda et al., 1988). Salient findings with this TABLE I REGIONAL DISTRIBUTION OF NAAG AND NAA
IN
ADULT RAT"
Region
NAAG
NAA
Spinal cord Medulla Pons Cerebellum Tectum Tegmentum Hypothalamus Thalamus Septum Hippocampus Pyriform cortex Striatum Parietal cortex Frontal cortex Retina Pituitary Superior cervical ganglion Dorsal root ganglion Dorsal root Ventral root Sciatic nerve Heart Liver
23f3 16fl
42f3 40f4 35f4 42f4 51 f 5 46f5 45f5 48f4 45f5 54f3 46f4 43f4 46f4 67f6 5 fl 1 +0.3 23k3 43*4 14f2 llfl 12fl 0.3 f O . 1 1.7f0.6
7 f l 650.5 l0fl 12fl 9f l 7 f 1 5f1 4f0.3 4f0.5 2fO.2 3f0.3 4 f 0.2 4f0.3 0.8f0.2 7.8f1.5 10.5 f 1 . 1 9.5 f 1.5 12.0f1.8 15.5 f 1.2 NDb ND
*
"Thevalues (nmol/mg protein) are mean SEM of at least three separate determinations (Koller et al., 1984; Ory-Lavollee et al., 1987). 'ND. not detected.
NEUROBIOLOGY OF N-ACETYLASPARTYELUTAMATE
49
technique are (1) high correlation with previous measures of NAAG levels in CNS structures, (2) reasonable correspondence with regions possessing large numbers of NAAG-like immunoreactive neurons (see below), and (3) demonstration of significant NAAG-like immunoreactivity in rat peripheral tissues, including skeletal muscle, heart, lung, and kidney. These findings confirm the considerable enrichment of NAAG in neural tissues, as peripheral values fall 100- to nearly 1000-foldbelow the levels observed in the CNS. Further studies will be required to determine the contribution made to these levels in nonneural organs by peripheral nervous system (PNS) innervation. In this regard, the relative restriction of NAAG and NAA to neural tissues does not apparently extend to a dichotomy between peripheral and central neural tissues, although fewer studies have examined PNS tissues. NAA has been reported in low but measurable quantities in bovine peripheral tissues of neural origin including the superior cervical ganglion, adrenal medulla, and splenic nerve (Nadler and Cooper, 1972a). Ory-LavollCe et al. (1987), in studies of rat PNS tissues, found high levels of NAAG in sciatic nerve, confirming similar findings by Miyake et al. (1981) in rabbit. Notably, the levels of NAA in this tissue, as well as in ventral root, were considerably below the levels in the CNS, underscoring the lack of a quantitative regional correlation between NAA and NAAG levels (Miyake et al., 1981; Koller et al., 1984a). Both rodent dorsal roots and spinal ganglia (Ory-LavollCeet al., 1987; Cangro et al., 1987), as well as superior cervical ganglia (Ory-LavollCe et al., 1987), exhibit high levels of NAAG and NAA (Table I), clearly necessitating the development of functional hypotheses suitable for central and peripheral neural tissues. At present, glutamate is not a major neurotransmitter candidate in superior cervical ganglia, although a considerable dorsal horn innervation by putatively glutamatergic primary sensory neurons has been described (Jahr and Jessel, 1985; Schneider and Perl, 1985). Importantly for the present discussion, the marked concentration of NAAG in central and peripheral neural tissues and the nonuniform regional distribution of its levels are inconsistent with a role associated with constitutive cellular metabolism, but rather support hypotheses focused upon the special properties of specific neuronal cells, clearly consistent with a role as a neurotransmitter or neuromodulator. Furthermore, the higher levels of NAAG present in caudal brain structures suggests that such a role gains prominence in pathways arising from, or projecting to, spinal cord and brainstem. In addition, NAAG is not distributed as would be expected of a molecule simply involved in global, glutamatergic synaptic transmission (Cotman et al., 1987; Coyle and Robinson, 1987).
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RANDY D. BLAKELY AND JOSEPH T. COYLE
B. DEVELOPMENTAL PATTERNS
A corollary of the heterogeneous regional distribution expected of a neurotransmitter is a developmental expression consonant with the maturation of synaptic processes, which in rodents is considerably delayed into postnatal life (Benjamins and McKann, 1981). Indeed, many markers for neurotransmitter systems, such as biosynthetic enzymes, mRNA expression, uptake and inactivation processes, or the neurotransmitters themselves, are known to display a marked postnatal development (Coyle, 1977; Lanier et a l . , 1976; Strittmatter et al., 1986). The ontogenetic profile of a neurotransmitter should not only reflect the maturation of specific synaptic elements but should also follow the underlying regional heterogeneity of specific neuronal systems, leading to region-dependentvariations in both the p a t t e r n and steady-statelevels of expression. In this regard, rodent levels of NAA and NAAG display quite different ontogenetic profiles (Miyake and Kakimoto, 1981; Koller and Coyle, 1984a). NAA levels rise rather uniformly to adult levels in the first 3 postnatal weeks in the rat with no discernible pattern of regional variation. NAAG levels, on the other hand, display a region-dependent postnatal pattern of expression. The neonatal rat brain, in toto, exhibits levels of NAAG at birth that approach those of the adult; yet, when examined on a regional basis, the postnatal pattern appears as a complex mosaic of distinct temporal profiles. Levels of NAAG in the two regions of the rat brain with the greatest content of the peptide, brainstem and spinal cord, approach adult levels by 1 week after birth. Spinal cord expression, however, appears to be biphasic, with an initial peak around 1 week after birth, followed by a plateau or decline, which has reversed by the third week of life and thereafter rises substantially to attain adult levels. One plausible model to explain the pattern of spinal cord NAAG expression is the early maturation of an intrinsic cellular population, followed by the delayed arrival of NAAG-concentrating fibers, which in rodents probably arise from brainstem nuclei and/or sensory ganglia (Tracey, 1985). NAAG expression in forebrain regions shows a regiondependent postnatal rise (1-3 weeks), followed by substantial reductions thereafter. The latter effect has been suggested to result from the precocious maturation of NAAG-containing populations, subsequently diluted during the massive expansion of the telencephalon, as is observed for certain other neocortical markers of neurotransmitter systems, including tritiated GABA uptake (Coyle and Enna, 1976). Consistent with these biochemical data, few NAAG-like immunoreactive neurons are found in adult rat cortical structures, whereas brainstem and spinal
NEUROBIOLOGY OF N-ACETYLASPARTYLGLUTAMATE
51
cord possess many intensively immunoreactive perikarya, as do sensory ganglia (reviewed below). Thus, the developmental expression of NAAG levels in rodents is consistent with a role in central neurotransmission, especially for neurons whose somata and/or processes are contained within the brainstem and spinal cord. Clearly, the interpretation of data obtained from gross regional dissection is limited, necessitating more sensitive microassays for smaller regional samples and the direct identification of NAAGsynthesizing neurons. However, it is evident that NAA and NAAG are under differential developmental regulation, leading to the marked differences in their ontogenetic profiles. Along with the fact that NAA expression is delayed relative to NAAG, these findings argue for a role of NAA in facets of cellular metabolism other than the biosynthesis of NAAG. NAA may in fact play many roles in the vertebrate brain. Recently, NAA has been isolated as a heat-stable cofactor required for the @-oxidationof lignoceric acid to cerebronic acid and Glu in the pathways of ceramide and cerebroside biosynthesis (Shigematsu et al., 1982), a function consistent with early reports of its involvement in lipid biosynthesis (D’Adamo and Yatsu, 1966; D’Adamo et al., 1968), and its rather uniform regional distribution (Marcucci et al., 1966; Koller et al., 1984a). Thus, its functional and biosynthetic relationship to NAAG is suspect, although this issue remains unclear, reflecting the paucity of information pertinent to NAAG biosynthesis.
111. Cellular Localization of N A A G
A. VISUALIZATION OF NAAG-LIKEIMMUNOREACTIVITY IN VERTEBRATE NEURONS The most powerful method at present for determining the cellular localization of endogenous molecules is immunocytochemistry, such as that employed for the localization of neurotransmitter biosynthetic enzymes, including choline acetyltransferase( h e y et al., 1983), glutamic acid decarboxylase (Wu et al., 1982), and tyrosine hydroxylase (Pickel et al., 1975). Given the impact of immunocytochemical methods for the direct localization of neuropeptides (see reviews by Pickel, 1981; Cooper et al., 1986), recent investigators have adapted these strategies for the visualization of small amino acid-sized molecules, with several notable successes. In a number of cases, antibodies with high specificity for putative neurotransmitters, including serotonin (Steinbusch, 1981), GABA
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RANDY D. BLAKELY AND JOSEPH T. COYLE
(Somogyi et al., 1985), and acetylcholine (Geffard et al., 1985), have been produced and found to reveal sets of neurons identical to those visualized with antibodies against their biosynthetic enzymes or with histofluorescence approaches. Furthermore, when biosynthetic pathways are not unique to one product, specific enzymes are not purified, or where synthetic routes are largely undefined, as with NAAG, the localization of the product may be the only method available. Indeed, several recent studies have documented selective neuronal patterns of staining for Glu (Ottersen and Storm-Mathisen, 1984; Magnusson et al., 1986), Asp (Ottersen and Storm-Mathisen, 1985; Campistron et al., 1986; Saito et al., 1986), and glycine (Pourcho and Goebel, 1985), whose biochemical roles clearly extend far beyond neurotransmission (McGeer and McGeer, 1981). These studies provided optimism that the generation of specific antibodies for the immunocytochemical localization of NAAG would not only be advantageous, but practical as well. To the limits of immunocytochemical specificity, results with these techniques have considerably expanded our understanding of NAAG distribution and have delimited specific pathways for investigations of the peptides mechanism of action. 1. Technical Developments for the Visualization of NAAG-Like Immunoreactivity Several problems, however, had to be overcome before the production of specific NAAG antibodies and their first use in neuroanatomical studies (Anderson et al., 1986; Blakely et al., 198713) could be achieved. In general, amino acids and small peptides fail to generate substantial antibody titer without covalent attachment to a carrier protein (Erlanger, 1980; Maurer and Callahan, 1980), which then can serve the dual function of antigen presentation and preservation over the course of weeks between serial injections. As NAAG has a blocked amino-terminus, aldehyde coupling of the peptide to protein, a commonly used method (Reichlin et a l . , 1968; Frohman et al., 1970; Qttersen and StormMathisen, 1984) is ineffective, necessitating the introduction of bifunctional reagents capable of producing a covalent linkage with NAAG and protein through one or more of its three free carboxyl groups. In this regard, the peptide must be fixed in situ as well prior to immunocytochemical localization, and thus coupling strategies had to be developed that were extendable to immersion or perfusion furation techniques. In addition, quantitative assays capable of detecting potentially small levels of specific immunoglobulin are required for quantifying immune titer following purification and documenting specificity. Many direct peptide immunoassays utilize competition between unlabeled and
NEURO B I 0 LO GY 0F N-ACETY LAS PARTY LG LUTAM ATE
53
iodinated antigens (Yalow and Berson, 1960; Felber, 1974; Corrie and Hunter, 1981) with specific activities of labeled antigen orders of magnitude above that available in tritiated NAAG. Thus, indirect approaches detecting covalently bound NAAG with iodinated, fluorescinated, or enzyme-linkedimmunoglobulin probes were necessary. A water soluble carbodiimide, 1-ethyl-3-(3-dimethyl-aminopropy1)carbodiimide (EDAC), which can efficiently form peptide linkages between free carboxyl groups of soluble antigens and free amino groups of protein side chains, has been demonstrated to be a useful reagent for the generation of peptide immunization conjugates (Goodfriend et al., 1964; Bauminger and Wilchek, 1980; Erlanger, 1980) and has recently been used for the production of Glu-recognizing antibodies (Mad1 et al., 1986). Two groups have utilized this reagent to produce NAAG-protein conjugates suitable for injection into rabbits in the production of polyclonal antisera (Anderson et d . , 1986; Blakely et a l . , 198713). This carbodiimide has also proved useful in covalently attaching NAAG to amino-alkyl-agarose resins for the production of affinity gels for antibody adsorption (Blakely et a l . , 1987b; Kowalski et a l . , 1987). Finally, indirect solid-phase RIA and ELISA for NAAG have been developed based upon the ability of iodinated protein A (Blakely et al., 198713) or peroxidase-labeled anti-rabbit IgG (Cangro et al., 1987), respectively, to detect antibodies bound to protein-coupled antigens that have been previously adsorbed into plastic microtiter wells. Solid-phase RIA of the affinity-purified serum against equivalent concentrations of amino acids and peptides covalently linked to polyL-lysine revealed a distinct pattern of selectivity for N-acetylated acidic dipeptides. The highest signal was observed for NAAG, with significantly reduced binding to the N-acetylated amino acids NAA and N-acetylglutamate apparent. Notably, the amino acids Asp and Glu exhibit essentially no cross-reactivity. This is an important finding, especially in light of the hypothetical function of NAAG as a neurotransmitter in putative glutamatergic pathways, and permits immunocytochemical studies in the presence of an excess of fixed, acidic amino acids. Furthermore, these amino acids can be covalently attached to protein with aldehydes alone, unlike NAAG, and when adsorbed and probed have not been found to be immunoreactivewith carbodiimide-derivedpeptide antisera (Anderson et al., 1986; Blakely et a l . , 1987b; Cangro et al., 1987). Also, control immunocytochemical studies conducted with only aldehyde fixatives have demonstrated little or no specific NAAG-like immunoreactivity, as expected of the visualization of an N-blocked antigen. The present authors concur with previous reviewers (Schipper and Tilders, 1983; Hodgson et al., 1985) that the antigens to be
54
RANDY D. BLAKELY AND JOSEPH T.COYLE
recognized in neuroanatomical studies must be covalently bound to cellular protein during fixation and subsequent localization, and thus the specificity of antisera used in immunocytochemical studies should be tested against the covalently modified hapten and potential crossreactants, whenever possible. In this regard, NAA levels are high in brain and, thus, immunocytochemical studies will probably be unable to preclude NAA cross-reactivity on the basis of blocking studies alone. However, NAA levels are rather uniformly distributed across brain regions, whereas NAAG levels are concentrated in brainstem and spinal cord (Miyake et al., 1981; Koller et al., 1984a). Thus, a rather global immunoreactive profile should be suggestive of a mismatch between identified cells and the distribution of NAAG levels. Whereas immunoreactivity concentrated in hindbrain may have a legitimate reference in the cellular localization of the dipeptide. Indeed, the distribution of NAAGimmunoreactive neuronal somata visualized to date indicates high numbers of labeled neurons in medulla, pons, and spinal cord, with few, though distinct, immunopositive forebrain nuclei (see Table 11). 2 . NAAG-Like Immunoreactivity in Rodent Spinal Cord A major NAAG-synthesizing neuronal population is likely to be present within sensory ganglia. Consistent with the high levels of NAAG in dorsal root ganglia of rodents (Ory-LavollCeet al., 1987; Cangro et al., 1987), NAAG-like immunoreactivity (LI) has been detected in neurons of spinal ganglia of frogs (Kowalski et al., 1987) and rats (Blakely et al., 1986a; Cangro et al., 1987). In both amphibian and rodent ganglia, immunoreactivity was found to be present in relatively large neurons. In rat dorsal root ganglion (DRG), immunoreactivity was found in neurons greater than 35 pm in diameter, a distribution distinct from that reported for many other neuropeptides or the enzymatic markers cytochrome oxidase or glutaminase (Cangro et al., 1987). Thus, NAAGsynthesizing neurons may represent a distinct functional subset of primary afferent fibers to the spinal cord. As yet, the projection areas of these neurons within the spinal cord are unknown, although Cangro et al. (1987) have noted that neurons in the size range identified mainly utilize large diameter myelinated axons, classified as Aa, or
[email protected] of this type are thought to be involved in the conduction of mechanosensory information, rather than for thermoreception or nociception, and have been argued to utilize a Glu-like neurotransmitter (Jahr and Jessel, 1985). As noted previously, NAAG levels in the rodent are highest in the spinal cord (Miyake et a l . , 1981; Koller et al., 1984a) being particularly enriched in ventral horns and roots (Ory-LavollCe et al., 1987). Immunostaining of spinal cord sections (Ory-LavollCeet al., 1987; Blakely, 1987)
TABLE I1 DISTRIBUTION OF NAAG-LIKEIMMUNOREACTIVE NEURONS IN RATRegion Spinal cord Medulla-pons
Nuclei
Intensity
+++++
Lamina 7-9 Abducens Cuneate Dorsal motor vagus Dorsal cochlear External cuneate
Cerebellum
Anterior interposed
Midbrain
Dorsal raphe Median raphe Mesencephalic sensory trigeminal Oculomo tor
++++ ++ ++++ +++ ++ +++++ ++ +++++ +++ ++ +++ +++ +++++ +++ +++ ++ ++++ +++++
Forebrain
Diagonal band Medial septa1 nucleus
++ ++
Facial Gracilis Gigantocellular reticular Hypoglossal Lateral cervical Lateral reticular Lateral superior olive Lateral vestibular
Nuclei
Intensity
++ ++++ ~~
Lamina 5-6 Locus coeruleus Median superior olive Mediodorsal reticular Mediolateral reticular Mesencephalic sensory trigeminal Nucleus ambiguus Pontine reticular Spinal trigeminal Superior vestibular Trapezoid body Trigeminal motor Ventral cochlear Vestibular ganglion
++
++
++ ++++ ++ +++ ++++ ++ +++ +++++ ++ +++++ +++
Lateral cerebellar nucleus
+++++
Red nucleus Substantia nigra Superior colliculus internal gray Trochlear Ventral Tegmental
+ + ++++ ++++ ++ ++++
Motor cortex (IV-V) Olfactory mitral cells
"Distribution as observed with affinity-purified NAAG-BSA (bovine serum albumin) antiserum. Scale of immunoreactive intensity ranges from + + , all neurons of nucleus intensively stained, to , weak staining observed in scattered neurons within defined nuclei. Also see Table 111 for additional nuclei visualized in separate colocalization experiments.
+++
+
56
RANDY D. BLAKELY AND JOSEPH T. COYLE
with affinity-purified NAAG antiserum revealed an intense localization of immunoreactivity to large diameter (30-45 pm) ventral horn neurons (Fig. 2A), concentrated in laminae VIII and IX, after the divisions of Paxinos and Watson (1982). The cellular characteristic of immunostaining was also noticeably punctate (Fig. 2B) and was present throughout the cell body and dendrites, although absent from the nucleus, a hallmark of immunostaining with this antiserum (Blakely et al., 1987a,b). In these studies, immunoreactive axons or terminals were rarely seen, although a recent report describes NAAG-like immunoreactivity in ventral roots (Cassidy et al., 1987). Recently, choline acetyltransferase (ChAT) has been colocalized with NAAG-like immunoreactivity in rat ventral horn neurons within the same tissue section (Forloni et al., 1987), and thus it is likely that motoneurons are revealed by these procedures. Smaller cells were occasionally stained in the other layers of the ventral horn and in lamina X. The numbers of cells stained dramatically diminished in dorsal horn laminae, which was most apparent in thoracic cord sections (Fig. 2C). No stained cell bodies were apparent within the substantia gelatinosa and marginal zone. Preadsorption of antiserum with 1pg/ml NAAG-BSA (bovine serum albumin) (Fig. 2D) and incubations with nonimmune serum or with non-carbodiimide-fixed sections resulted in a marked diminution of immunoreactivity (Ory-Lavollee et al., 1987). The presence of NAAG-LI within motoneurons is consistent with findings in invertebrates, in which a number of peptides have been suggested to serve as cotransmitters in invertebrate motor fibers (Adams and O’Shea, 1983; O’Shea et al., 1985; Richmond et al., 1986). Until recently, however, cholinergic motoneurons were not thought to contain peptides involved in modulation of nicotinic function at the vertebrate neuromuscular junction (Hunt, 1983). Gibson et al. (1984) demonstrated the presence of calcitonin gene-related peptide (CGRP)-like immunoreactivity in vertebrate motoneurons. Subsequent investigators have FIG. 2 . NAAG-like immunoreactivity in rat cervical and thoracic spinal cord: 40-pm sections from carbodiimide-fiied rat spinal cord were incubated with 1 : 10 dilution of affinity-purified NAAG antiserum and processed for NAAG-like immunoreactivity by the avidin-biotin peroxidase technique. (A) Cervical spinal cord NAAG-like immunoreactivity. Bar, 250 pm. (B) Higher powered view of NAAG-like immunoreactivity of the ventral horn section displayed in A, demonstratinng the punctate, pennuclear immunoreactivity in soma and dendrites. Arrows point to same motoneuron (MN) in both sections. Bar, 50 pm. (C) Thoracic cord NAAG-like immunoreactivity. Note the virtual absence of immunoreactive neurons in the dorsal horns. Bar, 250 pm. (D) Absence of NAAG-like immunoreactivity when incubations were conducted in the presence of 1 pg/ml NAAG-BSA conjugate. Similar results were found in sections incubated with nonimmune serum or in noncarbodiimide fiied tissue. Bar, 250 pm.
NEUROB I 0 LO GY 0F N-ACETY L AS PARTY LG LUTA MATE
57
58
RANDY D. BLAKELY AND JOSEPH T. COYLE
documented the ability of CGRP to stimulate the surface expression of nicotinic receptors (New and Mudge, 1986) and to elevate muscle adenylate cyclase activity (Laufer and Changeux, 1987; Kobayashi et al., 1987). Coupled with data demonstrating complex regulation of nicotinic receptors by protein phosphorylation (for a review, see Huganir, 1987), these data represent strong indications of a capacity of motoneurons to chemically modulate nicotinic responses. Thus, the finding of NAAGlike immunoreactivity within rodent motoneurons and the presence of NAAG in vertebrate muscle, as detected by a highly sensitive radioimmunoassay (Guarda et al., 1988), suggest that the dipeptide may be present and released at motor end-plates, clearly an important area for future functional studies. 3. NAAG- Like Immunoreactim'ty in Rodent Brainstem and Cerebellum Many different functionally distinct nuclei within the rodent brainstem (Fig. 3-5) have been shown to exhibit NAAG-likeimmunoreactive neurons (Blakely et al., 1986a; Blakely, 1987). As was found in spinal cord, motoneurons of the medulla and pontine cranial nerve nuclei possess intense NAAG-like immunoreactivity. Virtually all cranial nerve motor nuclei were found to be stained with affinity-purified NAAG antisera, including the dorsal motor nucleus of the vagus (X), and hypoglossal (XII) (Fig. 3A), facial (VII) (Fig. 3B), abducens (VI) (Fig. 4D), trigeminal (V) (Fig. 3D), trochlear (IV), and oculomotor (111) (Fig. 3C) nuclei. Forloni et al. (1987) have recently described the colocalization of NAAG-LI with CUT-LI in many of these motor nuclei. In addition to cranial nerve motor subdivisions, two primary sensory nuclei at brainstem levels were observed to possess NAAG-LI. Thus, virtually all of the sensory neurons of the vestibular ganglion (Fig. 4A), embedded in the eighth cranial nerve, were found to exhibit immunoreactivity, as was also the case for the neurons of the mesencephalic sensory nucleus of the trigeminal nerve (Figs. 3D and 5B), the latter population representing the only primary sensory nucleus found within the CNS. Both of these sensory nuclei serve as initial relays for a single sensory modality (mechanoreception) and have large diameter, myelinated sensory afferents (Tracey, 1985; Carpenter and Sutin, 1983), as observed for the population of sensory neurons visualized with anti-NAAG antisera in spinal ganglia (Cangro et al., 1987). Thus, it is likely that most, if not all, sensory ganglia possess neurons containing a NAAG-likepeptide, especially those neurons processing information from peripheral mechanoreceptors. Colocalization studies with other peptides known to be involved with pain and temperature pathways should help to clarify this question.
NEUROBIOLOGY OF N-ACETYLASPARTYLGLUTAMATE
59
FIG. 3. NAAG-like immunoreactivity in rat brainstem: 40-pm sections from carbodiimide-fixedrat brainstem were incubated with 1 : 10 dilution of affinity-purified NAAG antiserum and processed for NAAG-likeimmunoreactivity by the avidin-biotin peroxidase technique. (A) Immunoreactivity in neurons of the dorsal motor nucleus of the vagus nerve (DMV) and hypoglossal nucleus (HN). Bar, 100 pm. (B) Localization of NAAG-like immunoreactivityin the facial nucleus (FN), accessory facial nucleus (AFN), and spinal trigeminal nucleus (STN). Bar, 500 pm. (C) NAAG-like immunoreactivity in the ocuolomotor nucleus (OC) and red nucleus (RN) of the rat midbrain. Bar, 500 pm. (D) Appearance of NAAG-like immunoreactivity in the locus coeruleus (LC), mesencephalic nucleus of the trigeminal nerve (MEV), and the trigeminal motor nucleus (TMN). Bar, 500 pm. Sections appearing in panels A and D were taken from animals administered 70 pg of colchicine intracerebroventricularly (ICV) 48 hr prior to perfusion.
Consistent with the hypothesis that NAAG may reside within putative glutamatergic sensory neurons, biochemical and autoradiographic studies support the candidacy of glutamate as a neurotransmitter for primary vestibular afferents in the cat (Dememes et al., 1984; Raymond et a l . , 1984). In addition to motor and primary sensory neurons of the brainstem, NAAG-like immunoreactivity is found in several nuclei with major descending projections to the spinal cord that exert considerable control over motor units in the rodent spinal cord. These nuclei include the
60
RANDY D. BLAKELY AND JOSEPH T.COYLE
NEUROBIOLOGY OF N-ACETYLASPARTY LGLUTAMATE
61
magnocellular red nucleus (Fig. 3C), origin of the rubrospinal tract (Flumerfelt and Hrycyshyn, 1985), lateral vestibular nucleus (Fig. 4B), origin of the vestibulospinal tract (Mehler and Rubertone, 1985), and pontine and medullary reticular neurons (Figs. 5D and 4D), whose efferents form the reticulospinal tract (Femano et al., 1984; Martin et al., 1985). These pathways are thought to exert excitatory control over flexor and extensor musculature and serve as direct and interconnected relays with corticofugal and cerebellar motor pathways. Thus, NAAG (or a NAAG-like peptide) is not only present in the final common pathway for motor control, but in functionally related, higher brain nuclei as well. This feature, described for certain sensory neurons as well (see below), represents one of the more intriguing findings of NAAG immunocytochemical experiments. Although vertebrate motoneurons possess receptors for excitatory amino acids (O’Brien and Fischbach, 1986), little conclusive data are available to designate Glu or Asp as the putative neurotransmitters of these pathways. Studies with antibodies raised against cross-linked Asp have demonstrated somewhat similar labeling patterns in the lateral vestibular nucleus (Kumoi et al., 1987), although the possible cross-reactivity of this antibody with acidic peptides such as NAAG has not been precluded. Nonetheless, these pathways may represent cases in which the hypothesis of NAAG neurotransmission at putative glutamatergic pathways is tenable. Furthermore, these nuclei are strong candidates for the sources of the NAAG-like immunoreactive fibers in the ventral and lateral funiculi of the rodent spinal cord (Cassidy et al., 1987), a clearly testable hypothesis. NAAG-like immunoreactivity can also be demonstrated within medullary sensory relay nuclei, including the spinal trigeminal nucleus pars oralis (Fig. 3B), superior olivary nucleus (Forloni et al., 1987), and the nucleus of the trapezoid body (Fig. 5C). The spinal trigeminal nucleus is concerned with processing somatosensory information from the facial FIG.4. NAAG-like immunoreactivity in sensory, motor, and relay nuclei of the rat brainstem: 40-pm sections from carbodiimide-fixed rat brainstem were incubated with 1 : 10 dilution of affinity-purified NAAG antiserum and processed for NAAG-like immunoreactivity by the avidin-biotin peroxidase technique. (A) NAAG-like immunoreactivity contained within large sensory neurons of the vestibular ganglion (8G) within the tract of the eighth cranial nerve (8N). Bar, 200 pm. (B) Staining of the rat lateral vestibular nucleus (LV), ventral to the cerebellum and lateral to the fourth ventricle. Bar, 500 pn. (C) NAAG-like immunoreactivity within the dorsal (DR) and median raphe (MR) and staining of the ventral tegmental nucleus (VT). Bar, 500 pm. (D) Neurons of the abducens nucleus (AN) dorsal to immunoreactivity pontine reticular neurons (PRN), and stained cells in the nucleus of the trapezoid body (NTZ). Bar, 500 pm. Sections appearing in panels C and D were taken from animals administered 70 pg of colchicine ICV 48 hr prior to perfusion.
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RANDY D. BLAKELY AND JOSEPH T. COYLE
FIG. 5. NAAG-like immunoreactivity in brainstem and cerebellar nuclei: 40-pm sections from carbodiimide-fixed rat brainstem and cerebellum were incubated with 1/10 dilution of affinity-purifiedNAAG antiserum and processed for NAAG-like immunoreactivity by the avidin-biotin peroxidase technique. (A) NAAG-like immunoreactivity in the lateral cerebellar nucleus ( E N ) and the anterior interposed cerebellar nucleus (AIN). Bar, 500 pm. (B) Staining of the mesencephalic sensory neurons of the trigeminal nerve (MEV), located medial to the mesencephalic tract of the trigeminal nerve (MTV) and the superior cerebellar peduncle (SCP).Bar, 100 pn. (C) NAAG-likeimmunoreactive neurons in the brainstem nucleus of the trapezoid body (NTZ). Bar, 250 pm. (D) Large gigantocellular reticular neurons (GRN) of the rat medulla possessing intense NAAGlike immunoreactivity. Bar, 250 pn. Section appearing in panel D was taken from animals administered 70 pg of colchicine ICV 48 hr prior to perfusion.
skin and oral mucosa (Tracey, 1985), whereas the latter two nuclei receive second-order acoustic stimulation from the cochlear nucleus. The apparent conservation of NAAG-like immunoreactivity along functionally organized sensory pathways warrants a further investigation of the presence of NAAG in primary auditory fibers and cochlear neurons. Notably, the auditory nerve has been suggested to use a Glu- or Asp-like neurotransmitter (Wenthold , 1985). To date, NAAG-likeimmunoreactive neurons in the cerebellar cortex have not been reported. Thus, the cerebellar granule cells, origin of the massive putative glutamatergic parallel fiber system, were unstained with affinity-purified NAAG antisera. These neurons represent one of many
NEUROBIOLOGY OF N-ACETYLASPARTYLGLUTAMATE
63
examples of the lack of global correspondence of NAAG-like immunoreactivity and putative glutamatergic pathways. The deep cerebellar nuclei, however, contained numerous NAAG-like immunoreactive neurons (Fig. 5A). As with many of the brainstem nuclei described above, neurotransmitters used by these nuclei have not been identified. Glulike immunoreactivity, visualized with antibodies raised against the EDAC-coupled dipeptide y-Glu-Glu, has also been reported to be present within neurons of the same nucleus (Mad1 et al., 1986; Monaghan et al., 1986). Determination of the extent of overlap between these two findings will require double-staining experiments with NAAG and Glu antisera. Although it remains possible that a common acidic antigen is being recognized by the two antibodies, the affinity-purified NAAG antisera utilized has no measurable affinity for Glu or non-N-blocked Glu conjugates tested (Blakely et al., 1987b). Of more interest for the present discussion, these neurons deliver extensive efferent projections from the cerebellum to brainstem and thalamic nuclei (Chan-Palay, 1977; Voogd et al., 1985), with several targets now known to contain intensely labeled NAAG-like immunoreactive neurons, such as the red nucleus and oculomotor nuclei. Examination of the extent of such interconnectivity between NAAG-LI neurons may contribute to the delineation of general mechanisms to describe the function of NAAG. Just as NAAG-LI was found in nonglutamatergic motoneurons and is undetectable in certain glutamatergic neurons, such as cerebellar granule cells, chemically defined brainstem populations with identified neurotransmitters were found to contain NAAG-LI. Principal among these were the serotonergic(5-HT) neurons of the dorsal and media raphe complex (Fig. 4C) and the noradrenergic locus coeruleus (Fig. 3D). Forloni et al. (1987) have extensivelymapped the colocalization of NAAGlike immunoreactivity with dopamine P-hydroxylase (DBH) in brainstem noradrenergic nuclei and with 5-HT-like immunoreactivity in the brainstem serotonergic nuclei (Table 111). NAAG-LI was found to be extensively codistributed with DBH, with the absence of NAAG-LI in the 14-2 nucleus (see Hokfelt et al., 1984) an~exceptionto the general pattern. The presence of other neuropeptides in the locus coeruleus has previously been reported; the list includes neuropeptide Y, somatostatin, met-enkephalin, and vasopressin (Charnay et al., 1982; Everitt et al., 1984;Johansson et al., 1984; Caffe et al., 1985). These neuropeptides, however, appear to be localized in distinct subpopulations of the noradrenergic cell bodies, in contrast to the apparent ubiquitous distribution of NAAG. A more restricted pattern of colocalization was noted for NAAG- and 5-HT-immunoreactivity, with the median raphe exhibiting the most extensive pattern of colocalization. Colchicine
64
RANDY D. BLAKELY AND JOSEPH T.COYLE
TABLE I11 NAAG COL~CALIZATION IN DIFFERENT NEURONAL SYSTEMS~ CNS regions
Nuclei
Antibody
NAAG-LI coexistence
Spinal cord Medulla-pons
Motoneurons Abducens Ambiguus Facial Superior olivary Trigeminus Raphe magnus Raphe pallidus Raphe pontis Locus coeruleus A-1 A-2 A-4 A-5 A-7 c-1 c-2 Oculomotor Parabrachalis Raphe dorsalis Median raphe Medial septum Diagonal band Nucleus basalis
ChAT ChAT ChAT ChAT ChAT ChAT 5-HT 5-HT 5-HT DBH DBH DBH DBH DBH DBH DBH DBH ChAT ChAT 5-HT 5-HT ChAT ChAT ChAT
++++ +++ +++ ++++ +++ +++ ++ ++ ++ ++++ +++ + ++++ ++ +++ ++ ++ +++ +++ + ++ + ++ ++
Midbrain
Forebrain
+
"The rating of coexistence was made as follows: , NAAG-LI was exhibited in a small percentage (0-25%) of cells positive for the other antibody: + , 25-60%; + 60-80%; virtually all cells stained for both antibodies. (From Forloni et al., 1987.)
+
+ +,
+ + + +,
administration markedly enhanced the deposition of NAAG-LI within noradrenergic and serotonergic nuclei, suggesting an accumulation of the peptide, an immunoreactive precursor, or its biosynthetic machinery after disruption of axoplasmic transport. These findings demonstrate the presence of NAAG-LI in neurons with identified neurotransmitters other than glutamate. Coupled with the demonstration of NAAG- LI in cholinergic motoneurons, these findings argue for a widespread influence of the dipeptide, more reconcilable with neuromodulatory function(s) rather than with a role in direct neurotransmission. As with all of the immunocytochemical data on NAAG, however, much caution must be taken in interpretation of these results in the absence of biochemical or physiological confirmation.
NEUROBIOMGY OF N-ACETYLASPARTYLGLUTAMATE
65
Recently, Guarda et al. (1988) have provided such independent evidence, via radioimmunoassay coupled to the Palkovits (1973) micropunch technique, for the presence of high concentrations of NAAG in several of the nonglutamatergic brainstem nuclei mentioned above. These data certainly broaden the potential physiological actions of NAAG beyond those expected of an excitatory peptide for subpopulations of putative glutamatergic neuronal pathways. 4. NAAG-Like Immunoreactivity in Forebrain Nuclei and Retina Consistent with the much lower levels of NAAG, measured by quantitative neurochemical techniques, in the rodent forebrain (Miyake et al., 1981; Koller et a l . , 1984a), few NAAG-LI neurons are found rostra1 to the midbrain, even after colchicine administration. Forloni et al. (1987), in examining the potential covariance of NAAG and ChAT immunoreactivity, found only a few of the cholinergic neurons of the medial septum, diagonal band, and nucleus basalis to contain NAAG-LI. Anderson et al. (1986) have also described the presence of NAAG-LI within the medial septum. Furthermore, Forloni et al. (1987) noted the absence of NAAG-LI from cholinergic neurons of the globus pallidus and neostriatum, areas totally devoid of immunoreactive perikarya. The latter finding is not consistent with previous findings of significant striatal NAAG reductions following excitotoxic lesions (Koller et al., 1984a), which were interpreted as reflecting an intrinsic striatal, NAAG-containing neuronal population. This disagreement may reflect the differential sensitivitiesof HPLC and immunocytochemical studies or the action of the neurotoxin at a distance upon extrastriatal neurons with basal ganglia afferents (Zaczek et al., 1981). Within the hippocampus, Anderson et al. (1986) noted the presence of very few NAAG-LI neurons, confined to the hilus fascia dentata and area CA3. No obvious laminar organization of immunoreactivity was observed with these neurons, and staining was absent from the dentate granule cells and the hippocampal pyramidal cells. These findings have been confirmed independently (R. D. Blakely, unpublished observations), and are puzzling in light of the suggestion of a NAAG-likeneurotransmitter in the hippocampal-septa1pathways (Koller et a l . , 1984a;Joels et al., 1987). As in striatum, these discrepancies may reflect levels of NAAG below the sensitivity of present immunocytochemical techniques or the involvement of a structurally related compound in this pathway which is itself not immunoreactive. The large number of intrinsic putative glutamatergic neurons within the hippocampus that fail to exhibit NAAG-like immunoreactivity underscore the dissociation of NAAG with all glutamatergic ,pathways.
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RANDY D. BLAKELY AND JOSEPH T. COYLE
Following colchicine administration, large pyramidal cells of layers IV and V concentrated in the rodent motor cortex have been observed to exhibit NAAG-LI (Fig. 6B). Dark punctate immunoreactivity is present in the outer two layers of rat cortex as well. In the entorhinal cortex, NAAG-LI has been reported in layers I1 and 111, concentrated in small (10-20 pm) neurons of lateral subdivisions (Anderson et al., 1986) and visual cortex (see below). These results reveal a sparse, though detectable, presence of NAAG-LI within the rodent cortex, consistent with the rather low peptide content of this tissue. The presence of NAAG-LI in pyramidal cells of the motor cortex is intriguing given the localization af NAAG-LI in motoneurons as well as in brainstem nuclei involved in
FIG.6 . NAAG-like immunoreactivity in the rat olfactory bulb and motor cortex: 40-pm sections from carbodiimide-fiied rat olfactory bulb and cortex were incubated with 1/10 dilution of affnity-purified NAAG antiserum and pmcessed for NAAG-like immunoreactivityby the avidin-biotin peroxidase technique. (A) Localization of neuronal NAAG-like immunoreactivity to a single cell layer of the rodent olfactory bulb, the mitral cell layer (M). Immunoreactivity was absent from olfactory nerve (0),glomerular (G), external plexiform (EP), internal plexiform (I), or granule cell (GR) layers of the bulb. Bar, 100 pm. (B) NAAG-like immunoreactivity in layer IV and V pyramidal cells of the rat motor cortex. Punctate staining was also observed in layer 11. Bar, 250 q. Section appearing in panel B was taken from animals administered 70 pg of colchicine ICV 48 hr prior to perfusion.
NEUROBIOLOGY OF N-ACETYLASPARTYLGLUTAMATE
67
motor output, and would further support a role common to functionally similar nuclei. In accord with electrophysiologicalstudies in pyriform cortex slices on rat olfactory bulb in vitro (ffrench-Mullen et a l . , 1985), immunocytochemical studies in the rodent olfactory bulb have provided compelling evidence for a NAAG-like neurotransmitter in the lateral olfactory tract (LOT) (Anderson et al., 1986; Blakely et al., 198713). Olfactory bulb immunoreactivity is localized exclusively to the mitral cells (Fig. 6A). The mitral cells convey processed olfactory information from the bulb to the pyriform cortex and amygdala via the LOT (Shepherd, 1979). Previous immunocytochemical studies have demonstrated the presence of neuropeptides, including cholecystokinin and luteinizing hormonereleasing hormone (LHRH), and of biosynthetic enzymes for amine neurotransmitter candidates within all cell classes of the rodent olfactory bulb, with the notable exception of the mitral cells (Macrides and Davis, 1983; Seroogy et al., 1985). However, Saito et al. (1986) have recently reported the presence of Asp-like immunoreactivity within the mitral cells of the rat olfactory bulb and argued that their results might reflect a neurotransmitter pool of Asp within mitral cells. Although these investigators did not rule out the potential cross-reactivity of their antiserum with Asp-containing peptides such as NAAG, it is unlikely that the NAAG results represent artifactual staining of Asp and Glu, given the negligible cross-reactivity of these amino acids with NAAG antisera. Electrophysiological studies have noted a pharmacological identity of NAAG with the endogenous neurotransmitter of the rodent LOT, as opposed to Asp or Glu (ffrench-Mullen et al., 1985). Thus, at present, the best evidence for a neurotransmitter role of the dipeptide has been obtained in studies with the olfactory bulb mitral cells and their projections. To date, however, the evoked release of the peptide from the LOT has yet to be demonstrated and remains an important objective for future studies. Recently, another putative glutamatergic population, the retinal ganglion cells, has also received immunocytochemical scrutiny as the origin of another NAAG-containing pathway. Anderson et al. (1986) first noted the presence of punctate immunoreactivity associated with the rodent lateral geniculate nucleus and upper layers of the superior colliculus, results attributed to the presence of NAAG-positive fibers in these nuclei. Upon investigation of the retina, NAAG-LI was found in retinal ganglion cells, and endogenous NAAG was identified by HPLC (Anderson et al., 1987). Furthermore, by both biochemical and immunocytochemical criteria, unilateral enucleation led to a marked reduction in optic tract NAAG levels. Guarda et al. (1988) have independently
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RANDY D. BLAKELY AND JOSEPH T. COYLE
confirmed the presence of high levels of NAAG-LI in rat optic tract by quantitative radioimmunoassay coupled to HPLC. In the amphibian retina, where NAAG-like immunoreactivity is observed in amacrine and bipolar neurons (Kowalski et al., 1987), synaptic vesicles have been found to contain NAAG-like immunoreactivity (Williamson and Neale, 1987). This important finding, although preliminary, supports current models proposing a synaptic function of the dipeptide. Furthermore, NAAGlike immunoreactivity has been identified in projection neurons of the cat and monkey lateral geniculate nucleus, pulvinar, and cortex, as well as retinal ganglion cells, suggesting the presence of the dipeptide at many levels of visual information processing (Tieman et al., 1987a,b). This finding is highly reminiscent of the presence of the dipeptide at many levels of motor information processing, as discussed above. At present, the neurotransmitter of visual pathways is unknown, although Asp and Glu are leading candidates as the neurotransmitter at several of these levels (Kemp and Sillito, 1982; Schliebs et al., 1984; Hicks et al., 1985); in addition, some findings suggest the involvement of NMDA-type receptors in the organization of cortical visual columns (Singer et al., 1986). Although NAAG has been suggested to have weak, but measurable, activity at NMDA-type receptors on cultured murine spinal neurons (Westbrook et al., 1986), the lack of covariance of NAAG levels with NMDA receptor distribution (Cotman et al., 1987),especially in the hippocampus, would not appear to support the hypothesis that the peptide is the endogenous NMDA receptor ligand. However, it is probably safe to assert that our understanding of the heterogeneity of brain acidic amino acid receptor subtypes is limited and awaits the introduction of more selective pharmacological tools and/or the direct identification of NAAG receptor binding sites. Thus, ideas about a functional role in these pathways await neurophysiological and release studies, as do hypotheses concerning the large majority of NAAG-containing pathways only recently identified with immunocytochemical approaches. In summary, the application of immunocytochemical techniques for the direct localization of NAAG in vertebrate neural tissues has provided evidence that the peptide is, in fact, neuronal in localization and has led to the elucidation of several functionally distinct pathways suitable for more refined functional studies. Among these are many pathways wherein glutamatergic neurotransmission is plausible, including the rodent lateral olfactory tract, spinal sensory projections, and corticofugal and descending brainstem pathways, as well as fibers of the rat, cat, and primate optic tract. Clearly, however, NAAG-like immunoreactivity is not a hallmark of all putative glutamatergic neurons, since hippocampal pyramidal cells and cerebellar granule cells are essentially devoid
NEUROB I 0 LO GY OF N-ACET Y LAS PARTYLG LUTAMATE
69
of immunoreactivity. Finally, neurons with other well-defined neurotransmitters, such as cholinergic motoneurons and noradrenergic and serotonergic brainstem neurons, possess NAAG-LI. The latter findings must broaden our concepts of NAAG function and suggest that neuromodulation,.in addition to (or as opposed to) neurotransmission, may be a mechanism of synaptic action of the dipeptide. If this is the case, a search for direct electrophysiological effects may often not be particularly revealing.
B. EXCITOTOXIC, MECHANICAL, AND PATHOLOGICAL LESIONS OF NAAG-CONTAINING PATHWAYS In support of regional, developmental, and immunocytochemical analyses, the results of lesion studies suggest a neuronal localization of NAAG. Using the excitotoxin kainic acid, a selective neurotoxin that kills many intrinsic neurons in the regions of injection, while sparing neuronal fibers of passage and intrinsic glia (Schwarcz and Coyle, 1977; Coyle and Schwarcz, 1983), Koller and co-workers (1984a) documented significant reductions in striatal and hippocampal NAA and NAAG levels. Hippocampal injections also significantly reduced septal levels, suggesting an involvement of NAAG in the excitatory hippocampal-septa1 projection, or the regulation of NAAG levels in an intrinsic septal population by this pathway. It is also notable that decortication, which removes a substantial neuronal input to the striatum, significantly lowered NAAG and NAA levels ipsilateral to the lesion. Thus, these studies provided important support for the neuronal localization of NAAG, though they were performed in brain regions with relatively low levels of the peptide. A similar conclusion can be drawn for NAA, consistent with a report of its absence in gliomas (Nadler and Cooper, 1972a). These studies also first suggested the identity of specific NAAG-containing neurons such as (1) intrinsic striatal neurons; (2) cortical neurons with striatal projections, such as layer V pyramidal cells; and (3) intrinsic hippocampal neurons and/or septal neurons (Table IV). Of these areas, NAAG-like immunoreactivity can be demonstrated in cortical pyramidal cells (see above) and intrinsic medial septal neurons (Forloni et al., 1987), though not in intrinsic striatal neurons nor in hippocampal pyramidal cells. As previously mentioned, absence of immunoreactivity cannot be taken to indicate absence of peptide in these neurons and may simply reflect the insufficient sensitivity of current immunocytochemical methods. In the spinal cord, where NAAG levels approach those of NAA, midthoracic transection resulted in substantial (up to 50%) reductions of
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RANDY D. BLAKELY AND JOSEPH T. COYLE
TABLE IV EFFECTS OF LFSIONS ON THE LEVELS OF NAAG" Lesion Spinal cord transection Striatal excitotoxin lesion Decortication Hippocampal excitotoxin lesion Olfactory bulbectomy Enucleation of eye
Assayed region
Decrease in NAAG content (%)O
Proximal cord Distal cord Striatum Striatum Hippocampus Septum Pyriform cortex Lateral geniculate Superior colliculus
40 * 50* 31* 28* 37* 29* 22* 64* 79*
"All lesions were carried out in rats. The results are summarized from Koller et al. (1984), ffrench-Mullen et al. (1985), and Anderson et al. (1987). '*, p < 0.05 versus control.
NAAG in segments both above and below the transection (Koller et al., 1984a). Importantly, spinal levels of GABA, a putative neurotransmitter of intrinsic spinal circuits, were unaffected, an important control for local tissue damage induced by the lesion. As the NAAG reductions were still significant many segments away from the lesion and were most pronounced caudal to the transection, they suggested the transection of a major descending NAAG-containing input to the spinal cord and/or the interruption of ascending primary sensory afferents. With a more refined understanding of the probable cellular origin of NAAG in brainstem, spinal cord, and sensory ganglia drawn from immunocytochemical studies, it becomes reasonable to argue that spinal cord transection-induced decrements in NAAG levels represent the interruption of NAAG-containing rubrospinal, vestibulospinal, reticulospinal, and descending monoamine pathways, coupled with lesions to ascending primary sensory pathways. In addition, 50% or more of the NAAG, relative to controls, was still present 1 week after the lesion, and presumably reflects the presence of a major intrinsic, NAAG-containing spinal cord population and/or NAAG-containing spinal sensory afferents with localized, segmental innervation. In contrast to kainate and decortication results, the transection-induced decrements of NAA were not as pronounced as those for NAAG, with significant losses limited to segments nearest the lesion. Unilateral bulbectomy, which deprives pyriform cortex of a major excitatory input (the LOT), resulted in significant ipsilateral reductions
NEUROBIOLOGY OF N-ACETY LASPARTYLGLUTAMATE
71
in NAAG and NAA levels, consistent with electrophysiological findings of a NAAG-like neurotransmitter in this pathway (ffrench-Mullen et al., 1985) (see below). These results predict that NAAG should be synthesized and transported by olfactory mitral or tufted cells, the principal projection neurons of the rodent olfactory bulb (Shepherd, 1979). Thus, lesion data support the findings of a highly localized distribution of olfactory bulb NAAG-like immunoreactivity in the mitral cells. Similarly, Anderson et al. (1987) have reported that unilateral enucleation results in a loss of both HPLC-identified NAAG and NAAG-LI in rat optic nerve, supporting the suggestion that NAAG may be a neurotransmitter for retinal ganglion cells. Thus, lesion studies support a neuronal localization of NAAG and NAA, although populations expressing these molecules may not be coextensive. More importantly, they offer independent biochemical support for immunocytochemical studies, further establishing the specific cellular organization of NAAG in brain. Interestingly, specific reductions of NAAG in several regions of dystrophic mouse brains have been described, including nearly 50% reductions in the cortex, diencephalon, and medulla-pons, with significant reductions also observed in spinal cord (Blakely et al., 1987a). Given the presence of intense NAAG-LI within cortical pyramidal cells, motor relay nuclei of brainstem, and spinal motoneurons, it is tempting to speculate that these reductions represent a biochemical pathology of these pathways that may contribute to the characteristic phenotype of this mouse strain. Levels of NAA in the dystrophic mouse nervous system were not found to be significantly reduced, in contrast to the report of Marcucci et al. (1986). These studies also suggest the utility of other mutant mouse models for the examination of NAAG-containing pathways. To date, only one report exists of NAAG measurements from pathological human tissue. Antuono et al. (1985) found an elevation of NAAG levels in the cortex of patients with Alzheimer’s disease. In light of the partial colocalization of NAAG-like immunoreactivity in rodent basal forebrain cholinergic neurons, the reported increase of NAAG in postmortem Alzheimer’s cortex raises questions concerning the possible sparing of the subpopulation of NAAG-containing cholinergic neurons in the human basal forebrain. However, immunocytochemical studies also demonstrate an intrinsic cortical population of NAAG-like immunoreactive neurons, which may themselves contribute to a large fraction of cortical NAAG levels. In addition, the profile of immunoreactive neurons in the human basal forebrain or cortex has yet to be determined.
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RANDY D. BLAKELY AND JOSEPH T. COYLE
IV. Metabolism of NAAG in Neural Tissues
A. BIOSYNTHESIS: ENZYMATIC OR RIBOSOMAL? Despite the more than 20 years that have passed since the original isolation of NAAG, little conclusive information exists with which to mechanistically describe and manipulate its biosynthesis; only recently has a mechanism for its degradation been uncovered. With major disparities in their regional (Miyake et al., 1981; Koller et al., 1984a), ontogenetic (Miyake and Kakimoto, 1981; Koller and Coyle, 1984a), and phylogenetic (Miyake et al., 1981; Koller et ad., 1984b) profiles, NAA and NAAG are likely to be subject to differential cellular regulation. This appears to be reflected in the dynamic alterations of their levels as well, demonstrated by the ability of amygdaloid-kindled seizures to elevate rat entorhinal cortex levels of NAAG but not those of NAA (Meyerhoff et al., 1985). Thus, biosynthetic hypotheses that relate the two compounds must consider their lack of covariance across regions, development, and phylogeny. Figure 7 presents a summary of potential biosynthetic and catabolic pathways involving NAAG in neural tissues. In contrast to the mechanism of synthesis of bioactive peptides from mRNA transcripts (Mains et al., 1983), several observations appear suggestive of a nonribosomal synthesis of NAAG from amino acid precursors. Low amounts of incorporation of radiolabel from [U-14C]glutamate, [U-'*C]aspartate, and [l-14C]acetateinto NAAG within 1 hr after incubation in murine brain slice preparations were first described by Reichelt and Kvamme (1967). Both the peptide-bound Glu and Asp were found to be of highest specific activity when radiolabeled Glu was the precursor. As the peptide-bound Asp was of lower specific activity than for NAA-bound Asp, a plausible model would entail the formation of NAAG from Glu and NAA, with the large intracellular NAA pool ansing only partially from free Asp. These data could also reflect the involvement of NAA in multiple metabolic compartments, each bearing specific precursors and with different rates of turnover (Nadler and Cooper, 197213). Radiolabeled acetate entered NAAG-bound aspartate carbon to a greater extent than it did NAA aspartate carbon, arguing that, if NAA is a precursor to NAAG, it arises from a small pool separated from a larger, more metabolically stable NAA pool involved in other cellular processes. Such a compartmentation of NAA would help to explain the aforementioned disparities observed for NAAG and NAA, although other explanations remain tenable. Recently, Cangro et al. (1987) have conducted similar experiments with isolated rat spinal ganglia. These investigators observed a small
NEURO B I 0 LO GY 0F N.ACETY LAS PARTY LGLUTAM ATE
NAA 4
+
73
Glu
I
3
FIG. 7. Scheme of potential metabolic pathways for NAAG biosynthesis and catabolism. Boxed labels represent enzymatic activities suggested to be involved in NAAG metabolism. Darker arrows represent pathways that appear most plausible in light of experimental findings. NPM, neuronal plasma membrane; GPM, glial plasma membrane.
incorporation of radiolabel from [3H]glutamate and [3H]glutamine, with the latter molecule serving as the most efficient precursor. Interestingly, [3H]aspartate failed to give detectable incorporation. Hydrolysis of newly synthesized NAAG demonstrated all incorporation to be with NAAG-bound Glu. Coupled with findings that the incorporation was not inhibited by the protein synthesis inhibitors anisomycin OT cyclohexamide, as is the synthesis of other DRG peptides such as substance P (Harmar et al., 1980), these data support the model of enzymatic NAAG biosynthesis from NAA and Glu. Other explanations for these results include the enzymatic formation of non-N-blocked, glutamate-containing peptides that might serve as NAAG precursors in a slightly more baroque mechanism. Glu and
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RANDY D. BLAKELY AND JOSEPH T. COYLE
Gln are known to be incorporated into small y-glutamyl peptides via the y-glutamyl transpeptidase reaction (Meister, 1973), whereas y -glutamyltaurine has been described as a major acidic peptide in brain synaptosomes. Also, the possibility of acetylation of the free N-terminus of a peptide of the sequence Asp-Glu-X,, in a manner like that observed for many other neuropeptides (Mains et al., 1985), rather than the utilization of NAA as a precursor in NAAG biosynthesis, cannot be presently eliminated. However, Truckenmiller et al. (1985) have identified an enzyme activity capable of forming NAA from Asp and acetyl coenzyme A, which is reported to be distributed roughly in accordance with NAAG (but not NAA) levels, enriched in DRG, and with no activity for the acetylation of acidic peptides such as Asp-Glu. Clearly, distinguishing among these biosynthetic possibilities will require more mechanistic studies involving the introduction of specific inhibitors, the identification of subcellular compartments involved in biosynthesis, and the reconstitution of NAAG biosynthesis in Vitro. Several groups have explored NAAG biosynthesis in broken cell preparations. Reichelt and Kvamme (1973) reported the incorporation of labeled NAA into larger peptides, including NAAG, after incubation with mouse brain homogenates, a reaction reported to be dependent upon ATP, stimulated by exogenous CAMP and histamine, and unmodified in the presence of the protein synthesis inhibitors puromycin, cyclohexamide, or chloramphenicol. Based on acid hydrolysis fragments, the structures of NAAG and several much larger forms were deduced. Though it may be tempting to conclude from these and more recent, though similar, experiments (Reichelt et al., 1976; Sinichkin et al., 1977; Uhdesmaki and Timonen, 1982) that indeed NAAG biosynthesis proceeds enzymatically in concert with the enzymatic formation of larger acidic peptides, the products were meager in quantity and have not always been unequivocally identified. Similarly, positive controls, such as the demonstration of alterations in protein synthesis following treatment with protein synthesis inhibitors or the use of RNase to digest contaminating mRNA, have not been described. In attempts to replicate the findings described for broken-cell NAAG biosynthesis, we and others (M. A. Namboodiri, personal communication) have been unable to demonstrate NAAG biosynthesis in the manner described. This may point toward an instability of biosynthetic compartments to the homogenization conditions used or result from the introduction of analytical tools, such as HPW, with greater capacity for resolving products. As a caveat, many of the data available on NAAG biosynthesis mirror early results from studies reporting nonribosomal synthesis for the tripeptide TRH (Jackson and Reichlin, 1973), now known to be synthesized by excision from a larger mRNA template-derived precursor (Rupnow et al., 1979; Richter
NEUROBIOLOGY OF N-ACETY LASPARTY KLUTAMATE
75
et al., 1984; Jackson et al., 1985). Similarly, the bioactive dipeptide GlyGln arises from the proteolytic digestion of @-endorphin(Parish et al., 1983; Plishka et a l . , 1985), and thus even a single peptide bondcontaining molecule must undergo intense scrutiny before its mechanism of biosynthesis can be established.
B. SUBCELLULAR COMPARTMENTATION OF ENDOGENOUS NAAG Only one attempt has been made to biochemically characterize the subcellular distribution of endogenous NAAG (Reichelt and Fonnum, 1969), with results that favor the conclusion of a distribution of NAAG and NAA that might be expected of small, highly soluble, cytoplasmic molecules. However, differences between the subcellular compartmentation of NAA and NAAG were noted, and NAA appeared to be more easily released from particulate preparations by hypo-osmotic conditions than NAAG. One explanation for these findings would postulate a large cytoplasmic NAA pool and a vesicular NAAG pool that also may contain NAA. As yet, the isolation of synaptic vesicles that sequester NAA, in analogy to those reported for Glu (Naito and Ueda, 1985), has not been reported. However, a preliminary report has provided evidence for NAAG-like immunoreactivity in association with subpopulations of synaptic vesicles in the inner plexiform layer of the frog retina (Williamson and Neale, 1987). This finding, if substantiated by biochemical observations, suggests the presence of a discrete compartment within synaptic terminals suitable for NAAG release, potentially serving as a compartment for biosynthesis and/or catabolism. In addition, a NAAGcontaining peptide, N-acetylaspartylglutamyl-taurine,has been biochemically identified as a constituent of bovine synaptosomes and synaptic vesicles (Marnela and Ehdesmaki, 1983; Marnela et al., 1984). The involvement of this latter peptide in the previously discussed routes that are likely for NAAG biosynthesis will need to be investigated, although at minimum, it does mandate caution in the interpretation of immunocytochemica1 findings. C. DEGRADATION OF NAAG BY AN N-ACETYLATED, (11-LINKED, ACIDIC DIPEPTIDE-PREFERRING PEPTIDASE (NAALADAsE)ACTIVITY: A MECHANISM OF NAAG INACTIVATION? Considerably more is known of the likely mechanisms of NAAG catabolism than of its biosynthesis. One of the initial suspicions regarding the neurotransmitter candidacy of small Asp- and Glu-rich peptides,
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RANDY D. BLAKELY AND JOSEPH T. COYLE
like NAAG, was that degradation by brain peptidases might generate free Asp and Glu observed in evoked release paradigms, as well as lead to difficulties in the direct demonstration of evoked NAAG release (Blakely et al., 198613; Coyle et al., 1986; Robinson et al., 1987a). In addition, it was suspected that for NAAG to act rapidly and locally at central synapses, as suggested by electrophysiologicalfindings (Bernstein et al., 1985; ffrench-Mullen et al., 1985), effective mechanisms of inactivation in addition to diffusion must be present (Blakely et a l . , 1986b; Coyle et al., 1986). Little evidence existed, however, to discern whether NAAG could be removed from synapses by a sodium-dependent, highaffinity uptake system as suggested for other putative transmitters, including Glu and Asp, the catecholamines, and GABA (Cooper et a l . , 1986) or was subject to degradation by extracellular peptidases, as is the putative route of inactivation for many neuropeptides (Griffiths and Kelley, 1979; Loh et al., 1984; McKelvey and Blumberg, 1986). Blakely et al. (1986b) provided the first experimental indications, although indirect, of specific NAAG degradation. Incubations of synaptosomes with [3H]glutamate-labeled NAAG under conditions suitable for the measurement of sodium-dependent, high-affinity Glu uptake (SDHAGU) resulted in the accumulation of radiolabel into an osmotically sensitive compartment in a time-, temperature-, and sodium-dependent manner. HPLC analysis of the accumulated radiolabel demonstrated only background levels of radiolabeled NAAG within synaptosomes, whereas a time-dependent increase in sequestered [3H]glutamate was apparent. Although these experiments could not rule out the degradation of NAAG immediately following transport, subsequent studies with [14C]acetate-labeled NAAG demonstrated an inability of the intact peptide to enter the synaptosomes. The apparent mechanism, that of a peptidase degrading NAAG to NAA and Glu, followed by SDHAGU, received support from a pharmacological dissection of these processes. Two groups of compounds were found to inhibit the transport of radiolabel from [3H]NAAG into synaptosomes. A number of amino acid analogs were found to inhibit the transport of [3H]glutamate and of radiolabel from [3H]NAAGwith equivalent potency, suggesting that free and peptide-derived Glu are transported by the'same SDHAGU process. However, a separate set of compounds were found to selectively inhibit the transport of radiolabel from [3H]NAAG. These were all acidic dipeptides, structurally related to NAAG, except for the conformationally restricted analog of Glu, L-quisqualic acid (Quis). As these compounds failed to block the uptake of free Glu, they were proposed to selectively inhibit a peptidase targeting NAAG for degradation. Due to complications associated with an indirect, energy-dependent assay of NAAG hydrolysis in synaptosomes, the direct characterization
NEUROBIOMGY OF N-ACETYLASPARTYULUTAMATE
77
of this peptidase activity required its demonstration in lysed synaptosomal membranes. Robinson et al. (1987a) have recently characterized this activity, termed NAALADase after its apparent specificity for hydrolysis of N-Acetylated a-Linked Acidic Dipeptides, and have reported its partial purification (Robinson et al., 1987b). NAALADase activity is membranebound and enriched in synaptic plasma membranes compared with myelin and mitochondria1subfractions (Blakely et al., 1988). The activity was found to be optimal at neutral pH and at 37OC in vitro with a K , value of 140 nM and V,, value of 180 pm/mg protein/min in rat whole brain. NAALADase displays an absolute requirement for monovalent anions such as C1-, whereas the polyvalent anions phosphate and sulfate inhibit activity at submillimolar concentrations. Since the divalent metal ion chelators EGTA, EDTA, and o-phenanthroline completely abolished activity, which could be restored with manganese, NAALADase is probably a metallopeptidase. Characterization with a large number of generic and specific peptidase inhibitors supports the conclusion that NAALADase is a previously uncharacterized aminopeptidase and is insensitive to antagonists of acetylcholinesterase (EC 3.1.1.7) (physostygmine), angiotensin-converting enzyme (EC 3.4.15.l) (MK-522, a Captopril analog), enkephalin convertase (EC 3.4.17.10) (GEMSA); or enkephalinase (EC 3.4.24.11) (phosphoramidon) (Robinson et a l . , 1987a). Investigation of inhibitor specificity demonstrated a virtually identical pharmacology for NAALADase activity in lysed, synaptosomal membranes as was observed in studies with intact synaptosomes, including potent inhibition by specific acidic dipeptides and by Quis (Robinson et al., 1986, 1987a). A summary of structure-activity relationships inferred from inhibitor profiles is presented in Fig. 8 and Table V. The marked potency of Quis (IC50 = 480 nM) for inhibition of NAALADase represents a previously unrecognized activity of this convulsant Glu analog and suggests that the compound may exert certain of its effects indirectly, by inhibition of a peptidase that cleaves a bioactive brain peptide. In analogy to Captopril and angiotensin-converting enzyme (Patchett and Cordes, 1985), structural analogs of Quis may also provide important tools for the pharmacological manipulation of NAALADase and assist in the determination of its functional relationship with NAAG in vim. In this regard, NAAG has recently been shown to undergo degradation along similar paths in Vivo as in vitro (Blakely et al., 1988b). Thus, intrastriatal coinjections of Glu-labeled [3H]NAAG and [14C]acetatelabeled NAAG are followed rapidly by the appearance of compounds co-chromatographing with [3H]glutamate and [14C]NAA, respectively (tl/z = 10 min). However, further studies are required to definitively assess the degree of participation of NAALADase in NAAG hydrolysis in vivo.
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RANDY D. BLAKELY AND JOSEPH T. COYLE
Alpha-Linkage Increases Potency
0
FIc
CH-C-NH-CH-C-NH-CH-W
8 GIU &eases
Potency
C
FIG.8. Schematic illustration of NAALADase structure-activity relationships inferred from inhibition studies with acidic peptides and their analogs. (From Robinson et al., 1987.)
Examination of regional (Robinson et al., 1987a) and developmental profiles (Blakely et al., 198813) of rat brain NAALADase activity suggest that NAALADase may play a role in the degradation of NAAG along with other structurally related acidic peptides in vivo. Regional studies have revealed an apparent restriction of NAALADase activity to two tissues, kidney and brain (Table VI). This distribution stands in marked contrast to the localization of previously reported acyl-amino acidreleasing activities (Tsunasawa et al., 1975; Marks et al., 1983; Schoenberger et al., 1986) and of peptidases preferring acidic peptide substrates (Meister, 1972; Lojda and Gossrau, 1980; Kelly et al., 1983). Furthermore, this striking regional restriction to two tissues suits an enzymatic function other than that concerned with constituitive protein metabolism. Although a role for renal NAALADase activity is not presently clear, both NAAG and NAA have been reported in human urine (Miyake et al., 1982), and recent findings with a sensitive NAAG radioimmunoassay demonstrated low levels of NAAG in many peripheral tissues, including kidney (Guarda et al., 1987). Within the brain, NAALADase activity is distributed nonuniformly, with highest specific activity found in membranes obtained from cerebellum, medulla-pons, and midbrain, whereas lowest specific activity occurs in telencephalic structures and the spinal cord. The specific activity in the spinal and pontine regions is likely to be deceptively low due to the high content of myelin in these tissues, since myelin membranes exhibit substantially lower NAALADase specific activity compared to synaptic plasma membrane elements (Blakely et al., 198813). NAAG levels, as described above, show a high concentration in caudal brainstem structures and are reduced in forebrain structures. Developmental studies (Blakely et al., 1988b) revealed a marked
NEURO BIO LOGY OF N-ACETYLASPARTY LGLUTAMATE
79
TABLE V AMINOACIDAND PEFTIDEINHIBITORS OF NAALADAsE ACTIVITY’
Amino acid analogs Quis Glu Serine-0-sulfate N -Acetyl-Glu Peptid es N-Acetyl-Glu-Glu N-Acetyl-Asp-Glu (NAAG) Glu-Glu Gly-Gly-Glu Asp-Glu Ala-Glu Gly-Glu y - Glu - Glu y-Glu-Cys-Gly (glutathione) N -Acetyl - GI u -Asp Val-Gly-Asp-Glu Glu-Glu-Glu Phe-Glu
0.48
31 42
58 0.31 0.54
0.75 0.98 2.4
6.1 8.0 9.5 19 36 36 62 77
“NAALADase assays were conducted with increasing concentrations of inhibitor and the IC,, values were calculated from log concentrationpercentage inhibition curves. The reported values are weighted means of IC,, values calculated from each experimental value. All of the data conformed to theoretical inhibition curves with a Hill coefficient of 1. If one assumes competitive inhibition, ICaovalues would be equal to 1.05 of their respective Ki values at the substrate concentration used. Except where noted, all experiments were conducted with L-isomers and a-linked peptides. Results are the means of at least three experiments performed in duplicate. (From Robinson et a l . , 1987.)
postnatal rise in NAALADase activity in most brain regions, with peak activity observed in a region-dependent manner. This pattern of differential temporal expression of rat brain NAALADase activity is expected of an enzyme involved in postnatal synaptic peptide degradation and may reflect the maturation of intrinsic peptidase-synthesizing soma and/or their processes. It is intriguing that the greatest delay in expression (cortex, cerebellum) occurs in regions with pronounced postnatal synaptogenesis (Molliver and Van der Loos, 1970; Altman,
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RANDY D. BLAKELY A N D JOSEPH T.COYLE
TABLE VI REGIONAL DISTRIBUTION OF NAALADAsEACTIVITY-
Region
Activity f SEM (pmol/mg proteidmin)
Activity relative to forebrain (regional activity/forebrain activity)
CNS tzjsues Cerebellum Hyopthalamus Midbrain Olfactory bulb Medulla-pons Thalamus Septum Cervical cord Forebrain Hippocampus Striatum Cortex Peripheral tissues Kidney Sciatic nerve Adrenal Lung Heart Liver Small intestine Pancreas Testis Skeletal muscle
15 f 1.9 12f1.6 12f1.7 8.8 f 1.3 8.7 f 1.0 7.9 f 0.9 7.7 f 0 . 5 6.5 f 0.5 6.4fl.l 6.2 f 0 . 7 5.4f0.8 5.2f0.4
0.8 0.8
25f0.3 1.6f0.2 0.9 0.1 0.5 f 0 . 4 0.2 f 0 . 0 3 0.1 f 0.2 0.1 f 0.2 -0.1 f 0 . 2 -0.1 f O . l - 0.4 f 0.4
4.0 0.2 0.1 0.1 0.03 0.02 0.02 -0.01 - 0.01 -0.1
*
2.4 1.9 1.8 1.4
1.4 1.2 1.2 1.o 1.o 1.o
"NAALADaseactivity, measured as the amount of ['Hlglutamate-like material liberated from ['Hlglutamate-labeled NAAG, was quantitated in membrane preparations of various CNS and peripheral tissues. Parallel assays containing Quis (100 ~ L Mrevealed ) that all of the observed metabolism from all regions was Quis-sensitive. Data are the mean f SEM of duplicate assays on at least three different membrane preparations. (From Robinson et al., 1987.)
1972). Furthermore, both brainstem NAALADase activity and NAAG levels peak at day 8 in rat and plateau thereafter, whereas cerebellar NAAG levels and NAALADase activity show a delayed postnatal expression. However, a developmental correspondence is not evident in other regions and may reflect the involvement of NAALADase with other substrates or association of the two with independently regulated cell populations. Until the sites of expression of NAALADase activity and the site of NAAG biosynthesis and storage are more precisely determined,
NEUROBIOLOGY OF N-ACETYLASPARTYLGLUTAMATE
81
one cannot infer too much (or little) from correspondence between the regional and developmental profiles of NAAG levels and NAALADase activity. Indeed, recent lesion studies (Blakely et al., 1988b) suggest that glial membranes may express NAALADase activity. Obviously, immunocytochemical (Shine and Haber, 1981; Pickel et al., 1986) and autoradiographic (Strittmatter et al., 1984; Lynch et al., 1984) approaches are needed to further define the cellular localization of NAALADase. These studies document the most likely route of NAAG degradation, both in vitro and in vivo, and suggest a testable mechanism for NAAG inactivation upon release. Its ionic requirements, sensitivity to reducing conditions, and inhibition by Glu, suggest that NAALADase activity is likely to exhibit optimal activity in an extracellular environment or within an intracellular organelle with extracellular characteristics, such as the inside of synaptic vesicles. Thus, the possibility that NAALADase might cleave NAAG prior to release, liberating neurotransmitter Glu, remains a possibility, although the apparent involvement of NAAG in nonglutamatergic neurons such as motoneurons suggests that NAAG is neither a replacement for transmitter Glu nor its precursor. Rather, NAAG is more likely to have its own intrinsic activities, which dictate a unique regional and cellular distribution.
V. The Search for NAAG Receptors and Their Mechanism of Action
A. ELECTROPHYSIOLOGICAL EFFECTS OF NAAG Of paramount importance for the establishment of NAAG as a neurotransmitter or neuromodulator is the identification of specific physiological responses linked to its interaction with brain receptors. Although modern concepts of neuromodulation by synaptically released molecules involve electrically silent, receptor-mediated signal transduction systems (for several examples, see Kaczmarek and Levitan, 1987), direct electrophysiologicalresponses are expected of exogenous molecules argued to be identical to endogenous neurotransmitters directly mediating rapid, frequency-coded information processing. Glu and Asp are thought to be involved in such pathways throughout the CNS; thus, if NAAG (or NAA, for that matter) is a neurotransmitter at certain of these synapses, it would be expected that specific, quantifiable electrophysiological responses, similar to those observed after physiological stimulation, should be observed. Indeed, recent findings from diverse systems haee suggested intrinsic activity of the dipeptide at subclasses
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of glutamate receptors (ffrench-Mullen et a l . , 1985; Westbrook et al., 1986; Mori-Okamoto et a l . , 1987; Joels et al., 1987; Sekiguchi et al., 1987), consistent with the original suggestion of NAAG neurotransmission in subsets of glutamatergic pathways. At present, however, no concensus has been achieved as to the nature of the receptor with which NAAG may interact, as both NMDA-type (Westbrook et al., 1986; MoriOkamoto et a l . , 1987; Sekiguchi et a l . , 1987) and non-NMDA-type (ffrench-Mullen et al., 1985; Joels et al., 1987) excitation has been reported. Furthermore, the presence of NAAG-like immunoreactivity in nonglutamatergic neurons and the low potency noted by several investigators in certain biochemical indices of glutamatergic receptor stimulation (Luini et al. , 1984; Riveros and Orrego, 1984; Nicoletti et al., 1986; Westbrook et al., 1986), warrants the introduction of other models for the mechanism of NAAG action. In this regard, it should become apparent that all reported activities of the peptide have arisen from studies in which NAAG was applied alone. Just as a novel activity of glycine was recently uncovered when the amino acid was applied with NMDA or Glu (Johnson and Ascher, 1987), coadministration of NAAG and synaptic agonists may lead to a new understanding of the peptide’s function in neural tissues. Such a paradigm may also reflect more upon in Vivo findings, reviewed below, especially if modulation of synaptic responses is a principal mechanism of NAAG action. 1. In Vivo Studies
Shortly after the discovery of NAAG, Curatolo et al. (1967) described the ability of both NAA and NAAG to depress cortical multiunit activity after local application and to antagonize the effects of locally applied Glu or Asp, respectively. Similarly, Avoli and co-workers(1976) reported similar alterations in the spontaneous activity of rat cortical, thalamic, mesencephalic, and cerebellar neurons after local NAA and NAAG application, with both facilitory and inhibitory effects on multiunit firing rates reported. Although these studies are difficult to interpret, the regional potency of NAAG was reported to be roughly parallel with the distribution of NAAG levels. The activity of NAA in these preparations is curious, given the structure-activity studies of Curtis and Watkins (1960), which revealed an inability of NAA to depolarize neurons within the cat spinal cord. In addition, Jacobson (1959) had previously found NAA unable to act on the crayfish stretch receptor. Cecchi et al. (1978), with an apparently unconfirmed finding, suggested that NAA might act @esymfitically at the squid giant synapse. In another in wvo preparation, Zaczek et al. (1983) reported the induction of cortical seizure activity
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following injection of 125 nmol of NAAG into the rat hippocampus, with a latency and potency similar to that of L-quisqualate. Injections of NAAG in vivo have been shown to be followed rapidly by hydrolysis at the Asp-Glu peptide bond, generating NAA and Glu, and thus the delayed seizure responses noted are probably not due to the continued presence of the dipeptide (Blakely et al., 1988b). Importantly, the cortical seizures caused by NAAG are not characteristic of intrahippocampal Glu injections, even at much higher concentrations, a control apparently overlooked by certain critics (Riveros and Orrego, 1984), although the coinjection of Glu with NAA has not been reported. Nonetheless, a critical test of the direct effects exerted by NAAG in this paradigm awaits the development of specific NAALADase inhibitors, which, by blocking NAAG degradation, should increase the peptide’s potency. No attempt was made in these initial studies to characterize the type of receptor at which NAAG might be exerting direct effects. Joels et al. (1987) have recently reported NAAG-induced excitation of lateral septal neurons in anesthetized rats in vivo. Neuronal sensitivity to NAAG was enriched in the lateral septum in contrast to the medial septum; the lateral septum is known to receive excitatory glutamatergic hippocampal projection (Zaczek et al., 1979). Glutamate diethyl ester (GDEE) blocked NAAG responses, Quis responses, and those recorded previously from fimbria stimulation. The specific NMDA receptor antagonist (Davies et al., 1981) D-aminophosphonovalerate(APV), however, was a much weaker antagonist of NAAG-induced excitation than of NMDA responses. Consistent with this action of NAAG in the septum, Koller et al. (1984a) found a reduction in septal NAAG levels following hippocampal kainate lesions. In contrast, Henderson and Salt (1988), in extracellular recordings of the ventrobasal thalamic neurons of the urethane-anaesthetized rat, found little intrinsic excitatory activity of iontophoretically applied NAAG, although the neurons could be activated by somatosensory inputs as well as by Glu, kainate, and Quis. However, over half of the units exhibited inhibitory effects of concurrent application of NAAG with kainate or N-methyla,L-aspartic acid; fewer than a quarter of the units showed enhancement of the excitatory effects. Although all of these studies suffer from complexities inherent with in vivo analyses, such as the potential proteolysis of the dipeptide and an inability to distinguish pre- and postsynaptic action, they represent the few neurophysiological studies conducted in mvo in brain (also see Mori-Okamoto et al., 1987) and suggest a relatively unexplored direction for future studies in NAAG physiology.
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2. In Vitro Studies
More recent studies conducted with acute brain slices or neuronal cells in culture have demonstrated NAAG-induced depolarizations of single neurons that in vivo receive well-characterized excitatory innervation, presumably mediated by an Asp- or Glu-like neurotransmitter (ffrench-Mullen et al., 1985; Bernstein et a l . , 1985; Westbrook et al., 1986; Mori-Okamoto et al., 1987; Sekiguchi et al., 1987). Synapses of the LOT in rat pyriform cortex have been argued to utilize neither Glu nor Asp as a neurotransmitter (Hori et al., 1981), despite supporting biochemical evidence from several rodent species (Harvey et al., 1975; Bradford and Richards, 1976; Yamamoto and Matsui, 1976; Collins and Probett, 1981). This surmise resulted from the inability of the antagonist D,L-APBto block the excitation of exogenous Asp and Glu at concentrations that abolish synaptic excitation produced by direct LOT stimulation (Hori et al., 1981). In contrast, responses evoked by iontophoretically applied NAAG were consistently abolished by D, L-APB(ffrench-Mullen et al., 1985) (Fig. 9). D,L-APV,which lacked inhibitory activity on LOT synaptic responses, failed to block NAAG-induced excitation, although antagonism of Glu and Asp effects was observed. Thus, NAAG exhibited a pharmacological sensitivity to antagpnists similar to the endogenous neurotransmitter of the LOT, whereas Glu and Asp did not. NAA was found to be electrophysiologicallyinactive with both bath perfusion and iontophoresis onto single neurons excited by aspartate, suggesting that the rapid effects observed with NAAG were direct. In addition, Hearn et al. (1986) have noted a heterogeneity in the evoked LOT response that is only partially inhibited by L-APB.These authors suggested that their preparation may contain the projections of olfactory bulb tufted as well as mitral cells, which may potentially utilize different neurotransmitters and/or affect different receptors. This line of reasoning is supported by the restriction of NAAG-like immunoreactivity to olfactory bulb mitral cells (Anderson et al., 1986; Blakely et al., 198713). Furthermore, these results support the proposal of a more limited target of NAAG-induced effects than either Asp or Glu, similar to that of the endogenous mitral cell LOT transmitter. Bernstein et al. (1985) have also reported a more confined microanatomical localization of NAAG responses (restricted to dendrites of CAI hippocampal pyramidal cells) than of those obtained with Glu, although the pathway through which these effects might be mediated has yet to be identified. In contrast to the non-NMDA receptor-mediated responses observed for NAAG in the LOT and lateral septa1 nucleus, Westbrook et al. (1986) have recently demonstrated NAAG-induced depolarization of dissociated spinal neurons in culture, at high concentrations, with electro-
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Control
APB (10% M )
Wash
NAAG
I
'
IOOVV
1
FIG. 9. Reversible antagonism of responses to iontophoretically applied NAAG (100 nAmp) and stimulation of the LOT by APB (100 p M ) with no effect on the response to L-glutamate (130 nAmp). Iontophoretic current pulses were of l-sec duration, and each barrel had a 2.5-nAmp retaining current. After APB perfusion for 8.5 min, the NAAG and LOT responses were completely blocked. In the LOT trace, an upward shock artifact precedes the extracellular spike; in the presence of APB, only the LOT shock artifact remains. After a 12-min wash, the NAAG and LOT responses have returned. (From ffrench-Mullen et al., 1985.)
physiological properties characteristic of NMDA receptors, namely voltage-dependent activation, sensitivity of responses to Mg2 , antagonism by D, L-APV,and similar changes in channel conductances (opening of 50- to 60-pS conductance channels) to those induced by NMDA itself. However, because of the low apparent potency of NAAG in inducing these depolarizations and the lack of regional correspondence of NAAG levels with NMDA-sensitive [3H]glutamate-binding sites (Monaghan and Cotman, 1985), these authors have concluded that +
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NAAG is unlikely to exert neurotransmitter action through NMDA receptors, if it has a synaptic function at all. They argue for an examination of other potential functions, such as a role in the biosynthetic regulation of a neurotransmitter pool of glutamate. It is presently unclear how such a role could explain the skewed regional distribution of NAAG, as extensive cortical and hippocampal pathways are thought to be glutamatergic as well as spinal pathways (Fagg and Foster, 1983; and above discussion of NAAG metabolism). Alternative conclusions possible from these studies would be that NAAG acts with higher potency on a class of non-NMDA receptors absent or altered on these cultured spinal cord neurons, although Quis and kainate type responses can be demonstrated (Mayer and Westbrook, 1984). Notably, culture conditions have been shown to affect the expression of Glu receptors on motoneurons (O’Brien and Fischbach, 1986). Alternatively, NAAG may act synaptically on NMDA-type receptors with higher potency under conditions not replicated in culture. The latter possibility would presuppose a neuromodulatory rather than a neurotransmitter role for NAAG on these cells, an activity not unwarranted for brain NMDA receptors. Despite clear controls for free Glu in the NAAG samples applied, these authors have little control over the amount of peptide that actually reaches the receptor intact. Similarly, potency of NAAG in vivo, relative to Glu, on these receptors may not be as low given the activity of highaffinity synaptic Glu uptake processes (Fonnum, 1984) for which NAAG is not an immediate substrate (Blakely et al., 1986b). NAAG has also been found to excite cultured chick cerebellar neurons, identified by the authors as Purkinje cells, with a potency roughly equivalent to Asp but less than that of Glu (Mori-Okamoto et al., 1987). In agreement with the findings of Westbrook et al. (1986), NAAG elicited a Mg2+-sensitive, voltage-dependent depolarization of these neurons with a reversal potential identical to that observed with Asp or Glu. Antagonists (APV, DAA, APB, GDEE) blocked NAAG-induced excitation in a similar manner to that elicited by Asp, whereas Glu appeared to have a broader pattern of action than either NAAG or Asp. In contrast to findings from rodent preparations, APB appears to be a preferential NMDA-type receptor antagonist whereas APV is much weaker in the chick preparation. Thus, these investigators use the greater ability of APV over APB to block NAAG responses to argue that NAAG is not acting through chick cerebellar NMDA receptors. These findings may represent species-specific pharmacology of excitatory amino acid receptors or the presence of a unique (NMDA receptor-like)NAAG receptor with properties, other than antagonist sensitivity, very similar to the rodent NMDA-gated channel. Notably, levels of NAAG are extra-
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ordinarily high in chick brain (Miyake et al., 1981; Koller et al., 1984b), making this preparation a very useful one for further characterization. In summary, studies examining the direct effects of NAAG on vertebrate neurons have demonstrated intrinsic activity of the dipeptide on at least one subtype of excitatory amino acid receptor, although the low potency of the peptide in these paradigms is a consistent finding. It is difficult to argue against compounds as neurotransmitters simply on the basis of potency, given our ignorance of their synaptic concentrations and difficulties replicating in uivo conditions in m'tro. Nonetheless, these findings may reflect the involvement of NAAG in synaptic roles unrelated to glutamatergic neurotransmission, although it is still possible to reconcile them with activation of subpopulations of acidic amino acid receptors. B. INVITROBIOCHEMICAL APPROACHES TO NAAG FUNCTION With regard to present models of NAAG neurotransmission at glutamatergic synapses, it should be noted that NAAG has been reported to be inactive in three putative biochemical tests of excitatory amino acid receptor stimulation, namely agonist-stimulated phosphatidyl inositol turnover (Nicoletti et al. , 1986), agonist-stimulated Ca2+ flux (Riveros and Orrego, 1984), and agonist-induced Na+ flux (Luini et al., 1984). No information is available regarding the ability of NAAG to stimulate or inhibit the production of CAMP,cGMP, or arachidonic acid metabolites, second messengers linked to responses of other bioactive neuropeptides. Interestingly, NAA has been reported to elevate cAMP and cGMP in minced forebrain preparations (Burgal et al., 1982). Although these effects were observed at rather high millimolar concentrations, they suggest a potential biochemical index for NAAG function in which a structurally related molecule is active. Furthermore, NAA was more potent than Asp or Glu at elevating cAMP production and may reflect the presence of receptors specific for N-blocked acidic peptides. It is with the recognition of NAAG hydrolysis by NAALADase activity, under conditions considered optimal for the demonstration of binding sites labeled by [3H]NAAG, that evidence of NAAG interaction with Glu receptor subtypes must presently be regarded as suspect (Blakely, 1987; Blakely et al., 1988a). Based upon constants determined from quantitative kinetic studies of peptide hydrolysis in 50mM Tris-HC1(previous binding media, Zaczek et al., 1983; Koller and Coyle, 1984b; Koller and Coyle, 1985), a subset of [3H]glutamate sites is predicted to be displaced
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by peptide-derived Glu with higher apparent affinity than observed with exogenous free Glu itself, without imputing intrinsic displacement activity for the dipeptide. Coupled with the growing appreciation that sites labeled by [3H]glutamateunder these conditions do not have the kinetic properties expected of a Glu “receptor” (Pin et al., 1984; Fagg and Lanthorn, 1985; Zaczek et al., 1987), the association of NAAG with Glu receptors cannot be supported by present biochemical evidence. Clearly, the in vitro identification of biochemical activities and membrane binding sites specific for the dipeptide may profit from the development of NAALADase inhibitors and the synthesis of nonhydrolyzable peptide analogs and is critical for synaptic models of NAAG action, glutamatergic or other.
VI. Conclusions
Information regarding the role of NAAG in the vertebrate brain has grown considerably since its reisolation from rodent brain as an endogenous ligand for putative brain membrane glutamate receptors (Zaczek et al., 1983). Although present evidence argues strongly against a deduction of NAAGs synaptic role from [3H]glutamate “receptor” displacement studies (Blakely et al., 1988a), the hypothesis of a neurotransmitter role for the dipeptide, especially within glutamatergic pathways, has provided specific and testable explanations for its mechanism of action. Although not without experimental support, especially in the rodent lateral olfactory tract (ffrench-Mullen et al., 1985; Anderson et al., 1987; Blakely et al., 1987b), the glutamatergic neurotransmitter hypothesis would not appear to be inclusive enough to cover most recent biochemical and immunocytochemical findings. It appears reasonable, then, at this juncture, to summarize the cogent points expanded upon in the preceding pages with the specific aim of underscoring the unresolved questions that must be answered as more detailed hypotheses are generated. Although the restriction of NAAG to neural tissue has been clear for some time (Curatolo et al., 1965; Miyake et al., 1981; Koller et al., 1984), the description of NAAG as a neuropeptide has rested upon interpretations of its differential regional distribution, developmental expression, and response to mechanical and neurotoxic lesions. Immunocytochemical studies, which have demonstrated the presence of intense NAAG-like immunoreactivity in neuronal somata in both the CNS and PNS, have provided the first direct evidence for the peptide’s neuronal
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localization in the vertebrate nervous system (Anderson et al., 1986; Blakely et al., 1987b; Ory-Lavollee et al., 1987; Kowalski et al., 1987; Forloni et al., 1987). Immunoreactive neurons are distributed in accordance with NAAG levels, with large numbers of neurons stained in brainstem and spinal cord. The cumulative data from independent laboratories with different antibodies make it highly probable that NAAG is synthesized by specific sets of neurons in the vertebrate CNS, restricting hypotheses of its functions to those features characteristic of neurons. The neuronal function most often ascribed for NAAG is neurotransmission in putative glutamatergic pathways (Zaczek et al., 1983; Koller et al., 1984; Coyle et al., 1986). In this regard, NAAG-like immunoreactivity is present within mitral neurons of the rodent olfactory bulb (Anderson et al., 1986; Blakely et al., 198713). These cells give rise to fibers that course through the lateral olfactory tract (LOT) and synapse upon dendrites of pyriform cortex pyramidal cells (Shepherd, 1979). In previous electrophysiological studies, a NAAG-like neurotransmitter has been proposed for synapses of this pathway (ffrenchMullen et al., 1985), previously considered to use Glu or Asp (Collins et al., 1981; Fagg and Foster, 1983). In addition, unilateral bulbectomy significantly reduces the NAAG concentrations of the ipsilateral pyriform cortex (ffrench-Mullen et al., 1985). Thus, neurochemical, electrophysiological, and anatomical studies support a role of NAAG as a neurotransmitter in the lateral olfactory tract, lending credence to the glutamatergic neurotransmission hypothesis. Recent electrophysiological findings with other neurons receiving well-described excitatory innervation, attributed to a Glu- or Asp-like neurotransmitter, have found NAAG to have activity at pharmacologically identifiable subtypes of excitatory amino acid receptors (Westbrook et al., 1985; Bernstein et al., 1985; Joels et al., 1987; Mori-Okamoto et al., 1987). However, evoked release of the peptide from LOT fibers, or any other neural pathway, has yet to be demonstrated. NAAG-like immunoreactivity has been described to be associated with secretory vesicles of the amphibian retina (Williamson and Neale, 1987) and NAAG levels can be significantly lowered by transection of putative glutamatergic pathways (Koller et al., 1984a; ffrench-Mullen et al., 1985; Anderson et al., 1986). With the application of immunocytochemical strategies, the list of putative glutamatergic neurons wherein NAAG may act synaptically is now much larger, although others such as hippocampal pyramidal cells and cerebellar granule cells are unlikely candidates. Thus, if NAAG does act as a neurotransmitter at any of these synapses, a determination of the important structural or functional features that distinguish these from glutamatergic pathways that do not utilize the dipeptide may
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provide important tools for excitatory amino acid pharmacology and therapeutics. The biochemical isolation of NAAG-containing synaptic vesicles and their comparison with the Glu-sequesteringvesicles reported by Ueda is a potentially fruitful line of research with direct bearing upon the specific relationship of NAAG with neurotransmitter Glu in synaptic terminals. Until the evoked release of the peptide is demonstrated, however, the hypothesis that NAAG is a precursor for transmitter Glu remains a possibility (Westbrook et al., 1985), although here again, present data suggest that such a precursor role would not be exclusive for all glutamatergic terminals. Although the most plausible biosynthetic routes involving NAAG can be outlined, and present data point toward enzymatic pathways (Reichelt and Kvamme, 1973; Sinichkin et al., 1976; Cangro et al., 1987), the understanding of NAAG biosynthesis remains rudimentary. Thus, the elucidation of the specific routes by which NAAG is synthesized is of utmost priority, as such findings will undoubtedly lead to the production of antibodies against specific biosynthetic enzymes or RNA probes for in situ hybridization with specific peptide precursors to complement the direct visualization of NAAG-like immunoreactivity. If, indeed, NAAG is synthesized enzymatically, the isolation and reconstitution of specific enzyme activities may lead to strategies for the regulation of the peptide’s function via the development of site-directed synthesis inhibitors, with importance for basic and clinical neurochemistry. Many neurotransmitters have complementary inactivation processes that can limit the duration of their actions. The monoamines and acetylcholine possess defined catabolic enzymes and selective transport processes suitable for synaptic inactivation (Cooper et al., 1986). Neuropeptides are generally considered to be inactivated by extracellular peptidases (McKelvey and Blumberg, 1986). NAAGs reported electrophysiological responses are relatively brisk (ffrench-Mullen et al., 1985; Bernstein et al., 1985) and may reflect a rapid mechanism of extracellular inactivation. In this regard, a C1- -dependent, membrane-bound metallopeptidase activity with high specificity for small N-acetylated, Glu-containing acidic peptides is present in brain membranes (Robinson et al., 1987) and concentrated in subcellular membrane fractions enriched in intact or fragmented junctional complexes (Cotman and Taylor, 1972). The peptidase can rapidly cleave NAAG to NAA and Glu, a catabolic route exhibited in vivo as well (Blakely et al., 1988b). The peptidase activity is expressed postnatally during a major period of synaptogenesis in the rodent brain and, in regions in which NAAG levels are highest, proceeds in parallel with the ontogenetic increase in NAAG levels
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(Blakely et al., 1988b). Although this peptid.ase, termed NAALADase, may have other substrates in vivo, it is likely that extracellular NAAG would be subject to NAALADase hydrolysis upon release. Any Glu produced from NAAG by NAALADase would probably be removed from the extracellular space by the high-affinity Glu transport system, in analogy with the efficient transport of choline from hydrolyzed acetylcholine. Thus, a route by which synaptic NAAG could be efficiently inactivated upon release has been identified, although an extracellular site of NAALADase activity remains to be demonstrated. The identification of selective inhibitors of this activity should bring this hypothesis to a test. In this regard, L-quisqualate, a conformationally restricted Glu analog, has been identified as a potent inhibitor of NAALADase activity. Indeed, one may wonder whether certain of the neurobiological effects of quisqualate are mediated indirectly, through an inhibition of the degradation of an acidic dipeptide. Modifications of the structure of quisqualate or those of acidic dipeptides may produce the tools necessary for an experimental investigation of this hypothesis, without the present concern for agonist activities, and, as well, illuminate the role of NAALADase activity in synaptic NAAG inactivation. Furthermore, the purification of this enzyme (Robinson et al., 1987b) should lead to its visualization by immunocytochemical strategies and clarification of its relationship with NAAG-synthesizing neurons. If NAAG acts as a neurotransmitter or neuromodulator, the peptide should possess identifiable receptors through which its effects are mediated. These receptors have been argued not to be those of excitatory amino acids coupled to phosphatidyl inositol turnover (Nicoletti et a l . , 1986), Na+ flux (Luini et al., 1984), or Ca2+ uptake (Riveros and Orrego, 1984) systems. The synaptic responses of the endogenous transmitter (Hori et al., 1981) and of NAAG in the terminal fields of the LOT are selectively inhibited by L-APB(ffrench-Mullenet al., 1985). Thus, it is expected that NAAG recognition sites in membranes that are APB-sensitive should exist and be revealed in radioligand-binding studies. Indeed, previous radioligand-binding studies (Koller and Coyle, 1984; Koller and Coyle, 1985) identified such sites and found them to possess a pharmacological specificity suggestive of a labeling of synaptic receptors. However, these sites are likely linked to a chloride-dependent transport process of as yet undefined function (Pin et al., 1984; Zaczek et a l . , 1986), and thus their identity with synaptic receptors must be questioned. Furthermore, the previously described competition with, and labeling of, these sites by NAAG can be accounted for on the basis of peptidase hydrolysis of Glu from NAAG in C1- -containing buffers, without imputing an intrinsic activity for the intact dipeptide. Thus,
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the in vitro biochemical demonstration of NAAG receptors must be presently regarded as undocumented. Here again, the development of nonhydrolyzable NAAG analogs may permit the direct demonstration of NAAG receptors in radioligand-binding paradigms. The identification of NAAG receptors is clearly critical for specifying an extracellular function for NAAG. Westbrook and colleagues (1986) have documented a low, though measurable, potency of NAAG at NMDA-type Glu receptors on cultured spinal cord neurons, effects argued to be direct and unrelated to NAAG hydrolysis. In the LOT studies, however, NAAG was shown to have activities pharmacologically distinguishable from those of NMDA, Glu, or NAA, whereas Joels et al. (1987) and Mori-Okamota et al. (1987) have recently demonstrated electrophysiological effects of the dipeptide suggestive of interactionswith distinct subtypes of excitatory amino acid receptors. Clearly, further electrophysiological and radioligand-binding studies, under conditions of NAALADase inhibition, are required to determine the specificity and potency of NAAG for putative excitatory amino acid receptors. Here again, NAAG release must be demonstrated before an extracellular role can be established with certainty, as an electrophysiological finding can always be regarded as a pharmacological curiosity unless a definitive match is made between agonist and endogenous ligand. The advent of very sensitive radioimmunoassay methods for the measurement of picomolar NAAG concentrations (Guarda et al., 1988) and the synthesis of selective NAALADase inhibitors should prove instrumental in elucidating this latter problem. Just as the synaptic receptors that can convey NAAG's responses may not coincide with those activated by Glu, the pathways within which NAAG functions would not appear to be exclusively glutamatergic. One intriguing finding that may yield functional information is the presence of immunoreactivity along consecutive levels of sensory and motor pathways. Cholinergic motoneurons, noradrenergic locus coeruleus neurons, and serotonergic raphe neurons, among others, exhibit intense NAAG-like immunoreactivity (Blakely, 1987; Ory-LavollCeet al., 1987; Forloni et al., 1987) and are distributed throughout the spinal cord and brainstem, in which NAAG levels are at their highest (Koller et al., 1984; Ory-LavollCe et al., 1987). Although they agree with quantitative neurochemical studies, immunocytochemical results may not unequivocally identify the antigens visualized. Thus, independent validation, such as the documentation of enriched levels of NAAG in isolated motoneurons, should clarify the presence of NAAG in nonglutamatergic neuronal populations. If these immunocytochemical findings truly reflect the cellular localization of NAAG, they demand a search for alternative
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hypotheses unrelated to the postulated roles in excitatory synaptic neurotransmission. Thus, a virtually unexplored area for future studies is the ability of NAAG to modulate the synaptic responses of other neurotransmitters, especially in light of its reportedly weak potency for excitatory amino acid receptors and the presence of NAAG-like immunoreactivity in chemically defined, nonglutamatergic neurons. Investigations of this type should involve the coapplication of NAAG with specific neurotransmitters on potential targets suggested by immunocytochemical studies. Broadening concepts of NAAG synaptic action, focused within the specific pathways only recently identified and utilizing new information regarding routes of NAAG metabolism, portend an exciting, and potentially surprising, area of neurobiological studies.
Acknowledgments
T h e authors thank Alice Trawinski for her secretarial assistance and appreciate the contributions and comments of their coinvestigators, including M. Robinson, G. L. Forloni, L. Ory-Lavollee, R. Zaczek, and K. Koller. Some of the research described in this review was supported by USPHS Grant NS 13584, a McKnight Scholar Award, and the Surdna Foundation.
References
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NE U ROPEPTIDE- PROCESS ING, - CONV ERT ING, AN D -INACTIVATING ENZYMES IN HUMAN CEREBROSPINAL FLUID By Lars Terenius and Fred Nyberg Department of Pharmacology Uppsala University 751 24 Uppsala, Sweden
I. Introduction A. Neuropeptide-TransformingEnzymes in CSF B. Isolation of Neuropeptide-TransformingEnzymes from CSF 11. Individual Enzymes in CSF A. Angiotensin-Converting Enzyme B. Substance P-Converting Endopeptidase C. a-Amidating Monooxygenase D. Dynorphin-Converting Enzyme E. Membrane Metallo-Endopeptidase (EC 3.4.24.11) (Enkephalinase) F. Aminopeptidases G . Acetylcholinesterase H. Potential Significance of Proteases-Peptidases in CSF 111. Concluding Remarks References
1. Introduction
Analysis of the constituents in cerebrospinal fluid (CSF) has a relatively long tradition in clinical chemistry. Also, enzymes have been measured in CSF, including “peptidases” or “proteases” of unknown character that have been considered as potential indicators of tumors (e.g., Green and Perry, 1958), purulent meningitis (Buchler, 1938), or demyelinization in multiple sclerosis (Rinne and Riekkinen, 1968). More recently, candidate markers of more specific processes have also been measured. This review will focus on proteolytic enzymes that more or less specifically degrade neuropeptides. Such enzymes could well have important regulatory roles. The biosynthesis of biologically active peptides is stereotypical and common for both neurons producing neuropeptides and endocrine cells. The peptides derive from precursor proteins called prohormones. By limited and selective proteolysis, the active peptides are released. INTERNATIONAL REVIEW OF NEUROBIOLOGY. VOL. 30
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Copyright @ 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.
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Sometimes the products of proteolysis are further modified by enzymatic acetylation or a-amidation, or by secondary proteolysis. Such secondary transformations may be called conversions and the enzymes collectively called converting enzymes. Finally, transformations, particularly by proteolysis, may lead to inactivation. These metabolic steps are schematically outlined in Fig. 1. It should be emphasized that a particular enzyme may serve in several different capacities; for instance, angiotensinconverting enzyme, in addition to converting angiotensin I to angiotensin 11, which is the active peptide, also degrades enkephalin and enkephalyl peptides into inactive fragments (Lantz and Terenius, 1985). During the past two years, the structures of the protein precursors of most of the major neuropeptides have been identified. The active peptide sequences are usually flanked by basic amino acids, frequently arranged in doublets (Fig. 2). The number and location of such sequences may, however, vary. Although it was originally postulated that one or a very few enzymes are involved in the excision, it is now clear that there are several enzymes with this capacity that are mechanistically distinct (Cohen, 1987). Cleavage may also occur at single basic amino acids, particularly arginine, but in such cases other structural requirements are also important (Schwartz, 1986).
Prohormone Processin enzyme (sj
Neuropeptide A Conversion enzyme (s) Peptidases Neuropeptide B
4T
Inactive fragments
c
FIG.1. Steps in the enzymatic peration of neuropeptides from prohormones and their enzymatic inactivation. h e s i n g enzymes excise peptide fragments from the prohormones; converting enzymes transform an inactive or active peptide to another peptide with biological activity; inactivating enzymes transform an active peptide into inactive fragments. FIG.2. Schematic representation of three neuropeptide prohormones: preproenkephalin A, the enkephalin (enk) precursor; preproenkephalin B, also call prodynorphin, the precursor of dynorphins (Dyn) A and B and a-neoendorphin; and a-preprotachykinin, the precursor of substance P.
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PREPROENKEPHALIN A 1 I
20
40
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I
60 I
80 I
100 I
120 I
160 J
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Putative signal peptide d
P
PREPROENKEPHALIN B 1
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Putative signal peptide
o
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ALPHA-PREPROTACHY KlNlN
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_ l u
Putative signal peptide
Substance P
P P P E
a a
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Alpha-neoend.
? P a a
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_
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260 I
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The secondary conversion step may involve several kinds of enzymes. Carboxypeptidases may be exemplified by enkephalin convertase, which trims off a C-terminal basic amino acid left at processing. Other transformations involve acetylation or C-terminal a-amidation. The amidation process is essential for activity in a large group of neuropeptides (Mains et al., 1983). Further proteolytic degradation that generates active fragments is well known in several systems and will be discussed below. These observations coincide with an increasing awareness that neuropeptides may have several “message”sequences (e.g., Schwyzer, 1977). Finally, proteolytic activity may lead to inactivation. Since there is no convincing evidence that inactivation occurs by other mechanisms (sesquestration, conjugation, neuronal reuptake, etc.), enzymatic inactivation has been studied extensively and probed pharmacologically.
A. NEUROPEFTIDE-TRANSFORMING ENZYMES IN CSF The CSF is in constant exchange with extracellular fluid of the central nervous system (CNS). Macromolecules produced within the CNS will leak into CSF and be passively distributed with the bulk flow. The protection provided by the blood-brain barrier effectively restricts the entrance of blood-borne macromolecules into the CNS under nonpathological circumstances. Thus, only very abundant plasma proteins such as albumin will appear in substantial quantities in CSE A simple kinetic model is presented in Fig. 3. Provided contributions from plasma can be disregarded, the concentration of a macromolecule in the CSF compartment will be a function of the rate of release from the CNS and the rate of CSF turnover. Provided the CSF turnover is the same in different individuals, the direct measurement of enzyme would therefore be a valid index of enzyme
Rate release of
\ / . &
Rate of passage
of 000
Rates of dilution and excretion
FIG.3. Factors governing the level of enzymes in the CSF compartment. An enzyme in the CSF may be derived from central neurons or glial cells or from other tissues within the CNS, such as the epithelial cells of the choroid plexus, through release. An enzyme could also be derived from plasma by passage through the blood-brain barrier (BBB).
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released. Ideally, an independent marker of CSF turnover should also be measured, or the ratio between different enzymes calculated. The CSF compartment is not homogenous, and contributions to its content of neuropeptides will derive from all parts of the neuroaxis. Concentration gradients of neurotransmitters or their metabolites have been described (Wood, 1980). However, 0-endorphin, a neuropeptide with 31 amino acids that is largely produced within the cerebrum, shows no ventriculo-cisternal-spinalgradient (Fachinetti et al. , 1987). @Endorphin is relatively stable metabolically and would therefore be able to mix better than neurotransmitters or their metabolites, which are subject to regional differences in metabolism or outward transport. To our knowledge, comparable studies have not been carried out with enzymes. It seems likely, however, that they would follow the pattern of 0-endorphin rather than that of neurotransmitters and metabolites. Clinically, CSF can usually be obtained only by lumbar puncture. The enzyme levels in the lumbar CSF are probably representative for those of the total CSF compartment.
B. ISOLATION OF NEUROPEFTIDE-TRANSFORMING ENZYMES FROM CSF Most previous studies of CSF enzymes have largely concerned their measurement as potential clinical markers. Enzyme kinetics, sensitivity to inhibitors, and composition of incubation buffers or their pH values are variables that have been investigated and compared with those of enzymes isolated from tissue. Only rarely have these enzymes been characterized by physicochemical criteria. This chapter will also review the opposite approach, in which CSF is a source for preparative work. Since the CSF has a very low protein content (about 600 mg/liter), relatively large CSF volumes are needed. Nevertheless, CSF has been found to be a good source of neuropeptidetransforming enzymes with concentrations on the order of 1-5 mg proteidliter. By the use of a serial separation procedure, the same batch of CSF can be fractionated to yield several enzymes (Fig. 4). These separations are run on 200- to 500-ml CSF batches. Clearly, samples from several individuals have to be pooled to obtain these volumes.
II. Individual Enzymes in CSF
A. ANGIOTENSIN-CONVERTING ENZYME Angiotensin-converting enzyme (ACE) is present in high concentrations in endothelial cells, particularly in the lung. It is also present in plasma. One important function of this enzyme is to convert angiotensin
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DEAES e ph aro se
I
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a-amidating enzyme
Phenyl-
Sephacryl s-200
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CL-48 Sephadex G- 100 1 Break-
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PPDAP
through
SP-converting
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endopeptidase
FIG.4. Outline of a procedure for the isolation of enzymes from human CSF. ACE, angiotensin-convertingenzyme; DCE, dynorphin-convertingenzyme; PPDAP, postproline diaminopeptidase; SP,substance P.
I to angiotensin 11, which is a vasoconstrictor that stimulates aldosterone activity. Inhibitors of the enzyme, such as captopril and enalapril, are clinically effective drugs in hypertension. ACE is present in the mammalian brain. The distribution is nonuniform, with particularly high levels in basal ganglia (Yang and Neff, 1972). There is no apparent overlap between the distribution of ACE, angiotensin nerve terminals, or areas of the CNS sensitive to microinjection of angiotensin (Brownfield et al. , 1982). A striatonigral ACE pathway has received considerable attention (e.g., Arregui et al., 1978; Strittmatter et al., 1984). ACE is a carboxydipeptidase that cleaves C-terminal dipeptides from a variety of substrates including enkephalins; Met-enkephalin-Arg-Pheis, for instance, cleaved to Met-enkephalin (Yang et al., 1981; Norman et al., 1985). However, ACE also acts as an endopeptidase with high affinity. Thus, substance P is split at the 8-9 position
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in addition to the 9-10 position (Skidgel et al., 1984). It is therefore possible that the function of ACE in the CNS may be different from its major actions in the periphery. The observation by Strittmatter et al. (1985)of a separate ACE isoenzyme in brain gives further support for a different function of brain ACE. The brain enzyme has a lower molecular weight and, interestingly, not only cleaves substance P, but also neurokinin A, a tachykinin originating from the same gene as substance P. A potential marker role of ACE in neuropathology was suggested by the finding of reduced levels in the substantia nigra of patients with Huntington’s disease (Arregui et al., 1978). ACE has also been found in human CSF and measured in various clinical conditions. Beckman et al. (1984)found low levels of ACE in the CSF of schizophrenics. There was no effect of neuroleptic therapy. Interestingly, an increase in angiotensin I and low angiotensin I1 levels have been reported in schizophrenics (van Kammen et al., 1981). Schweisfurth and Schioberg-Schiegnitz(1984) found elevated levels in patients with various neuropathological disorders including infections and sarcoidosis. In individuals with multiple sclerosis, levels were slightly elevated. Also, Oksanen et al. (1985)observed elevated ACE levels in patients with neurosarcoidosis. In an extensive study, Zubenko et al. (1985)measured ACE in CSF of patients with various kinds of dementia. Demented patients with Alzheimer’s disease, Parkinson’sdisease, or supranuclear palsy all showed decreased ACE levels. This decrease contrasted with an increase of ACE in normal aged subjects. In a multivariate study (Zubenko et al., 1986), the levels of ACE in CSF were found to correlate with acetylcholinesterase in Parkinson’sbut not Alzheimer’s patients; neither enzyme showed any correlation to total CSF protein. Apparently ACE is not a particularly specific marker for neurologic or psychiatric disorders. In patients with Parkinson’s disease, low ACE levels are accompanied by low 3,4-dihydroxyphenylaceticacid (DOPAC) and homovanillic acid (HVA) levels, suggesting that there is some association with the dopamine system and that the source of ACE could be the basal ganglia. However, ACE is also present in epithelial linings of the choroid plexus and blood vessels, although in humans, in contrast to, for instance, rats, the levels in these tissues are relatively low (Arregui and Iversen, 1978). The enzyme present in the choroid plexus is similar to that found in other epithelial cells and different from that observed in brain. The broad substrate specificity of ACE and its relatively high concentrations in CSF could influence the concentrations of several neuropeptides. This has been confirmed indirectly by a study of the
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metabolic stability of enkephalins and various proenkephalin- and prodynorphin-derived peptides in CSF (Lantz and Terenius, 1985). The major peptides from these opioid families in the CNS (i.e., enkephalins and dynorphins) occur in lower concentrations in the CSF than, for example, enkephalyl hexa- and heptapeptides (Nyberg et al., 1986a). Paradoxically, however, these enkephalyl peptides are less stable in CSF than the enkephalins. The degradation of enkephalyl peptides was blocked by captopril and EDTA, which inhibit ACE, but was not affected by the enkephalinase inhibitor phosphoramidone. The estimated molecular weight of the enzyme was 140,000, not very different from that reported for ACE in brain tissue (Lantz and Terenius, 1985). Apparently, ACE is a major enzyme in CSF. Its origin could be in plasma, the choroid plexus, or the CNS. The most probable source is the CNS. Since the enzyme derived from nerve tissue appears to be different from the enzyme in epithelial cells, a chemical characterization of the CSF enzyme would likely establish its origin.
B. SUBSTANCE P-CONVERTING ENDOPEPTIDASE
Several peptidases that cleave substance P are known and some have been ascribed a specific role in substance P fragmentation. These include several enzymes acting on the N-terminal end, releasing substance P(3-11) or substance P(5-11) (Orlowski et al., 1979; Blumberg and Teichberg, 1979; Kato et al., 1980). Endopeptidases acting at the Gln6-Phe7, Phe7-Phe8,and Glyg-Leu10positions have also been described (e.g., Lee et al., 1981). The substance P pharmacology and the relevant receptors are complex; deletion in the C-terminal end invariably leads to loss of typical substance P activity, whereas N-terminal truncation is more acceptable, but not beyond the 4-5 bond. Thus, all the metabolic transformations described here would have consequences for activity. Using substance P as a substrate, we have found several enzymes in CSF that degrade the peptide (see Fig. 4). Enzymes acting from the N-terminal end have not yet been characterized. However, an endopeptidase that cleaves substance P at the Phe7-Phe8and the Phe8-Glygpositions has been isolated and characterized (Nyberg et al., 1984). The major component showed an apparent molecular weight of 43,000 and occurred at a concentration of approximately 1 &ml. The enzyme is not inhibited by captopril or phosphoramidone. EDTA and dithioerythritol give partial inhibition, suggesting that it is a metallothiolpeptidase. When compared with a previously described enzyme in human brain tissue (Lee et al., 1981), it shows several similarities (Table I). The main difference, which is that the tissue enzyme
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TABLE I PROPERTIES OF A SUBSTANCE P ENDOPEPTIDASE ISOLATEDFROM HUMAN CSF"
Molecular weight pH optimum Essential SH groups Kmb
Product formationb Enzyme protein/ total protein
43,000 7.5 Yes 20 pM 3.7 pmol/min/ml CSF 0.14%
"From Nyberg et al. (1985) and unpublished results of F. Nyberg. bReleaseof substance P(1-7).
also cleaves substance P at the Gln6-Phe7bond, might be explained by an impurity. Compatible with the existence of this enzyme in the CSF is the presence of the substance P(1-7) fragment in CSF (Rimon et al., 1984). The substrate specificity of this CSF endopeptidase has been studied (Nyberg et al., 1986b). No substrate is more rapidly converted than substance P itself; C-terminal fragments (3-11, 5-11) show substantially less conversion. Other tachykinins, such as neurokinin A and B, as well as enkephalins and 0-casomorphin, are not cleaved. Substance P(1-8) is not cleaved but gives some inhibition of substance P conversion. These data on the enzyme's effects on structurally related (neurokinin) or unrelated peptides suggest a high selectivity. However, more recently, this enzyme has also been found to cleave calcitonin gene-related peptide (CGRP) and somatostatin, two peptides with no sequence homology and no homology with substance P. CGRP acts as an inhibitor of substance P fragmentation (LeGreves et al., 1985), which can explain why CGRP is able to potentiate the action of substance P at the level of the spinal cord, as evidenced by behavioral analysis (Wiesenfeld-Hallin et al., 1984) or from electrophysiologicalrecordings (Woolf and Wiesenfeld-Hallin, 1986). There is also evidence for an interaction between somatostatin and CGRP (Wiesenfeld-Hallin, 1986). At doses at which CGRP potentiates substance P activity, it is inactive by itself. It is now established that in addition to substance I?, CGRP is a major neuropeptide in thin nonmyelinated primary afferent fibers, which are known to transmit noxious impulses to the spinal cord. To a large extent, these two neuropeptides coexist in the same neuron (Gibson et al., 1984; Wiesenfeld-Hallin et al., 1984). It is therefore an attractive hypothesis that the two coexisting peptides share a common degradation step. Since the enzyme apparently is present in CSF, we have speculated (Hokfelt
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and Terenius, 1987) that it also acts as a scavenger for these peptides in the CSF compartment. Several other peptidases are known to cleave substance P into fragments, including ACE (discussed previously) and enkephalinase (Matsas et al., 1984). ACE is present in a striatonigral projection, and there is a major striatonigral projection containing substance P (e.g., Brownstein et al., 1977; Gale et al., 1977; Hong et al., 1977). It is not known to what extent these systems overlap or whether there is coexistence. Given the large quantity of ACE in the projection, it seems probable that ACE also has a role in the fragmentation of substance P.
MONOOXYGENASE C. ~-AMIDATING
A large number of hormonally active peptides have their C-terminals a-amidated. Most commonly, this transfonnation is necessary for biologic activity (Mains et al., 1983). Amidation occurs in peptides having a C-terminal Gly residue. Enzymes converting peptides with the X-Gly sequence into the a-amidated products (X-NH2) have been isolated from several tissues (Bradbury et al., 1982; Emeson, 1984; Kizer et a l . , 1984). a-Amidation activity has also been found in CSF (Wand et al., 1985a) and in plasma (Wand et al., 1985b). There is also a-amidation activity in the brain, with the highest enzyme levels in the hypothalamus, lower activity in the cerebral cortex, and barely detectable levels in the cerebellum and pons (Wand et al., 1985a). The high levels in the hypothalamus are in agreement with the high levels of amidated peptides, including various releasing factors, in this brain area. More recently, we have isolated an a-amidating enzyme from human CSF that generates substance P from the precursor, substance P-Gly12. This enzyme was obtained in sufficient quantity for chemical characterization (Table 11) (Vaeroy et al., 1987). TABLE I1 PROPERTIES OF AN WAMIDATING ENZYME FROM HUMAN CSF" Molecular weight pH optimum Essential SH groups Cofactors required Product formationb Enzyme protein/total protein
25,000 7-8 No Cu*+, ascorbic acid 0.16 pmol/min/ml CSF 0.93%
"From Vaeroy et al. (1987). bThe substrate was substance P-Gly'2.
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It is yet unclear whether there is one a-amidating enzyme common to all peptide systems or whether there are several. So far, all investigators have found that a-amidation is stimulated by the presence of Cu2+ and ascorbic acid. However, the reported molecular weights differ considerably. The activity in bovine pituitary separated into two components of 38,000 and 54,000 with somewhat different properties (Kizer et al., 1984). In human plasma, the enzyme activity had an estimated molecular weight of about 60,000 (Wand et al., 1985b). The CSF enzyme, however, had a much smaller apparent molecular weight, around 25,000 (Vaeroy et al., 1987). Whether this represents a subunit of a larger, native form (dimer?)or a separate protein is not known. A critical issue is the substrate specificity of the a-amidating monooxygenases, which has not been given much attention. Bradbury and Smyth (1983) demonstrated the mandatory requirement for glycine in the C-terminal position by comparing the enzymatic conversion of various tripeptide substrates. Substitution in the position next to the C-terminal Gly residue was also important, since replacement of neutral with basic or acid amino acid residues greatly slowed the conversion. An a-amidating monooxygenase is secreted from a mouse pituitary corticotropin cell line (AtT-20) by a number of secretagogues including corticotropin-releasing factor (CRF), a-adrenergic agonists, or barium ion. Secretion of proopiomelanocortin (POMC) peptides rose in parallel, indicating that the monooxygenase and the prohormone are in the same cellular compartment (Mains and Eipper, 1984). By analogy, secretion of the enzyme into the CSF may also occur in parallel with neuropeptides that require amidation, such as substance P and other neurokinins, neuropeptide tyrosine (NPY), or the hypothalamic releasing hormones. So far, there have been no attempts to measure levels of the enzyme as compared with those of neuropeptides in individual CSF samples.
D. DYNORPHIN-CONVERTING ENZYME During the studies of dynorphin peptides in CSF, it was observed that levels fell rather rapidly following storage of CSF at room temperature. Chromatographic factionation of the CSF proteins identified one component that very actively converted dynorphin A into Leu-enkephalin-Are [dynorphin(l-6)] and the corresponding C-terminal fragment, dynorphin A(7-17). Further studies indicated that the Leuenkephalin-Arg6 fragment was released also from dynorphin B and a-neoendorphin (Nyberg et al., 1985). Thus, the enzyme has a similar
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action against all opioid peptides deriving from prodynorphin (the dynorphins) and the enzyme has therefore been called dynorphin-converting enzyme (DCE) (Table 111). The potential significance of this enzyme in CSF (and potentially the CNS) is supported by the observation that levels of Leu-enkephalin-Are in CSF greatly exceed those of dynorphin A and dynorphin B (Nyberg et al., 1986a); a-neoendorphin has, to our knowledge, not been measured in CSF. Other enzymes have been isolated that fragment dynorphin A. One enzyme isolated from bovine brain generates dynorphin A(l-8) and the (9-17) fragment. Dynorphin A(l-8) is a major peptide in the prodynorphin system in rat CNS (Devi and Goldstein, 1984). The importance of this conversion in human brain is not known. Dynorphin A(l-8) cannot be detected in human CSF (Nyberg et al., 1986b). Both dynorphins and the enkephalyl peptides have opioid activity, but with different receptor selectivity. Whereas the dynorphins are selective for x receptors, the enkephalin peptides are selective for 6 receptors (Paterson et al., 1983). For some time it was believed that processing in the prodynorphin neurons stopped at the dynorphin stage. More recent work, however, showed that there is considerable conversion to enkephalins in certain pathways, including the striatonigral pathway (Zamir et al., 1984; Christensson-Nylander et al., 1986). Thus, the enzyme discussed here could be involved in this conversion. In fact, Leu-enkephalin-ArgGis a major peptide in the striaturn and the substantia nigra. Pharmacologic stimulation or inhibition of D1 or Dz dopamine receptors subchronically produces characteristicchanges in the levels of dynorphin A, dynorphin B, and Leu-enkephalin-Are in the striatum
TABLE I11 PROPERTIES OF DYNORPHIN-CONVERTING ENZYMES ISOLATED FROM HUMAN CSF AND CHOROID PLEXUS
Characteristic
CSF enzyme
Molecular weight pH optimum Essential SH groups
43,000 6.5-8 No
Km"
10
Product formation" Enzyme protein/ total protein
w
15.3 pmol/min/ml CSF
Choroid plexus enzyme Soluble
Membrane bound
55,000 7-8
58,000 7-8 No 6PM
No 3 PM -
1.7%
"Release of Leu-enkephalin-Arg6from dynorphin B.
-
-
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NEUROPEPTIDE ENZYMES IN HUMAN CSF
and substantia nigra, further emphasizing the functional relevance of the conversion (Christensson-Nylander et al., 1986; Nylander and Terenius, 1987). The CNS may not be the only contributor of the enzyme isolated from CSF. Recent experiments have shown that an enzyme with similar properties can be isolated from human choroid plexi (see Table 111). Both the CSF and the plexus enzymes are inhibited by EDTA and phenylmethylsulphonylfluoride (PMSF) whereas diphenylisopropylfluorophosphate (DFP) only inhibits the CSF enzyme. Enzyme activity isolated from the CNS (spinal cord) is also sensitive to DFP, suggesting that the CSF enzyme is mainly derived from the CNS. It is also apparent that the CSF enzyme occurs in a major form of apparent M, 43,000 and a minor form of M,-80,000. The latter form may represent a dimer, since it cannot be distinguished by kinetic or inhibitor sensitivity criteria from the major form. Table IV illustrates the structure specificity of the enzyme. All dynorphin peptides are good substrates. The N-terminal fragment, dynorphin A(l-8), is less actively transformed. The activity of DCE is significantly lower in the CSF of women who are in late pregnancy than in nonpregnant women (Lyrenas et al., 1987). In contrast, prodynorphin-derived polypeptides are elevated, which would make sense if DCE activity in CSF is related to the CNS activity. TABLE IV E F F E COF ~ ~PROTEASE INHIBITORS AND SYNTHETIC PEPTIDES ON THE CONVERSION OF '251-LABELEDDYNORPHIN B TO LEU-ENKEPHALIN-AR~"
Inhibitor or Peptide EDTA (1 mM) Iodoacetate (1 mM) Dithiothreitol (1 mM) Phenylmethylsulfonyl fluoride (1 mM) DFP (1 mM) Dyn A (20 BM) Dyn B (20 PM) Dyn A(1-8) (20 ILM) a-Neoendorphin (20 pM) Leu-enk-Arg6(20 p M ) Leu-enk (20 pM) "Control level = 100%.
Conversion by CSF enzyme
(%I
Conversion by choroid plexus enzyme (%) Soluble
Membrane bound
90 90 95
46 87 95
49 95 69
45 10 47 45 80 69 95 100
85 79 26 37 79 85 92 90
84 92 39 38
77 84 100 100
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The DCE activity in CSF is very high, even if compared with ACE or a-amidating activity. It may therefore be an important marker of neuronal activity, perhaps not restricted to the prodynorphin systems. E. MEMBRANE METALLO-ENDOPEPTIDASE (EC 3.4.24.11) (ENKEPHALINASE) The trivial name of this enzyme, enkephalinase, suggests a very specific function. However, this enzyme is able to degrade several neuropeptides, some even with higher affinity than enkephalin, such as substance P or neurotensin. The enzyme cleaves rather generally on the amino side of hydrophobic amino acids (e.g., Matsas et al., 1984; Hooper et al., 1985). The distribution of endopeptidase (EC 3.4.24.11) in the CNS partly matches that of enkephalin and substance P (Matsas et al., 1986; Waksman et al., 1986). Inhibitors of endopeptidase (EC 3.4.24.11) have been synthesized by several groups. They have definite pharmacologic actions, including hypoalgesia, if introduced intracerebrally (Roques et al., 1980; Zhang et al., 1982). Despite these efforts, there is very little information about endopeptidase (EC 3.4.24.11) in CSF. Spillantini et al. (1986) observed that an enkephalinase inhibitor, thiorphan, reduced enkephalin degradation in CSF. The estimated Ki value for Met-enkephalin was about 20 pLM for both CSF and plasma. Superfusion of the spinal cord in the presence of another endopeptidase (EC 3.4.24.11) inhibitor, kelatorphan, also increased Met-enkephalin output (Bourgoin et al., 1986). In our own work, endopeptidase (EC 3.4.24.11) appeared considerably less important in CSF for enkephalin degradation than aminopeptidases (Lantz and Terenius, 1985).
F. AMINOPEPTIDASES
Among the exopeptidases, aminopeptidases have been particularly well studied with regard to CSF. Thus, Hazato et al. (1983) described degradation of the N-terminal Tyr of Met-enkephalin. The removal of the N-terminal Tyr inactivates almost every opioid peptide. In this study, formation of free tyrosine was suppressed by bestatin. There was no chromatographic fractionation of the CSF prior to testing and, obviously, contributions from both aminopeptidases and dipeptidyl aminopeptidases, as well as from carboxypeptidases, were observed. The complexity of the degradation pattern was partly resolved by chromatographic
NEUROPEPTIDE ENZYMES IN HUMAN CSF
115
fractionation of CSF prior to testing of enkephalin degradaton. Two major aminopeptidases were identified. Aminopeptidasel showed a pH optimum at 6.0 and was inactivated by EDTA or parachloromercuribenzoic acid but not by amastatin. Aminopeptidasez had a pH optimum of 7.5 and was inactivated by EDTA and amastatin (Hazato et al. , 1985). Aminopeptidasez therefore shows similarity to aminopeptidase M. Work in our laboratory has confirmed the presence of an amastatin-sensitiveaminopeptidase in CSF. A very similar enzyme is also present in plasma (Nyberg et al., 1988). Since aminopeptidase M has been localized exclusively to blood vessels in the brain (Hersh et al., 1987), it is therefore entirely possible that the amastatin-sensitive enzyme in CSF has an extraneuronal origin. An aminopeptidase with apparently higher specificity is a pyroglutamate endopeptidase described by Prasad and Jayaraman (1986). This enzyme has a pH optimum at 6.0-7.4 and shows high affinity for the tested substrate, thyrotropin-releasing hormone (TRH), with a K , of 16 p M , as well as for luteinizing hormone-releasinghormone (LHRH). Both peptides have an N-terminal pyroglutamate. The enzyme is inactivated by heavy metals, EDTA, and thiol-oxidizing agents. A similar enzyme is present in serum, which may also be the origin of the CSF enzyme. In CSF, the major metabolic route is via action of this enzyme. The dipeptide that forms from TRH, cyclo(His-Pro)is biologically active and even more potent than TRH in mediating several biological functions (Prasad et al., 1982). The pyroglutamate endopeptidase could therefore have an important natural regulatory function.
G. ACETYLCHOLINESTERASE Acetylcholinesterase has been shown to have a complex peptidase activity: it is able to act both as an exopeptidase and as an endopeptidase. The exopeptidase activity can involve both N- and C-terminal amino acids, as shown for enkephalin degradation (Chubb et al., 1983). Endopeptidase activity is trypsinlike (Small et al., 1987). These two activities are probably associated with two separate sites on the molecule, neither of which is the site of esterase activity. Since the combination of activities is required for processing of prohormones, Chubb and co-workers postulated that this enzyme may handle processing, for instance, of enkephalin precursors. It has been shown that acetylcholinesterase is present in CSF of rabbits (Chubb et al., 1976) and in cats (Vogt et al., 1984). In cat preparations, strong sensory input and psychotropic medication increased
116
LARS TERENIUS A N D FRED NYBERG
acetylcholinesterase CSF levels. It was not decided, however, whether this was due to increased nervous activity as such or due to associated phenomena such as altered circulation or metabolism. Acetylcholinesterase is also present in human CSF and there are several reports on measurements in patient populations. Reports by Deutsch et al. (1983) of levels in patients with schizophrenia, depression, or Alzheimer’s disease and by Singer et al. (1984) on patients with Gilles de la Tourette’s syndrome failed to show differences from controls. Recently, the primary sequence of acetylcholinesterase has been indirectly deduced by applying molecular genetic techniques. It was found that the enzyme isolated from Torpedo calqornaca has partial sequence homology with thyroglobulin (Schumacher et al.,1986), adding to the potential polyfunctionality of this enzyme.
H. POTENTIAL SIGNIFICANCE OF PROTEASES-PEFTIDASES IN CSF Historically, CSF proteases-peptidases were investigated as potential disease markers, particularly in disorders known or assumed to involve neuronal degradation. Such enzymes are probably mainly of lysosomal origin. They could definitely be influencing levels of neuropeptides in CSF and, more intimately, levels in the extracellular environment. Another source of CSF enzymes is blood, and passage of blood-borne enzymes would depend on the integrity of the blood-brain barrier. A defective barrier might then also lead to consequences for signal transmission by neuropeptides in the CNS. This review has focused on proteases-peptidases that are probably derived from normal processes in the CNS and its adjacent structures. Sources of enzymes other than neuronal and glial cells are the choroid plexus and other so-called circumventricular organs and cerebral microvessels, as well as plasma. The uncertainty as to cellular or tissue origin of these enzymes calls for studies in which the localization of the enzyme is studied by means of immunohistochemistry. This approach has been particularly enlightening with regard to angiotensin-converting enzyme and enkephalinase, as reviewed above. Another example that shows the need for histological studies is that of aminopeptidase M. The powerful nature of aminopeptidases in peptide degradation is well known. The enkephalins and other opioid peptides are easily attacked by such enzymes, which remove the N-terminal Ty. This leads to almost complete loss of opioid activity. It is also well known that opioid peptide analogs with D-amino acids in position 2 are metabolically protected and show powerful biological effects under conditions in which the natural compounds are inactive or poorly active (Morley, 1980). Also,
NEUROPEPTIDE ENZYMES IN HUMAN CSF
117
aminopeptidase inhibitors can protect Met-enkephalin from degradation in tissue slices (Giros et al., 1986). All these data suggest that aminopeptidase M is physiologically relevant for enkephalin inactivation. Recently, however, it has been shown by immunohistochemical studies that this enzyme is localized almost entirely in blood vessels in the brain (Hersh et al., 1987; Solhonne et al., 1987). This suggests that enzymatic inactivation of opioid peptides is far from complete in the synaptic area and that diffusion away from the synapses occurs. It also indicates that, in fact, enzymes in CSF may derive from brain microvessels and may have a biological function as “scavengers” of peptides. Extensive studies in this laboratory have identified enkephalyl peptides with “remaining” C-terminal (or N-terminal) basic amino acids in CSF in higher concentrations than those of the enkephalins themselves (Nyberg et al., 1986a). This gives additional evidence that enzymes in the nerve terminals or in the synaptic area are not fully capable of completing the conversion to what usually is assumed to be the active peptide(s). Certain of the enzymes described above, such as dynorphin-converting enzyme or substance P endopeptidase, occur in such concentration in CSF that it is very likely that they are actively secreted. According to the present views on peptide biosynthesis, the peptide prohormone as well as processing-conversion enzymes are packed into vesicles in the Golgi apparatus. Processing-conversion is probably a gradual, partly statistical process. It is not unlikely that during exocytotic release, the enzymes are coreleased with the peptides, as schematically illustrated in Fig. 5 . Supporting this concept is the fact that peptide precursors up to the size of the prohormones are also present in the CSF, as has been shown for proenkephalin and prodynorphin (Nyberg and Terenius,
Prohormone (precursor) Vesicle
Neuropeptide (s) Enzyme (s)
FIG. 5. Model for neuronal secretion of enzymes (as well as neuropeptide prohormones and various peptides) by exocytosis from vesicles. The figure shows a nerve terminal with vesicles as well as a single (enlarged) vesicle.
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LARS TERENIUS AND FRED NYBERG
1985) and a-protachykinin (F Nyberg and P. Le Grkes, unpublished results). If this model is correct, the concentrations of processingconversion enzymes in CSF could serve as markers for activity in peptidergic neurons and be of potential significance as activity markers. Critical to this approach is the actual demonstration of the enzyme under consideration in the relevant pathways. At an empirical level, it would be of strategic advantage to perform measurements on both enzyme and peptide levels in the same CSF sample.
111. Concluding Remarks
There are considerable concentrations of certain enzymes with more or less specific peptide-processing, -converting, or -inactivatingproperties in human CSE Several of these enzymes are likely to be of neuronal origin. With regard to such enzymes we may consider the following points. 1. These enzymes have a critical role in protein-peptide transformations. 2. These enzymes derive from specific neuronal pathways and exert their actions in particular pathways. 3. The release of these enzymes into the extracellular fluid and eventually into CSF is regulated by neuronal activity in the appropriate pathways. Several enzymes discussed in this review have been studied with regard to one or two of these points. Most enzymes meet the conditions of the first point. There is less information with regard to the second and practically no information regarding the third point. However, it seems reasonable that some of the enzymes discussed here are actively secreted. This is a possibility open to testing. The continued search for enzymes in the CSF may therefore lead to the discovery of more candidates for mediators of protein-peptide transformations. Such enzymes, and others already discovered, are possible candidates as markers for the turnover rate in peptidergic synapses. More work is needed to identify an essential role for CSF enzymes as markers in clinical diagnosis.
Acknowledgments
This work is supported by the Swedish Medical Research Council and the Magnus Bergvall Foundation.
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TARGETING DRUGS AND TOXINS TO THE BRAIN: MAGIC BULLETS By Lance L. Simpson Departments of Medicine and Pharmacology Jefferson Medical College
Philadelphia, Pennsylvania 19107
I. Introduction 11. Protein Toxins 111. Multicomponent Toxins A. ADP-Ribosyltransferase Toxins B. RNA N-Glycosidase Toxins C. Toxins with a n Unknown Mechanism of Action IV. Toxins as Magic Bullets V. Broadening the Concept of Magic Bullets VI. Chimeric Toxins VII. Chimeric Neuroactive Compounds A. Neural Tumors B. Pheochromocytoma C. Blockers of Transmitter Release D. Clostridial Chimeras E. Chimeric Antibodies F. Import of Drugs G. Myasthenia Gravis VIII. Concluding Remarks References I. Introduction
The pursuit of magic bullets has been one of the consuming passions of medicine and science. Unfortunately, the success stemming from that search has not been equivalent to the effort invested. This is due in part to technical difficulties, but it is also due to the increasing stringency given to the definition. The purpose of this review is to propose that, at least in one area of research, the technical difficulties may be surmountable. However, this proposal hinges on a definition of magic bullet that is historically accurate and conceptually feasible, as opposed to a definition that is idealistic and unattainable. The term magic bullet was introduced many decades ago by Paul Ehrlich, but it is one that can be articulated in INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 30
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the language of modern science. The term refers to a bifunctional molecule in which both functional domains play an essential role in the biochemical or pharmacological actions of the intact substance. One of the domains is envisioned as a tissue-targeting moiety that guides the molecule to vulnerable cells, and the second domain is seen as a biologically active moiety that exerts effects on vulnerable cells. When Ehrlich coined the term he had a specific idea in mind. He imagined the tissue-targeting domain to be something akin to an antibody. A targeting domain that possessed an affinity for receptors equal to that of an antibody for an antigen would be site-directed and selective. In terms of the biologically active domain, his thoughts were quite practical. He planned to use agents that could arrest or reverse the effects of disease. Thus, a magic bullet was a bifunctional substance that was directed with high specificity to diseased cells and that possessed a medication to overcome disease. The likelihood of creating an authentic magic bullet is dependent on the stringency of expectations. If the desire is that the agent go to a single cell type, with absolutely no other sites of binding, that is unrealistic. If, however, the desire is to create an agent with selectivity, for which the ratio of specific to nonspecific binding approaches two orders of magnitude, that is a realistic goal. Indeed, even greater ratios of selectivity may be attainable. In the recent past there has been a surge in the effort to build clinically useful magic bullets. The area of research that has shown greatest promise is somewhat remote from neurobiology, and therefore the methods that have been developed and the successes that have been achieved are not widely known to neuroscientists. The work is largely an outgrowth of the disciplines of toxicology and oncology. In deference to this, the present review will highlight the relevant findings in toxin and cancer research, and then it w ill introduce the now burgeoning effort to develop magic bullets as antineoplastic drugs. This will serve as a backdrop to discussing the modest accomplishmentsthat have been made in utilizing magic bullets in neurobiology and to proposing the future accomplishments that could be realized from a fuller application of the technology.
II. Protein Toxins
There are a host of relatively well-characterized protein toxins that possess remarkable potency (Gill, 1978; Alouf et al., 1984; Foster and Kinney, 1984). These toxins are typically of microbial or plant origin,
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and their biological activity is so extraordinary that it can be expressed in terms of numbers of molecules. There are data that suggest that a single molecule, productively internalized, can disrupt the function or produce outright death of a vulnerable cell (Yamaizumi et al., 1978b). Most investigators regard diphtheria toxin as the prototype for the group (Collier, 1977; Pappenheimer, 1977; Uchida, 1983). This toxin is synthesized by the microorganism Corynebacterium diphtheria, and it is a protein with a molecular mass of approximately 62,000 daltons. The toxin has an affinity for eukaryotic as opposed to prokaryotic cells, and it acts intracellularly to block protein synthesis. When the blockade of protein synthesis is significant, cell death ensues. The clinical presentation of diphtheria involves the upper respiratory tract and the cardiopulmonary system. During infection, the microorganism lodges in the oropharyngeal path, where it can produce and release toxin. When the toxin enters cells locally, its poisoning effect causes respiratory and and cardiopulmonary dysfunction. In serious cases of poisoning, the toxin enters the general circulation and is transported to a variety of cells, including the myelin sheath of peripheral nerves. One of the more well-known aspects of the disease is demyelination that leads to peripheral neuropathies. In myelin, as in other tissues, the toxin is internalized, blocks protein synthesis, and eventually causes cell death. This brief description of the toxin and the disease appears to have little to do with magic bullets, but that is deceptive. A closer inspection of the molecule and its interaction with vulnerable cells begins to reveal an important point. The toxin is synthesized as a single-chain polypeptide that possesses negligible biological activity. When the molecule is exposed to certain proteolytic enzymes, it is nicked to yield a dichain structure in which a heavy chain ( M , -40,000) is linked by a disulfide bond to a light chain ( M , -20,000). The two chains were originally sequenced by the techniques of protein chemistry (DeLange et al., 1976; Falmagne et al., 1985), and the entire molecule has since been sequenced by the techniques of molecular biology (Greenfield et al., 1983). The diphtheria toxin molecule has at least three functional domains, each of which has been tentatively assigned to a portion of the intact toxin. The carboxy-terminus of the heavy chain plays an essential, but perhaps not exclusive, role in specifying the target tissue for the toxin. This portion of the molecule is responsible for attachment to receptors on the plasma membrane of vulnerable cells (Ittelson and Gill, 1973; Zanen et al., 1976). The amino-terminus of the heavy chain is essential for the internalization process, and the underlying mechanism will be considered shortly. The light chain of the toxin possesses ADP-ribosyltransferaseactivity, and the substrate is elongation factor 2, a polypeptidyl-tRNA translocase
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that is essential for protein synthesis (Honjo et al., 1971). The light chain of the toxin, in the presence of its substrate, hydrolyzes NAD to yield ADP-ribose and nicotinamide. The latter is released, and the ADP-ribose moiety is covalently attached to substrate. The attachment site is a unique amino acid given the trivial name diphthimide; its only known occurrence in nature is in elongation factor 2 and its only known function is to serve as an acceptor for diphtheria toxin-induced ADP-ribosylation (Van Ness et al., 1980). The most perplexing of these three steps is the internalization process. The binding step is essentially the same as that of any ligand-receptor interaction, and the catalytic step is no different from that of any other enzyme-mediated process. Indeed, eukaryotic cells are known to possess their own ADP-ribosylating enzymes, and the intoxication process has certain similarities to ongoing physiological mechanisms in nucleated cells (Moss and Vaughn, 1978; Lee and Iglewski, 1984; Sitikov et al., 1984). The issue that has proved difficult to resolve is that of membrane penetration. It is self-evident that the toxin must enter the cell because the substrate, elongation factor 2, is located in the cytoplasm. However, this self-evident point conveys nothing about the sequence of events involved in internalization. For argument’s sake, one might consider three possible schemes for internalization. The first of these is a form of direct injection. After binding to the plasma membrane, the toxin could act alone or in concert with the membrane to create a channel or pore. The entire toxin molecule, or some portion of it, could then penetrate directly from the cell exterior to the cell interior. The next two schemes are more elaborate, due to the number of membranes that must be crossed. There is the possibility that the toxin binds to the cell surface, but rather than undergoing direct injection, it is imported by the process of receptor-mediated endocytosis. This places the toxin inside the cell but still within a membrane-delimited structure. To exit the endosome, the toxin could do one of two things. It could create channels or pores or, alternatively, it could produce a more disruptive effect, such as membrane breakage or endosome lysis. In either case, the toxin would leave the endosome and enter the cytoplasm. There is a consensus among investigators that receptor-mediated endocytosis brings the toxin into the cell, and most believe that channels or some other nonlytic mechanism convey the toxin into the cytoplasm (Neville and Hudson, 1986). An appreciation of these events is immensely important to the design of magic bullets, but that point will be momentarily deferred. Attention here will be directed to the evidence that indicates that endocytosis followed by membrane penetration is a plausible mechanism.
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Two lines of research are central to the discussion, and the first is pharmacological. There are a host of agents, often referred to as lysosomotropic agents, that are known to antagonize the actions of substances that reach the cell interior by endocytosis. Among these agents, the most thoroughly studied are ammonium chloride, chloroquine, and methylamine hydrochloride. It is of interest that these agents are very effective antagonists of diphtheria toxin (Kim and Groman, 1965; Leppla et al., 1980). They do not inhibit tissue fixation by the toxin, nor do they inhibit ADP-ribosylation of elongation factor 2 . They act at an intermediate step, impairing the transit of the toxin to the cytosol. The second line of evidence concerns channel formation and/or membrane perturbation. There is a prevailing belief that the toxin molecule has a pH-sensitive domain, and under the appropriate conditions this domain (namely, the amino-terminus of the heavy chain) creates pores or perturbations in the endosome membrane (Sandvig and Olsnes, 1980; Donovan et al., 1981; Kagan et al., 1981). The toxin molecule then uses these pores or perturbations to reach the cytosol. More precisely, the proposal is viewed as follows. The toxin enters an endosome that has a pH of approximately 7.4. However, the endosome has a proton pump, and within a short time the internal pH falls to 5 or below. In addition, certain endosomes are destined to fuse with lysomes that also have a low internal pH. Under the influence of acidic conditions, the toxin molecule is induced to insert into the membrane. The insertion is either the initial step in channel formation or the initial step in membrane perturbation, and the end result is that the molecule (or a portion of it) exits the endosome to reach the cytosol. It is important to note that channel formation does not occur at physiological pH. This accounts for the fact that diphtheria toxin is not a surface-active molecule that distorts the ionic gradient at the plasma membrane. There is a hypothetical question that stems from this, and the answer helps to establish the credibility of the proposed scheme for internalization. What would happen if a vulnerable cell were to be exposed to the toxin under acidic conditions? Would the toxin inject directly across the plasma membrane into the cytoplasm and bypass the endosome? In point of fact, this is what happens. When tissues are bathed in medium at pH 4.5, the toxin causes poisoning, and the lag time that is normally associated with endosomal importation is largely abolished (Sandvig and Olsnes, 1980; Draper and Simon, 1980; Sandvig et al., 1986). There is an additional observation that adds to the weight of the evidence. As discussed above, drugs like chloroquine are potent diphtheria toxin antagonists. Their presumed mechanism of action is that they enter endosomes and lysosomes, and due to their basic nature they raise internal pH. Under these conditions
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the pH-induced channel formation does not occur, and the action of the toxin is markedly inhibited. By contrast, drugs like chloroquine have no antagonistic effect when cells are bathed in acidic medium (Sandvig and Olsnes, 1982; Draper and Simon, 1980). By virtue of injecting directly into the cytoplasm, the toxin is no longer affected by the internal pH of endosomes or lysosomes. To summarize, the toxin is endocytosed by normal cells in physiological medium. There follows a pH-dependent event, during which the toxin inserts into the endosome membrane. Using channels or some other membrane-penetrating device, the toxin reaches the cytosol and catalytically inactivates elongation factor 2. In essence, the toxin molecule displays two of the cardinal features of a magic bullet. It has a binding domain that positions the molecule on target tissue, and it has an intracellularly acting domain that alters tissue function. In these respects, it is a molecule that can serve as a model. The precise mechanism for transit into the cytosol has not been established, but as we shall see later the task of effectively linking the two components of a putative magic bullet and retaining the capacity for productive internalization is highly challenging.
111. Multicornponent Toxins
There are a number of potent protein toxins that are similar to diphtheria toxin. Three groups are important here, and they can be identified as: (1) the ADP-ribosylating toxins, (2) the RNA N-glycosidase toxins, and (3) the toxins of unknown mechanism of action. A. ADP-RIBOSYLTRANSFERASE TOXINS The ADP-ribosylating toxins can themselves be divided into three classes. The first of these is composed of diphtheria toxin and Pseudomonas aerugznosa exotoxin, both of which covalently attach an ADPribose group to elongation factor 2 (Honjo et al., 1971; Iglewski and Kabat, 1975). In spite of the fact that these two toxins have an identical mechanism of action, they do not possess sequence homologies, nor are they antigenically similar. The evolutionary mechanisms that gave rise to two toxins so closely related in function but so unrelated in structure remains a mystery. The second class of ADP-ribosylating toxins is composed of cholera toxin, E. coli enterotoxin, and pertussis toxin. Each of these agents
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attaches an ADP-ribose group to a regulatory protein that governs adenylate cyclase. Cholera toxin (Cassel and Pfeuffer, 1978; Gill and Meren, 1978) and E . coli enterotoxin (Moss and Richardson, 1978) covalently modify a protein commonly referred to as N, (N, nucleotide binding protein; s, stimulatory). Pertussis toxin ADP-ribosylates Ni (i, inhibitory) (Katada and Ui, 1982). The pathophysiological consequences of enzymatically modifying N, and Ni have been reviewed frequently (e.g., Hayaishi and Ueda, 1982). There is a modest amount of homology within the class of toxins that alters N, and N;. Cholera toxin and E. coli enterotoxin have structural and immunological similarities, but pertussis toxin is unique. There is no known structural overlap between the toxins that modify regulatory proteins and those that modify elongation factor 2, nor do these two overlap with the next class of toxins, the binary toxins. This last class is the one most recently identified, and it has been found to have special properties. The identification of this class resulted from the discovery that a previously recognized toxin had been misclassified. Botulinum neurotoxin, which is discussed below, occurs in several serotypes. Until the late 1970s investigators had thought that there were eight serotypes designated A , B, C1, C2, D, E, F, and G. The designations C1 and C2 were meant to convey that these two serotypes are often produced by the same strain of bacteria. However, work reported by Sakaguchi and his associates revealed that type C2 is unique in its structure (Iwasaki et al., 1980; Ohishi et al., 1980), and data from the laboratories of Eklund (Eklund and Poysky, 1972), Jensen (Jensen and Duncan, 1980), and Simpson (Simpson, 1982) showed that it has special biological properties. The botulinum C2 toxin, also called the botulinum binary toxin, is composed of two separate and independent polypeptide chains ( M , 50,000 and 100,000). The structure-function relationships of the toxin have been partially determined, and they are in many respects similar to those of diphtheria toxin. The heavy chain is known to bind to tissue receptors, and in doing so it appears to create acceptors for the light chairi (Ohishi and Miyake, 1985). The latter is an enzyme with ADP-ribosyltransferase activity (Simpson, 1984), and the substrate appears to be G-actin (Aktories et al., 1986; Ohishi and Tsuyama, 1986). Because the brain possesses relatively large amounts of G-actin, broken cell preparations of central nervous system origin are a rich source of substrate. Two additional binary toxins have been reported, a Clostridium perfnngens iota toxin and a Clostridium spiroforme iota-like toxin, and they share the structure-function relationship of the botulinum binary toxin. More precisely, each has a light chain that ADP-ribosylatesintracellular
-
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G-actin (Simpson et al., 1987; L. L. Simpson, unpublished findings). The primary structures of the binary toxins have not been determined, but there may be commonalities between iota and iota-like toxins. This conclusion is based on the finding that there is some cross-reactivity between toxin and heterologous antitoxin. B. RNA N-GLYCOSIDASE TOXINS The ADP-ribosylatingtoxins form one group of potent protein toxins; the next group is one that acts on ribosomes and possesses another form of catalytic activity. This group is in many respects like the ADPribosylating toxins, and therefore they will be discussed more superficially. The group has two major representatives; there are the plant lectins, as typified by abrin and ricin (Olsnes and Pihl, 1982)’ and there are bacterial products, as exemplified by Shiga toxin (O’Brien and Holmes, 1987). All of them reportedly act like hydrolytic enzymes that cleave the N-glycosidic bond of the adenine at position 4324 of 28 S rRNA (Endo et al., 1987;Endo and Tsurugi, 1987). Ricin can be taken as a prototype for the group. Ricin is obtained from the seeds of Ricinus communis, more commonly known as the castor bean. Like diphtheria toxin, it is an exquisitely potent substance; perhaps as little as one molecule will cause death of a susceptible eukaryotic cell. The ricin molecule is composed of two polypeptide chains, each weighing slightly more than 30,000,and the molecular weight of the holotoxin is 62,057.The ricin molecule has a binding domain that resides entirely within one of its two polypeptide components (M,-31,432). The receptor for the toxin has not been isolated and characterized, but one integral feature of the site has been determined. The binding component affixes itself to terminal galactose residues in complex carbohydrates and glycolipids. This finding has been exploited in several experimental contexts. For example, galaCt6Se and related sugars can be used to antagonize the poisoning effects of the toxin. As another example, a f f ~ t columns y that attach galactose or comparable sugars to the rigid support can be used to isolate the toxin. These data are highly suggestive that the molecule recognizes a (complex) sugar on the cell surface, but the true nature of the receptor remains to be clarified (Eidels et al., 1983). The second polypeptide component ( M , 30,625)has recently been shown to be an RNA N-glycosidase(Endo and Tsurugi, 1987). For many years investigators have realized that the toxin inhibits protein synthesis and that the local site of action is the ribosome. Furthermore, there have
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been clues that the toxin acts by a cleavage reaction rather than an addition reaction, because no cofactors are needed for the blockade of protein synthesis. Although the reported finding of N-glycosidase activity has not yet been reproduced, the general phenomenon is in accordance with expectations. Because the site of toxin action is intracellular, the toxin molecule, or at least the catalytic chain, must be able to penetrate the plasma membrane. An acceptable mechanism to account for this has not been established. There may well be an intervening step of receptor-mediated endocytosis, and indeed many workers favor this idea, but firm evidence has not been forthcoming. It must also be reported that the endocytosis step, should this prove relevant, is not identical to that employed by diphtheria toxin. The ricin molecule has not been convincingly shown to produce pH-dependent channels, nor can direct penetration through the membrane be induced by incubating cells in low-pH medium. Drugs like ammonium chloride, chloroquine, and methylamine hydrochloride do not antagonize the toxin and may even enhance biological activity (Sandvig and Olsnes, 1982). Shiga toxin appears to behave much like ricin, but there is one structural difference. The ricin molecule has a single polypeptide chain that mediates binding; the Shiga toxin molecule is thought to have five promoters that act in concert to form a tissue-targeting domain (O’Brien and Holmes, 1987). Aside from this, the theme is one of shared properties. A catalytic chain from Shiga toxin enters the cytosol, where it exerts an N-glycosidaseaction that culminates in blockade of protein synthesis.
C. TOXINS WITH
AN
UNKNOWN MECHANISM OF ACTION
The final group of toxins is the one whose mechanism of action is least well understood, but nevertheless this group is the one most familiar to neurobiologists. The group has two representatives: botulinum neurotoxin, which exists in seven serotypes (Sakaguchi, 1983; Simpson, 198l), and tetanus toxin, which has only one serotype (Wellhoner, 1982). Both toxins are known for their abilities to produce devastating neurological disease. Botulinum neurotoxin acts mainly at the cholinergic neuromuscular junction to block the release of acetylcholine; in patients this can produce flaccid paralysis (Gundersen, 1980; Simpson, 1986). Tetanus toxin acts mainly in the central nervous system to block release of inhibitory transmitters such as glycine and GABA (Habermann and Dreyer, 1986). By removing the influence of inhibitory fibers, the toxin leaves excitatory efferent fibers unchecked, and this leads to spastic
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paralysis. Although each of the toxins has a principal site of action (i.e., the neuromuscular junction versus the central nervous system), both will block the release of many types of transmitters, assuming that there is access to the synapse and that a sufficiently high concentration is tested (Simpson, 1986). Indeed, the toxins can block release of chemical mediators from nonneural tissues, such as the adrenal gland (Penner et al., 1986) and macrophages (Ho and Klempner, 1985). The clinical presentations of botulism and tetanus are very different, and this led investigators for years to assume that the underlying mechanisms of action were unrelated (Wright, 1955). There was also the belief that botulinum and tetanus toxins were fundamentally different from better characterized toxins, such as those described above, but this belief was based more on a narrowness of perspective than on clinical presentations or laboratory data. Researchers who study agents like diphtheria toxin or Shiga toxin are usually cell biologists or microbiologists who are not well versed concerning the nervous system, whereas botulinum and tetanus toxin workers are familiar with the nervous system but only rarely familiar with agents like diphtheria and Shiga toxins. Narrowness of perspective created the impression that botulinum and tetanus toxins were singular and unique, but that assessment is no longer deemed to be true. The two toxins are now thought to be rather similar to one another, and as a group they share many properties with the ADPribosylating and N-glycosidase toxins (Simpson, 1986). Botulinum toxin has long been known to exert an effect at the neuromuscular junction (Burgen et al., 1949), and more recent work has shown that tetanus toxin will produce a similar effect (Habermann et al., 1980). Botulinum toxin is active at very low concentrations, producing neuromuscular blockade at 10 -lo M and lower. The concentrations of tetanus toxin that are equiactive are about 200-fold higher. In spite of these concentration disparities, the neuromuscular junction is increasingly used as a system in which to compare the two toxins. There are highly suggestive data indicating that botulinum neurotoxin and tetanus toxin are multiple-domain molecules that proceed through a sequence of at least three steps in producing paralysis (Simpson, 1986). Both toxins have molecular masses of approximately 150,000 daltons, and like other toxins that have been discussed above they possess two polypeptide chains. The heavy chain ( M , -100,000) appears to play a prominent role in specifying the molecule’s target tissues, and it is the carboxy-terminus that has been implicated (Kozaki, 1979; Goldberg et al., 1981; Dolly et al., 1987; Bandyopadhyay et al., 1987). There is evidence that the toxin molecules must be internalized, probably by
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receptor-mediated endocytosis, and part of this evidence suggests that the amino-terminus of the heavy chain is involved (Boquet and Duflot, 1982; Hoch et al., 1985). Among the findings that support the idea of receptor-mediated endocytosis are the following. First, it has been shown that drugs like ammonium chloride and methylamine hydrochloride, drugs that antagonize many endocytosed substances, are clostridial toxin antagonists (Simpson, 1983). Second, investigators have shown that appropriately labeled toxin molecules can be localized inside nerve cells (Black and Dolly, 1986a,b). The data that point to the amino-terminus of the heavy chain as playing a role in endocytosis are derived from in vitro studies on lipid membranes. This portion of the botulinum neurotoxin and tetanus toxin molecule forms pH-induced channels (Boquet and Duflot, 1982; Hoch et al., 1985; Donovan and Middlebrook, 1986). The intracellular effects of the toxins in producing blockade of transmitter release have not been determined. There is a prevailing belief that the toxins are enzymes, based mainly on two lines of logic. The toxins are remarkably potent, and this would seem to suggest a catalytic rather than a stoichiometric effect. Additionally, the toxins have structures and presumed functions that resemble those of other potent protein toxins, and this by deduction seems to point to an enzymatic action. There is an aspect of the pharmacology of these toxins that may seem curious and that needs clarification. If both substances act at the neuromuscular junction to block acetylcholine release, why does one evoke flaccid paralysis whereas the other produces spastic paralysis? The answer is related to dose-response characteristics and, at least speculatively, it may also relate to the phenomenon of intracellular trafficking (Simpson, 1988). There is agreement that both toxins bind with high affinity to the cholinergic nerve ending. When low doses are administered, the toxins bind and are internalized, but they are routed differently. Botulinum toxin remains at the nerve ending, where it impairs transmitter release. Tetanus toxin undergoes retrograde axonal transport to reach the central nervous system, and there it acts to block inhibitory transmitter release and secondarily produces spastic paralysis (Wellhoner, 1982; Habermann and Dreyer, 1986). When high doses are administered, botulinum toxin continues to act mainly at the neuromuscular junction, although some of it may undergo axonal transport (Habermann, 1974; Black and Dolly, 1986a). Similarly, high doses of tetanus toxin are mainly routed to the central nervous system, but some is diverted to act locally, where it produces flaccid paralysis (see above). If this general scheme is correct, it suggests that tetanus toxin may act centrally the same way it acts peripherally. There may be binding, internalization,
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and poisoning steps, and these events may be related to the functional domains described earlier.
IV. Toxins as Magic Bullets
One does not ordinarily think of extremely potent toxins as therapeutic agents. However, work done during the past decade has shown that toxins, either in their native state or as modified proteins, have the potential to be used medicinally. The major thrust in this area has come from a merger of toxicology and oncology, and the product is a new class of antineoplastic drugs. In one sense the work can be traced to a straightforward series of experiments in protein chemistry. Investigators studying the structure-function relationships of potent toxins used procedures to dissociate the holotoxins into their constituent parts. For example, dichain molecules like diphtheria toxin and ricin, in which the heavy and light chains are linked by a disulfide bond, were reduced with dithiothreitol, and the respective polypeptides were isolated. The individual chains were essentially devoid of toxicity, but biological activity could be restored by reannealing the chains under oxidizing conditions. This set the stage for a more ambitious experiment. Individual toxins were dissociated into their respective parts, and then the component parts of heterologous toxins were joined. For example, the binding chain of diphtheria toxin was attached to the enzymatic chain of ricin, and the unnatural toxin that was formed was found to be biologically active (Sundan et al., 1982). This was one of the first of several unnatural toxins that have been constructed, and as a group these substances are called chimeric toxins (Olsnes and Pihl, 1982). In the course of this work the question arose whether it was necessary to use the constituent parts of a natural toxin in order to form a chimeric molecule. An obvious alternative was to discard the naturally occuring binding fragment in favor of another substance that would confer target specificity on the newly formed chimera. Because of their specificity of action, antibodies were among the substances tested as targeting moieties. Thus, antibodies directed against appropriate antigens were linked to the poisoning fragments of toxins, and these agents were tested for their abilities to kill cells (Pastan et al., 1986). A host of chimeras constructed of antibodies linked covalently to toxin enzymes have been made. A general statement is that most of these substances are toxic, but their potency is less than that of the native
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toxins. These chimeras behave in accordance with the properties of their components, meaning that the molecule binds to antigen rather than to the native toxin receptor. Once internalized, the enzyme component exerts its characteristic effect (i.e., ADP-ribosyltransferase,N-glycosidase, etc.). There are two immediate implications of this work. To begin with, antibody-toxin chimeras have precisely the structure that was envisioned by Ehrlich when he originally conceived magic bullets. Beyond this, the chimeras have the functional utility expected of magic bullets, that is, they can act as medicinal agents. When an antigen directed against a cell surface antigen on a cancer cell is linked to an ADP-ribosyltransferase or N-glycosidase toxin that blocks protein synthesis, the product is a drug that has antineoplastic properties. The drug is targeted to the cancer cells, where it can produce cell death (see, for example, Vitetta and Uhr, 1985).
V. Broadening the Concept of Magic Bullets
Although Ehrlich could not have realized it when he coined his famous phrase, the concept of a magic bullet is but one part of a larger scheme that is used throughout the life sciences. The scheme was implemented by nature long before it was conceived by laboratory scientists. The scheme can best be understood by examining two ideas that have recently been advanced, one of which is the supergene and the other of which is superfunction. Hood has coined the term supergene, and he has used it to explain a well-documented phenomenon (Hood et a l . , 1985). There are many proteins that share segments of sequence homology in spite of the fact that these proteins are seemingly unrelated and do not serve the same function. An apparent explanation is that the individual genes that encode the individual proteins are descendants of a single ancestral gene. Through a variety of mechanisms (e.g., gene duplication), segments of the ancestral gene have descended and become incorporated into many genes, including those that do not necessarily encode for proteins of similar function. Simpson has coined the term superfunction, and it is in some respects the corollary of supergene (Simpson, 1986). There are many proteins that have the same or similar function in spite of the fact that they have no sequence homology. The assumed explanation for this commonality of function in the absence of commonality of structure is that evolution
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is a selection process, and one of the properties for which evolution selects is efficiency. The concept of superfunction is easily grasped by dissecting the pieces of a protein toxin, such as diphtheria toxin. Described in broad terms, the molecule has three components. There is, as we have already seen, a tissue-targeting component (i.e., the carboxy-terminus of the heavy chain). This will now be described as the ligand component, meaning that it binds with high affinity to specific receptors on the cell surface. Its mechanism of action is stoichiometric. Another portion of the molecule (i.e., the amino-terminus of the heavy chain) plays a prominent role in the internalization process. This will now be referred to as the transduction component, meaning that it links the actions of the cellto-cell component, or ligand, with those of the intracellular component. This brings us to the third portion of the toxin molecule (i.e., the light chain) that acts within the cell to express ADP-ribosyltransferaseactivity. Thus, there is a ligand component, a transduction component, and an enzymatic component. This characterization of the diphtheria toxin molecule applies equally well to the other ADP-ribosyltransferases and N-glycosidases. Each has a ligand component that binds extracellularly, and each has a catalytic component that acts intracellularly. Irrespective of whether a transduction component has been identified, one can deduce that such a component must exist. Given that the substrates for the toxins are in the cytosol, there must be a vehicle for linking extracellular binding to intracellular expression of activity. The question of whether the clostridial neurotoxins fit the same mold has not been resolved, because a catalytic mechanism of action has not been determined, but there is a presumption that they too belong to the group. In sum, the toxins display three individual families of function: the ligand components, the transduction components, and the catalytic components. There is no sequence homology, for example, between the binding domain of diphtheria toxin and that of cholera toxin, yet they serve a similar function. Likewise, there is no sequence homology between the enzymatic domains of pseudomonas toxin and ricin yet they too serve a common function. It is interesting that these three-component models are not unique to toxins. To the contrary, they are rather widespread, and it is because of this that the concept of superfunction takes on value. Let us consider two examples, one of primitive origin and one that has evolved in complex eukaryotes. In essence, many viruses are built like toxins. Of necessity viruses have a ligand component that can attach to receptors on prokaryotic or eukaryotic cells. They also have internalization domains,
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which for the moment can be envisioned as transduction components that help translate the membrane-binding event into an intracellular event. (Parenthetically, certain viruses have served as models for the study of protein toxins. Semliki Forest virus, for instance, was shown to undergo a pH-dependent internalization process, and it was this work that stimulated the subsequent pH studies on diphtheria and other toxins.) Finally, the virus relies not on one but on an entire host of enzymes to exert its intracellular effects. As an illustration, this could include the enzymes for scission of host nucleic acid, insertion of nucleic acid from a temperate phage, and then repair of the spliced material. Microbial toxins and viruses may not be familiar to most neurobiologists, but the final example of the three-component system certainly is. Many cellular messenger systems are designed precisely as described above, and even the nomenclature is identical. Cell-to-cell mediators such as norepinephrine can act as neurotransmitters or hormones, and in either case they are ligands that bind to catecholamine receptors. The ligand-receptor interaction typically leads to a transduction event, either the opening of ion channels, the activation of regulatory proteins, or both. In the latter case, second messengers are created that initiate an intracellular cascade of enzymatic events. The intimate details of the several examples do vary, but the overall motif is strikingly similar. There exists a diverse group of molecules that are composed of at least three functional domains. One of these groups includes protein toxins such as diphtheria toxin, another includes virus particles such as Semliki Forest virus, and yet another includes first and second messenger systems such as norepinephrine and the CAMP initiated cascade. Within each group there are many representatives, some of which have sequence homologies and some of which do not. Across groups, however, there is as yet no evidence of genetic or primary sequence homology. Nevertheless, the family of ligand components in protein toxins play essentially the same role as the family of ligand components in viruses. Therefore, the various families of functionally equivalent molecules can be placed together under the concept of superfunction. By the same reasoning, the families of transduction components and enzyme components from protein toxins, viruses, and messenger systems can also be placed together under the concept of superfunction. The various implications of superfunction, including its potential to explain principles that govern cell biology and evolution, will be explored elsewhere. For the moment, there is one point that is of some importance. This article began with the premise that investigators have often sought magic bullets, but the search has only occasionally been rewarding. When one compares the original concept of a magic bullet
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with the thought inherent in the concept of superfunction, at least one reason for the rarity of successes comes to mind. A magic bullet is a bifunctional molecule, possessing a ligand component (namely, antibody) and a pharmacologically active component (namely, medicinal agent). Although Ehrlich could not have known it, this is an incomplete structure for a complex molecule intended to express an intracellular effect. The traditional concept of a magic bullet omits the premise that there must be a transduction component, something to link cell surface binding to intracellular actions. Another implication of the concept is that it should be possible, so to speak, to "mix and match" functional components, both within families and across families. An illustration of this has already been alluded to. One component of diphtheria toxin was linked to a heterologous component of ricin, and the product was a potent toxin. This is an example of linking within a group (i.e., protein toxins). Uchida et al. (1977) have shown that the principle can extend across groups. They have separated diphtheria toxin into its heavy and light chains, the latter of which represents the enzymatic component. They then created ghosts from Sendai virus (i.e., they emptied the virus of its nucleocapsid). The ghosts were subsequently loaded with the enzymatic chain from diphtheria toxin, and the assembly (ligand and transduction components from virus; enzymatic component from toxin) was shown to poison cells.
VI. Chimeric Toxins
During the past decade, there has been a resurgence of optimism that magic bullets can be constructed that will have genuine therapeutic value. The dominant area of investigation has been that relating to the development of antineoplastic drugs. This field of research has been extensively and well reviewed (Olsnes and Pihl, 1982; Vitetta et al., 1983). The premise that underlies this work is easily understood in the context of the earlier discussion. Investigators have separated ricin, diphtheria toxin and related substances into their constituent parts. The poisoning components have been retained, and they have been covalently attached to ligands that are targeted to cancer cells. In most cases, the ligand function has been served by an antibody. Thus the prevailing strategy has been to use an antibody directed against a cell surface antigen as one component, and the enzyme fragment of a toxin as the other component. There are two broad assessments that can be made of this work. First, the chimeric toxin typically possesses the expected specificity of action.
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The antibody moiety binds to the appropriate antigen rather than to the toxin receptor, and the enzymatic moiety exerts the predicted poisoning effect. Second, these chimeric molecules are usually less potent than the parent toxin from which the enzymatic domain was extracted. One of the most common explanations for this is the assumption that the chimeric molecule fails to achieve productive internalization. The latter point is a reiteration of the argument raised above. Magic bullets should have three domains rather than two, and in the absence of a transduction domain the chances of success are diminished. An examination of the literature reveals that more than 100 chimeric toxins have already been described. There is ample evidence that the fundamental concept is sound. The challenge now is to refine the technology in a way that will maximize specificity of tissue binding, ensure productive internalization, and allow full expression of intracellular activity.
VII. Chimeric Neuroactive Compounds
It seems timely to consider the ways in which the technology of creating chimeric drugs, until now confined mainly to antineoplastic drugs, can be broadened. One area that could benefit from the development of such drugs is neurobiolgy (and see Bizzini et al., 1980; Cohen, 1982). There is the obvious matter that chimeric drugs could be created that would have therapeutic benefit in the treatment of neuroblastoma. However, there are other and equally compelling areas that merit exploration. In the examples that follow, illustrations are given of the ways in which chimeric drugs have been used or could be used beneficially in neurobiology. A. NEURAL TUMORS Dozens of drugs have been described that have potential utility in the treatment of cancer, but these agents are mainly for tumors of nonneural origin. Only recently has the concept of a chimeric drug been applied to the problem of the treatment of malignant brain tumors. A recent study has focused on the use of an immunotoxin in the killing of human glioblastoma and medulloblastoma cells (Zovickian et al., 1987). The molecule was constructed of a mouse monoclonal antibody directed against human transferrin receptor and the plant lectin ricin. The rationale underlying the use of the antitransferrin receptor was that
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many cells contain this protein, but proliferating cells typically express larger amounts. Ricin was chosen because of its potent killing activity; the intact ricin molecule was used to ensure that both an enzymatic and translocating domain were available. In studies on cells in culture, Zovickian et al. (1987) demonstrated that their chimera was extremely effective against an SNB75 cell line (glioblastoma of multiform origin) and an SNB40 cell line (medulloblastoma cell line). In the former case, the IC50 for inhibition of protein synthesis was 1.7 X 10-lo M, in the latter case it was approximately the same. Furthermore, the chimera showed considerable selectivity: the difference in activity against tumor cells and normal brain cells was in the range of two to three orders of magnitude. Results like these are very encouraging. They suggest that immunotoxins may ultimately be used to treat tumors of the brain. This could be done by employing a technique for importing the chimera into the central nervous system (see below), or, alternatively, it could be done by using conventional means such as intrathecal or intraventricular injection.
B. PHEOCHROMOCYTOMA Pheochromocytoma is an adrenomedullary neoplasm characterized by excessive production and release of catecholamines. The disorder is usually benign, and it is customarily managed by surgical intervention. However, there are some patients for whom neither surgery nor the vigorous administration of a- and P-adrenergic blocking agents is appropriate. An alternate form of pharmacological intervention would be desirable, and chimeric drugs might be the solution. Agents like botulinum neurotoxin and tetanus toxin are known to block the secretory process in a variety of neurons, including cholinergic, glycinergic, GABAergic, and adrenergic cells (Habermann and Dreyer, 1986). In addition, when used in high concentrations, the toxins will block secretion from nonneural cells, including adrenomedullary cells (Knight et al., 1985; Knight, 1986). There is a belief that the toxins may have a somewhat universal effect in blocking release of chemical mediators. The reason for the differences in sensitivity from cell type to cell type may reflect the relative absence of receptors or means for internalization rather than an absence of intracellular substrate (Penner et al., 1986). The fact that clostridial toxins can block exocytosis of catecholamines from the adrenals may mean that the molecules could be modified to
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become therapeutic agents for the treatment of pheochromocytoma. The approach would be conceptually identical to that for the development of antineoplastic drugs. An antibody directed against a cell surface antigen could be linked to the portions of the toxin molecule that mediate internalization and later blockade of transmitter release. The key difference between this chimeric drug and the ones discussed earlier for treatment of neural tumors is that the latter kill cells whereas the former does not. Thus, the patient would be left with an adrenal medulla, albeit a tissue with a depressed capacity for catecholamine release. On the other hand, should removal of the tumor rather than mere blockade of transmitter release be required, then a chimeric antineoplastic drug (i.e., a cell-killing drug) would be in order.
C. BLOCKERS OF TRANSMITTER RELEASE As just noted, the clostridial toxins block mediator release from a variety of cell types. The presumed explanation for differences in cell sensitivity relate to the relative absence of appropriate receptors. Therefore, it seems reasonable to suggest that a new class of drugs could be created by discarding the binding domains of these toxins while retaining the internalization and enzymatic domains. The nature of the newly created ligand domain would hinge on the cell type being studied. If the cell in question had receptors for a polypeptide hormone, then the hormone itself might be the best ligand. If the cell was a neuron with a single transmitter, one might consider antibodies that recognize proteins unique to the cell (e.g., antibodies against the high-affinity uptake system.) There are compelling reasons for wanting to create drugs that are highly tissue-specific and that produce sustained blockade of chemical mediator release. One of these, the treatment of pheochromocytoma, was given above. Another might be the treatment of allergy or asthmatic disorders. The advantages of a drug that affected only mast cells and blocked histamine release are obvious. Yet another likely benefit of these drugs would be as research tools that could block release of specific transmitters at specific sites. Botulinum neurotoxin has already been exploited for its ability to block acetylcholine release at cholinergic nerve endings in the periphery. It has been used to study such phenomena as trophic influences of nerve on muscle, denervation supersensitivity, and nerve sprouting (Simpson, 1981). Drugs that blocked transmitter release at other sites might have equal utility. A drug that blocked dopamine release could be helpful in the study of Parkinson’s
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disease: a drug that acted centrally to block acetylcholine release might be of value to those studying Alzheimer’s disease; and tetanus toxin, a drug that blocks inhibitory transmitter release, is already being used in the study of epilepsy (Brace et al., 1985).
D. CLOSTRIDIAL CHIMERAS Botulinum neurotoxin and tetanus toxin are similar in their origins, macrostructures, and presumed mechanisms of action at neuronal terminals (Simpson, 1986). Both are thought to enter the cell and block the process of exocytosis. It may be that the two are evolutionary descendants of the same ancestral parent, in which case one would expect close parallelisms in structure and biological activity (Eisel et al., 1986). It is intriguing to consider the possibility of forming chimeric molecules that would help to clarify the relationship between these two toxins. One course of action might be the following. At very low doses, botulinum neurotoxin acts locally to produce flaccid paralysis and tetanus toxin acts in the central nervous system to produce spastic paralysis. Presumably, it is the heavy chains of the two molecules that confer the ability to bind and act locally or bind and be transported into the central nervous system. This suggests that one should dissociate the toxins into their respective heavy and light chains, and then the heterologous components should be linked. Conceivably, the heavy chain of botulinum neurotoxin, when linked to the light chain of tetanus toxin, might result in a molecule that paralyzes neuromuscular transmission and causes flaccidity. Conversely, the heavy chain of tetanus toxin, when bound to the light chain of botulinum neurotoxin, could be an agent that is transported into the central nervous system to cause spasticity.
E. CHIMERIC ANTIBODIES All of the magic bullets that have been described in the literature use an antibody as a tissue-targeting domain, if these agents employ an antibody at all. It is worth considering that there is an alternate way to envision chimeras, and the products might prove to be a new class of drugs with broad therapeutic utility (Simpson, 1981). For most of the potent protein toxins, conventional antibody therapy is of no practical value when vulnerable cells in the patient have internalized the poison. Antibody will of course neutralize any circulating toxin, but it cannot enter target cells to inactivate endocytosed toxin.
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It is conceivable that the toxin molecule itself could be modified to overcome this problem. The chimeric molecules discussed thus far have had in common that the binding or ligand domain of the native toxin has been discarded in favor of a novel ligand domain. Invariably, the enzymatic domain has been retained. The reverse situation would entail discarding the enzymatic domain while retaining the ligand and transduction domains. In place of the enzymatic domain, one could add a neutralizing antibody that is directed against the poisoning domain. A molecule like this might circumvent the barriers that normally exclude a free and circulating antibody from the cell interior. By virtue of having the toxin’s native ligand and internalizing domains, the chimeric agent would be directed to the poisoned cell, and by virtue of having a neutralizing antibody, the drug might arrest the intracellular effects of toxin. This is only one version of a broad plan. If an internalized antibody can exert a therapeutic effect, then there is the possibility that other medications might likewise be productively internalized. This could entail the administration of a drug to correct an endogenously or exogenously induced disorder, or it might involve the supplying of a substance whose absence is related to disease (i.e., overcoming a deficiency syndrome). There is encouraging evidence that a strategy like this might work. As reported above, Sendai viruses can be converted to ghosts and loaded with the poisoning domain of diphtheria toxin. The addition of this complex to cells with Sendai receptors leads to internalization of the enzyme, inactivation of elongation factor 2, and poisoning of the cell (Uchida et al., 1977). In a variation on this procedure, workers have loaded erythrocyte ghosts with antibodies directed against the light chain of the toxin. After vulnerable cells were exposed to the ghosts and had internalized the antibody, native diphtheria toxin was added to the tissue medium. Compared to control cells, the cells with internalized antibody had acquired a substantial amount of resistance (Yamaizumi et al., 1978a). In essence, the investigators had achieved what might be called a pharmacological ambush. The internalized antibody was waiting inside the cell, where it attacked toxin that entered by the normal route. F. IMPORT OF DRUGS
The discussion thus far has focused on agents that could produce selective neurobiological effects in the periphery. Selectivity might be achieved in the central nervous system by injecting material intrathecally or into a specific locus. How could one achieve central nervous system
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effects without injecting into the brain or spinal cord? There are two options that are worthy of consideration. The binding and internalization domains of tetanus toxin might be usable as an import device. There are convincing data to show that the toxin is normally routed to the spinal cord, using retrograde axonal transport and transynaptic movement as the means of trafficking. It seems plausible to suggest that these domains might be used as part of a vehicle to convey a therapeutic agent or a pharmacological research tool. The idea has been considered (Cohen, 1982), and modest evidence to support its feasibility has been published (Bizzini et al., 1980), but a true chimera possessing biological activity has not been reported. Alternatively, one might consider a virus ghost as a suitable vehicle. This is a concept that is being vigorously pursued in several laboratories, and the prospects for importing drugs into the brain and spinal cord appear good.
G. MYASTHENIA GRAVIS Relatively little work has been done in this area. However, the few papers that have been published show great promise (Killen and Lindstrom, 1984; Olsberg et al., 1985). Myasthenia gravis is an autoimmune disease in which antibodies are produced against the nicotinic cholinergic receptor. The antibody-antigen response leads to the muscle weakness that is characteristic of the disease. Both in uilro (Killen and Lindstrom, 1984) and a combination of in mlro and in Vivo experiments (Olsberg et al., 1985) show that immunotoxins may have therapeutic value. The latter study provided one particularly telling experiment. The investigators demonstrated, in confirmation of many previous studies, that immunization of laboratory animals with purified acetylcholine receptor led to muscle weakness. They also showed that lymphocytes exposed to the antigen in vitro and later injected into animals were sensitized to subsequent injection of the acetylcholine receptor, and here too there was onset of muscle weakness. However, when the immune lymphocytes were treated with receptor that was covalently linked to the enzymatic chain of ricin, the subsequent injection of these treated cells did not lead to sensitization such that later antigen produced myasthenia gravis. The implications of these findings for the treatment of the disease are clear. VIII. Concluding Remarks
A technology is emerging that allows for the creation of drugs that are a reasonable approximation of magic bullets. The success of this technology is due in part to the fact that increasing attention is being
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given to the functional domains that constitute such drugs. There is an emerging awareness that targeted drugs intended to act inside cells should have three domains (ligand, transduction, active) as opposed to the traditional two (ligand, active). Most of the successes with chimeric drugs have been in the field of antineoplastic therapy, although a notable recent accomplishment has been in the area of antithrombotic therapy (Bode et al., 1985, 1987). There is reason to believe that these advances can be put to good use in neurobiology. Most obviously, chimeric drugs could be designed that would attack neuroblastomas and pheochromocytomas. Beyond this, the potential therapeutic and research value of chimeric drugs is considerable. The limitations do not appear to be in the drugs, but rather in the ability of neuroscientists to adopt a new technology and exploit it fully.
Acknowledgments
This research was supported in part by NIH Grant NS-22153 and by DOD Contracts DAMD17-854-5285 and DAMD17-86-C-6161.
References
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Ne u ron-G Iia Int e r re Iat ions By Antonio Vernodokis Departments of Psychiatry and Pharmacology University of Colorado School of Medicine Denver, Colorado 80262
I. Introduction 11. Neuron-Glia Interactions during Ontogenesis A. Influence of Glial Cells on Neuronal Phenotypic Expression and Growth B. Influence of Neurons on Glial Cells 111. Neuron-Glia Interactions and Synaptic Events A. Morphological Evidence B. Ionic Regulation C. Neurotransmission Processes IV. Regeneration V. Neuron-Glia Interactions in Aging Processes A. Changes in Glial Cells with Aging B. Synapse Function VI. Summary References
1. Introduction
The neuron-glia functional partnership proposed by Hyden in 1961 has now been accepted and several reviews have been written describing the cellular events in neuron-glia interactions (Galambos, 1961, 1965, 1971; Kuffler and Nichols, 1966; Lasansky, 1971; Orkand, 1977; Somjen, 1975; Varon, 1975; Varon and Saier, 1975; Vernadakis, 1975; Watson, 1974). The review by Varon and Somjen in 1979 (summarizing a Neurosciences Research Program Work Session on neuron-glia interactions) is an up-to-date treatise of the state of affairs. More recently, several brief reviews have been focused on various aspects of neurons and glial cells and their interactions (reviews in Althaus and Seifert, 1987; Bunge and Waksman, 1985; Hatten and Mason, 1986; reviews in Sears, 1982; Treherne, 1981; and several chapters in Vernadakis et al., 1987). Finally, the publication of three volumes on Astrocytes (edited by Fedoroff and Vernadakis, 1986a-c) is a testimony to the enormous interest in the cells that Virchow in 1846 described as “nerve glue.” INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL 30
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Copyright @ 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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This review will focus on studies reported during the past 5 years and add an updated version to the saga of neuron-glia interactions. Although the reader might expect to find some background information on neurogenesis and gliogenesis as a prelude to a discussion of the neuron-glia partnership, I feel that these cellular events have already been recently described elegantly: gliogenesis has been reviewed by Fedoroff (1986), Roots (1986), and Sturrock (1986) and neurogenesis by Stent and Weisblat (1985),Jacobson (1985), Herrup et al. (1984), Rakic (1985), and, in an extensive series of papers, by Altman and Bayer (1980a-d, 1981, 1985a-c, 1987a-d). Another detailed description would be superfluous. Of course, certain key studies, although extensively reviewed earlier, do provide a foundation for conclusions drawn from more recent findings and, therefore, I will refer to them as appropriate.
II. Neuron-Glia Interactions during Ontogenesis
The role of cell-to-cell interactions involved in neurogenesis and neuronal growth and differentiation has been a major theme of developmental neurobiology in the last two decades. However, with few exceptions, most studies have focused on the interaction among various classes of neurons. For many decades the prevailing view was that glial cells are formed only after most of the neurons destined for a given structure complete their genesis. This view has now been challenged by Golgi and electron microscopic studies (Rakic, 1972, 1978; Schmechel and Rakic, 1979) and more recently by the use of glia-specific cell markers (Levitt et al., 1981; Levitt and Rakic, 1980; see reviews by Fedoroff, 1986; Sturrock, 1986). Considerable progress has been made in defining neurogenesis from the earliest formative stages; those studies have been reviewed by Rakic (1981). The coexistenceof neurons and glial cells during early neuroembryogenesis places these cells in a strategic position to interact with each other and thus influence their individual growth and differentiation. The recent selected in mvo and in mlro studies described in my review demonstrate (1) the influence of glial cells on neuronal growth and differentiation and (2) the influence of neurons on glial growth and differentiation. Such interactions appear to be mediated through cell surface components and cell-secreted factors in the microenvironment.
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OF GLIALCELLS O N NEURONAL PHENOTYPIC A. INFLUENCE EXPRESSION AND GROWTH
1. Phenotypic Expression Cell culture model systems have become increasingly valuable in studying mechanisms underlying neural growth (see recent books edited by Bottenstein and Sato, 1985; Vernadakis et al., 1987). Many aspects of neural development such as proliferation, differentiation, synaptogenesis, and myelination occur in culture with time courses remarkably similar to those in vivo. Peripheral autonomic ganglia provide an excellent source of neurons for tissue culture. Initially, all of the neurons appear to synthesize and store catecholamines (Landis, 1978, 1980; Mains and Patterson, 1973; Patterson and Chun, 1974, 1977a,b). The early expression of noradrenergic properties by dissociated sympathetic neurons occurs when they appear to receive an adrenergic signal from their environment during migration (Teillet et al., 1978). At present there is no evidence for comparably early expression of cholinergic function in rat sympathetic neurons or their precursors in vivo or in culture. Dissociated sympathetic neurons continue to differentiate to mature sympathetic neurons if cultured in the absence of nonneuronal cells (i.e., glial cells). It has also been possible to grow cultures of dissociated sympathetic neurons under conditions such that the cultures develop cholinergic as well as noradrenergic properties. Again, one condition that fosters the induction of cholinergic properties is the presence of certain nonneural cells (Patterson and Chun, 1974). This finding has been interpreted in several ways. First, it is possible that individual sympathetic cervical ganglion (SCG)neurons are able to express only one transmitter system, either adrenergic or cholinergic, and that the acquisition of cholinergic traits in culture actually represents a skill in the choice of transmitter production by the cell. Alternatively, it is possible that individual SCG neurons simultaneously develop both adrenergic and cholinergic transmitter-synthesizingmechanisms in culture, thereby acquiring at least the potential for dual functions. A dual neurotransmitter function has been shown by Iacovitti et al. (1981), who demonstrated using immunocytochemical methods that essentially all SCG neurons stain positively with antibodies to tyrosine hydroxylase, even at times when choline acetyltransferase levels in the culture are elevated significantly. This neuronal dual function expression in vitro has also been observed in znvo by Le Douarin and associates; neural crest cells destined to become catecholaminergic cells in the developing embryo may, if provided with an alternate site of residence, express cholinergic characteristics (for
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details, see Le Douarin, 1980). Recently, Iacovitti et al. (1985) have demonstrated both in witro and in vivo partial expression of catecholaminergic traits in cholinergic chick ciliary ganglia. Thus, the potential for a dual transmitter function appears to exist in both sympathetic and parasympathetic peripheral neurons. 2. Influence of Glial Cells on Neuritic Growth a. Glia-Enriched Substrata. Glial cells play an important function as substrate pathways during CNS development. As described early by Schmechel and Rakic (1979) and Levitt and Rakic (1980), the astrocyterelated radial glia are present in all brain regions during the peak of neuronal cell body migration, and these glial cells appear to offer the only class of cellular process with which migrating neurons are associated throughout the course of migration (Fig. 1). (See also discussion on migration. ) In uitro studies have provided further evidence of cell surfacemediated interactions in neuronal growth during development (Adler and Varon, 1981a,b; Sensenbrenner and Mandel, 1974; Varon, 1975; Wessels et al., 1980). That glia are a good substrate for the in mlro growth of CNS neurons has more recently been demonstrated by Noble et al. (1984). When cerebellar (7-day-old rat) or spinal cord (14-day-old rat embryo) cells were plated onto monolayer astrocyte-enriched or Schwann cell cultures, neurons generally grew as single cells and showed relatively little tendency to aggregate; similarly, neurites showed little tendency to fasciculate. In contrast, when plated onto fibroblast, heart muscle fibroblast, or astrocyte-free meningeal monolayers, neurons rapidly aggregated, and neurite outgrowth was primarily in large fascicles. Further experiments showed that whether or not aggregation and fasciculation occurred was due to surface properties of the glial and FIG. 1. Three-dimensional reconstruction of migrating neurons, based on electron micrographs of semiserial sections. The reconstructed cells are situated in the intermediate zone at the borderline between the optic radiation (OR)and the area occupied by the more variable and irregularly disposed corticocortical fiber system. Except at the lower portion of the figure, most of these fibers are deleted from the diagram to expose the radial fibers [striped vertical shafts (RF1-6)] and their relationships to the migrating cells (A, B, and C) and other vertical processes. The soma of migrating cell A, with its nucleus (N) and voluminous leading process (LP) is within the reconstructed space, except for the terminal part of the attenuated trailing process (TP) and the tip of the vertical ascending pseudopodium (PS).Cross-sectionsof cell A in relation to the several vertical fibers in the fascicle are drawn at levels a-d at the right side of the figure. The perikaryon of cell B is cut off at the top of the reconstructed space, whereas the leading process of cell C is shown just penetrating between fibers of the optic radiation on its way across the intermediate zone. (From Rakic, 1972.)
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nonglial cells. When neurons were added to astrocyte and nonglial monolayers growing in medium conditioned by a large excess of cocultured nonglia or astrocytes, respectively, the pattern of neuronal growth was determined by the type of monolayer with which the neurons were in contact. Thus, aggregates and fascicles formed on nonglia, even in the continual presence of astrocyte-conditioned medium: conversely, neither aggregation nor fasciculation was seen in neurons growing on astrocytes, despite their being bathed in medium conditioned by nonglia. The possibility that neurons adhere better to glial cells than to each other is noteworthy, since it would seem to be an exception to the general rule that cells are more adherent to like than to nonlike cells (Moscona, 1962). Reaggregation of dissociated cells demonstrates distinct preferences by cells for like cells. It has been debated whether such preferences reflect different degrees of an adhesive property common to all cells rather than qualitative differences across different cell classes (Moscona, 1974). The adhesive molecules recently described by Alliot and Pessac (1984), Edelman (1983a,b), Grumet and Edelman (1984), and Rutishauser (1984) promise to provide some clues to molecular components of this cellular phenomenon of homotypic cell tissue reconstruction. The influence of glial surfaces on aggregation and neurite fasciculation has also been indicated in studies by our laboratory using the 3-dayold chick embryo as an in mlro model (Vernadakis et al., 1986: Mangoura et al., 1988). We compared primary growth patterns in cultures prepared by dissociating 3-day-old chick embryos, either whole embryo (ESWE) or head only (E3H) and plating the dispersed cells on petri dishes coated with poly-L-lysine, collagen, or laminin. The culture medium was Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with either 5 or 10% fetal bovine calf serum (FCS). In E3WE cultures on poly-Llysine the primary neuronal growth patterns are aggregation with neuritic fasciculation, growth cones with microspikes, and very few flat cells. In contrast, in cultures on collagen or laminin, the distinctive growth pattern is one of extensive networks of isolated and differentiated neurons lying on acquired monolayers of flat cells. If 5% FCS is used, aggregates are fewer and smaller on poly-L-lysine:on collagen or laminin a tendency to aggregate has been noted. Several differences were observed between cultures derived from E3H cells and cultures from E3WE cells: (1) aggregates are less numerous, with the prevailing pattern being a weblike aggregate; (2) aggregates connecting with other aggregates or flat cells are rare and the aggregate adhesivity is minimized: (3) neurons on collagen or laminin form networks with the exception of a few, small aggregates, which do not display fasciculation; (4) flat cells do not form a monolayer but rather islets, which host the neuronal meshy networks.
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The fact that the poly-L-lysine coating favors aggregation whereas collagen and laminin promote cell differentiation strongly supports the view that the substratum, together with the presence of nonneuronal cells, plays a key role in this growth pattern. As discussed later, nerve cells are dependent for their survival, growth, and maintenance on the availability of specific trophic (growth) factors provided from various cells (see several reviews in Perez-Polo et al., 1983). In view of the fact that on the poly-L-lysine substratum proliferation of flat cells was inhibited, it may be suggested that fewer nonneuronal factors were present in the culture microenvironment. In addition, factors provided by the serum in the medium may not have been sufficiently attached to the poly-L-lysinesubstratum, a possibility supported by the observation that isolated neurons were present in cultures derived from E3W and grown in 10% FCS and virtually absent from similar cultures grown in the 5 % FCS. In contrast to poly-L-lysine, collagen and laminin favor the adhesion, growth, and proliferation of nonneuronal cells, probably resulting from factors in the serum (through attachment of growth factors to the substrata and through direct trophic action) and the adhesive cell-substratum interactions; the available nonneuronal cell surfaces and factors facilitate and accelerate neuronal adhesion and differentiation and inhibit aggregation and fasciculation. In agreement with Letourneau (1975a,b),we also found neuronal differentiationto be related to neuronal adherence. Neuronal adherence was pronounced on nonneuronal cell surfaces, less on collagen or laminin, and minimal on poly-L-lysine, where neurons formed aggregates and bundles rather than growing on this artificial substratum. Also, the adherence of neuronal aggregates and their processes on poly-L-lysine appeared to be determined primarily by the presence or the absence of flat cells and the serum content. We have concluded from these findings that substrata, nonneuronal cells, and factors in the serum are essential in modulating the outcome of the primary neuronal growth in culture and that all three elements mutually interact. These interplays are schematically represented in Fig. 2 and are as follows: (1) Substratum influences the neuronal growth directly, by allowing or preventing the proliferation and differentiation of nonneuronal cells or by allowing attachment of factors from serum. (2) The influence of nonneuronal cells is mediated by cell-cell contact, cell-secreted trophic factors, or by conditioning the substratum with factors. (3) The influence of serum content consists of supplying trophic factors for neurons and nonneuronal cells and of conditioning the substratum. (4) Neuronal factors also influence glial cells, as will be discussed later.
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Nonneuronal
I
Cell
(Surface/Factors)
,L,'..'-.;1 Substratum
b
NEURONAL
FIG. 2. Schematic representation of interactions influencing primary neuronal growth. (From Mangoura et al., 1988.)
The work by Fallon (1985a,b) has contributed further evidence on a neurite-glial cell surface interaction. Fallon has compared outgrowth of neurites from sympathetic and spinal sensory ganglia explants grown onto preformed monolayers of astrocytes derived from rat cerebral hemispheres, Schwann cells, or skin fibroblasts. This study confirms the prevailing view that peripheral nervous system (PNS) neurites grow on both glial and nonglial cell monolayers, but that glial cells are the preferred substrate. That factors associated with the glial cell surface are responsible for their superior neurite outgrowth-promoting effects was implied by (1) the rapid and finely fasciculated neurite outgrowth on the upper surface of the cells, (2) the distinctive morphology of the growth cones characteristic of growth on a highly adhesive substrate (Letourneau 1975a,b, 1979; Bray, 1982), and (3) the lack of effects by conditioned medium obtained from astrocytes. In contrast, the slow and fasciculated outgrowth of PNS neurites on fibroblast and Schwann cell monolayers was markedly different from that seen on glia cells. In a recent study, Lauder and associates (Lauder et al., 1982; see also Lauder and McCarthy, 1986) have reported a neuron-glia interaction using cocultures of dispersed transmitter-identified neurons (serotonin, dopamine) and monolayers of astrocytes or fibroblasts. As shown in Fig. 3, 5-hydroxytryptamine (5-HT) immunoreactive (serotonergic) neurons from raphe nuclei and tyrosine hydroxylase (TH) immunoreactive (dopaminergic) neurons from the substantia nigra are more highly differentiated when grown on astrocytes (compared to their essentially bipolar configuration) than when grown on fibroblasts. Using immunocytochemistry and high-affinity uptake of 3H-labeled transmitters, these authors found evidence of trophic influences of such neuron-glia interactions on both the morphological and biochemical differentiation of these monoaminergic neurons (Lieth et al., 1983). The enhanced morphological differentiation of monoaminergic neurons when
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FIG. 3. Morphological differentiation of 5-HT-immunoreactive (serotonergic) neurons from raphe (A and B) and TH-immunoreactive (dopaminergic) neurons from substantia nigra (C and D) of the El4 rat embryo grown in dispersed cell culture on a monolayer of postnatal astrocytes (A and C) or normal rat kidney fibroblasts (B and D).' Note that both types of monoamine neurons are much more highly differentiated when grown on astrocytes (A and C) compared to their essentially bipolar configuration when grown on fibroblasts (B and D), where they remain associated with clumps of embryonic cells or stretch out between clumps on what appear to be radial-like glial cells. Cultures have been double immunostained with antiglial fibrillary acidic protein (anti-GFAP) and either anti-5-HT (A and B) or anti-TH (C and D). X340. (From Lauder and McCarthy, 1986.)
grown on astrocytes as compared to fibroblasts appears to be reflected in their increased ability to take up their own 3H-labeled transmitters. Proschiantz and associates (see review Proschiantz, 1987) have demonstrated that glial cells from two different brain regions have distinct properties that may define neuronal polarity. When mouse embryonic mesencephalic dopaminergic (DA) neurons (embryonic day 13) are grown on monolayers of striatal flat astroglial cells, they adopt after 2 days in culture a simple morphology, the cell body being surrounded by several
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short neurites with the exception of one that is very long (up to 20 times the cell body diameter), thin, and lacking branch points; on mesencephalic flat astrocytes, this neuronal population can still be observed, but it only accounts for one-third of the cells. The other cells are more symmetrical, with several branched and varicose neurites. These observations contribute to the concept held by several investigators, including myself, of regional differences, the so-called regional specificity, of neuron-glia interactions. As also discussed in other sections, the identity of the glial cell surface molecules which may mediate the interaction with neurites remains unknown. One candidate is laminin, a component of the extracellular matrix which we, as well as others, have found, is a highly effective substrate for neurite outgrowth (see references in Mangoura et al., 1988) and is found on the surface of cultured Schwann cells (Cornbrooks et al., 1983). As mentioned, it is also possible that the neural cell adhesion molecule, N-CAM (Edelman, 1983a,b; Rutishauser, 1984), which has been reported to be a surface component of some astrocytes (Alliot and Pessac, 1984), or Ng-CAM (Grumet and Edelman, 1984) may be involved in these interactions. Another possibility that has been considered is that glial cells secrete a neurite-promoting factor which in turn binds to their surface (surface-bound molecules) and subsequently interacts with the neurite (for further discussion, see Section II,A, 3). b. Glia-Secreted Factors. In 1928 Ram6n y Cajal put forward the hypothesis that nonneuronal cells play an important physiological role in the trophic support of neurons. Later Hyden and associates (Hyden and Lange, 1962; H y d h et al., 1958) pioneered the concept of a metabolic partnership between glia and neurons. In the last 15 years the search for factors secreted from glial cells has been extensive. Several glial cell populations of peripheral and central nervous system (CNS) origin have been investigated and appear to produce neuroactive substances. Some of these factors have been shown by a variety of methods to be nerve growth factor (NGF) (Burham et al., 1972; Longo, 1978; Murphy et al., 1977; Perez-Poloet al., 1977; Reynolds and Perez-Polo, 1975; Schwartz et al., 1977; Varon et al., 1974; Varon and Somjen, 1979), whereas others do not interact with antibody against NGF or are capable of influencing NGF-insensitive cell populations (Banker, 1980; Barde et al., 1978; Ebendal and Jacobson, 1975; Helfand et al., 1978; Heumann et al., 1977; Lindsay, 1979; Lindsay et al., 1982; Monard et al., 1973, 1975; Schurch-Rathgeb and Monard, 1978). Arenander and de Vellis (1980, 1981a,b)have identified a glial-released protein obtained from clonal glial cells which is capable of altering morphology of both PC12 and PCG2 cells. In 1973 Monard et al. reported
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that cultured glioma cells release a macromolecular factor which promotes neurite outgrowth in neuroblastoma cells. Recently, these investigators (Monard, 1985; Monard et al., 1983) have presented evidence that inhibition of cell surface-associated proteases is involved in the gliainduced neurite extension in neuroblastoma cells. They have purified and characterized a 43-kDa glia-derived protein with both promoting activity and serine protease inhibitory activity. The glia-derived protein is able to inhibit the plasminogen activator activity associated with the neuroblastoma cells at a very low (10 -loM) concentration. Increased plasminogen activator activity has been associated with cell migration (Krystosek and Seeds, 1981a,b). It has been suggested that the protease inhibitor released by glial cells would modulate the extent of neuronal migration and also cause neuronal cell surface modifications which would be more favorable to the initial, target-independent phase of neurite outgrowth. Recently, Muller et al. (1984) and Varon and associates (see details in Manthorpe et al., 1986; Varon et al., 1987) have reported that serumfree medium conditioned over astroglial cells contains low-molecularweight ( < 1000) agents which will support the survival of cultured embryonic chick and rat CNS (but not PNS) neurons in the absence of recognized neurotrophic factors. Selak et al. (1985) found that (1) primary astroglial cells release pyruvate into their conditioned medium, (2) pyruvate added to unconditioned medium will equally support the survival of CNS neurons, and (3) pyruvate was the critical component within astroglial cell-conditioned media responsible for supporting CNS neuron survival. In subsequent studies, Beckh et al. (1987) separated neurotrophic factors with distinct biological activities from serum-free medium conditioned by rat brain primary astroglial cells. The biological activities of these factors include a survival-supporting low-molecular-weightneurotrophic activity which can be mimicked by pyruvate, a high-molecular-weightprotein which has neurite-promoting activity allowing rapid differentiation and which shares antigenic determinants with Puminin, and an as yet unidentified soluble component of conditioned medium which promotes neurite elongation and survival. 3. Influence of Glial Cells on Neuronal Migration and Guidance The migration of the postmitotic granule cell has served as a unique model to study the general issue of cell migration in the developing central nervous system. The cerebellar cortex contains only five neuronal types and most of their processes are distributed in geometrically rigid patterns. The several classes of cerebellar cortical cells arise at different times and places but eventually occupy a common territory. Thus, the
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differences in origin of the cell types have allowed the study of welldifferentiated cells in direct physical contact with newly generated ones. The migration of the postmitotic neurons from the external granular layer through molecular and Purkinje cell layers to the (internal) granular layer is well established from light microscopic observations (Ram6n y Cajal, 1911), autoradiography (Altman, 1969; Fujita et al., 1966; Miale and Sidman, 1961; and others), and electron microscopy (Larramendi, 1969; Mugnaini, 1969; Mugnaini and Forstronen, 1967; Rakic, 1970). In 1971 Rakic published a classic study on the relationship of the migrating granule cell to the Bergmann glial fibers in the rhesus monkey cerebellum. The close association of Bergmann fibers and granule cells was first noted by Retzius in 1894 (reference in Rakic, 1971) and later by Mugnaini and Forstronen (1967), Rakic (1970), and Rakic and Sidman (1970). The cytology of the postmitotic migratory granule cell and its relationship to Bergmann glial processes was examined with Golgi staining and electron microscopy in the three cardinal planes in the developing cerebellar cortex of Mucucus rhesus at various late fetal and early postnatal ages (Rakic, 1971). During the entire course of their migration across the molecular layer, granule cells are directly apposed to vertically oriented Bergmann fibers belonging to the Golgi epithelial cells (a type of protoplasmic astrocyte, radial glia). Numerous electron-lucent beaded enlargements were noted along the fiber, except at sites in which the surface was in contact with migrating cells. Lamellae expansions also were noted to project from the main shaft of the glial fiber and envelop the synaptic sites on spines of the Purkinje cell dendrites. Rakic stated that this neuron-glia relationship provides the necessary conditions for the migration of the young granule cell. As illustrated in Fig. 1 (Rakic, 1972), a young neuron setting out for the cortex has a bipolar shape, with a large leading process measuring less than 200 pm in length. The radial glial fiber provides a guide similar in principle to that available at earlier stages from the simple columnar organization of the neural tube. Strong surface affinity between radial glial fibers and migrating neurons is indicated by the fact that neurons remain apposed to, and follow precisely the course of, glial fibers even in regions where radial glial fascicles assume irregular, curved courses between ventricular zone and pial surface (Rakic, 1972, 1978). In 1974 Henrikson and Vaughn further advanced the concept of the neuron-glia relationship in a study of neurites and radial glia processes in developing mouse spinal cord. They describe three progressive patterns of neurite-glia relationships: a minimal complex involves a simple apposition of dendrites and dendritic growth cones to radial glia processes; a somewhat more specialized neurite-glia association is marked
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by the occurrence of puncta adhaerentia between neurites and radial glial processes; finally, the most elaborate morphological relationship observed is that of synapselike contacts between axons and radial glial processes (these synapselike structures, which are not seen after embryonic day 15, will be discussed later (see Section 11,A). Recently, Gadisseux and Evrand (1985), using particulate glycogen as an histochemical marker for radial glia, have reported some interesting findings from studies of developing mice. The presence of particulate glycogen in glial cells is one of the biochemical, morphological, and functional properties differentiating the glial lineage from most neurons (Borke and Nau, 1984; Bruckner and Biesold, 1981; Peters and Feldman, 1973; Sotelo and Palay, 1968). However, previous attempts to use glycogen as an ultrastructural marker of glial cells in developing nervous systems have been only partially successful (Henrikson and Vaughn, 1974) due to the unpredictable glycogen preservation in glial cells following the classical steps of fixation, embedding, and staining for electron microscopy. Postfixation with reduced osmium (Riemersma et al., 1984) combined with histochemical Thiery staining modified for embryonic nervous tissue has proven to be successful for the ultrastructural analysis of prenatal gliogenesis and neuron-glia relationships during development (Gadisseux and Evrand, 1985). Until embryonic day 16 (E16) in the normal mouse telencephalon, glial cells are always grouped in fascicles along their entire trajectory from the ventricular zone to the marginal layer. At E17, all glial cells appear to be defasciculated and isolated from one another within the cortical plate. The phenomenon of gradual defasciculation of glial fibers in the cortical plate begins at E14. Sequential studies demonstrated a strict parallelism between settling of latemigrating neurons in the cortical plate and glial defasciculation. Combined autoradiographic and glycogen labelings confirmed the constant association of migrating neurons with one or several glial fibers. The optic system has also been used as an anatomical model to examine neuron-target relationships and the role of glial cells. Silver and associates (Silver, 1984; Silver and Sidman, 1980; Silver et al., 1982) have been examining the involvement of astroglial cells in defining the routes of growing axons in the developing CNS. They were particularly interested in defining the forces that dictate how large groups of axons arriving at a known branch point could be separated into discrete bundles which then travel in different directions. They described (Silver and Sidman, 1980) the role of Muller cells (the counterparts of astroglia in the retina) in offering favorable growth surfaces to the mass of retina ganglion cell axons as they emerge from the undifferentiated mouse optic cup at E11.5-13 and are segregated into discrete portions of the optic
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stalk. After the optic axons are at the optic chiasm, they come into contact with two distinct primitive glial cell structures: one consists of tightly packed cells and seems to act as the “antithesis of a channel” by forming a barrier and directing all ventronasal fibers contralaterally into the opposing stalk; the other glial structure is a localized “pathway” of glial processes separated by large extracellular spaces: this serves to guide all ventrotemporal axons ipsilaterally toward the thalamus. Thus, the glial cells are again portrayed as mechanical guides to orient growth (Silver, 1984). In another report Silver et al. (1982) described how at E17, about 2 days before the appearance of callosal pioneer axons at the cerebral midline, a population of astroglial cells (identified by their possession of glycogen granules and intermediate filaments) forms a dense bridgelike structure or “sling” suspended below the longitudinal cerebral fmure and attached at the two ventricles. By E19, the incoming callosal fibers grow along the surface of this bridge and travel contralaterally. In contrast, the anterior commissural fibers are guided in a different direction by an aligned system of glial processes with large extracellular spaces. The glial bridge will disappear postnatally. The same group has further reported (Silver and Ogawa, 1983) that at El6 a small piece of Millipore filter could be placed in the location and with the orientation of the original tissue sling, after surgical transection of the latter. By 9-14 days postimplantation (P4-9), the Millipore filter had become covered with astroglial cells and a variable, occasionally massive axonal growth had emerged from the preexisting neurons and grown well into the contralateral hemisphere. The implication here again is that neuroglial cells are providing cues to developing axons. As will be discussed in another section, this phenomenon is also related to the reactive synaptogenesis described by Cotman and Scheff (1979), in which again the glial cells may play a variety of roles. Recent in vitro studies have provided some cellular clues to the problem of neuronal migration and the role of glial cells. As discussed earlier, we have been using cultures derived from 3-day-old whole chick embryo (body and head) as a model to study cell-cell relationships during early neuroembryogenesis (Mangoura et al., 1988; Vernadakis et al., 1986). The characteristic growth pattern of neuronal aggregation and neuyite fasciculation was noted when the dispersed cells were plated on a polylysine substratum. An unusual growth pattern, shown in Fig. 4 (from Vernadakis et a l . , 1986), was a long neurite process aligned with a continuous chain of interconnected cells. These cells were not stained positive for cholinesterase using the Karnovsky and Roots method (1964). We are proposing two possibilities for this cellular phenomenon: (1) these nonneuronal cells are Schwann cells beginning to ensheath an axonaltype neurite or (2) these cells are radial glial cells guiding the neurite.
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FIG. 4. Aggregates formed from cells dissociated from a 3-day-old chick embryo and plated on poly-L-lysine-coatedculture dishes, 4 days in culture. (a) Aggregate with long radiating neuritic outgrowth; arrowheads indicate possible contacts of the extending neurites with isolated flat cells. Bar, 71 pm. (b) Aggregates sharing an elaborate network of interconnected neurites. One of the aggregates also exhibits an independent circular pattern of outgrowth. Bar, 74 pm. (c) Aggregates interconnected with a long neuritic process that appears to be ensheathed by a continuous chain of interconnected cells (arrow, Schwann cells?). Bar, 71.6 pm. (d) Another type of aggregate exhibiting a less interconnected outgrowth consisting of single neurites. It appears that the diameter of the core of the aggregates is analogous to the density and the degree of organization of the outgrowth. Bar, 44.8 pm. (From Vernadakis et al., 1986.)
Hatten and Liem (1981), Hatten (1984), and Hatten et al. (1984a,b), using cultures derived from cerebella of mice 5-7 days after birth, have described cell migration along the arms of Bergmann-likeastroglia; most neurons associated with astroglia were granule neurons. In contrast to neurons observed in other studies, neurons did not migrate along the arms of the stellate astroglial forms but rather remained in place and
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extended growth cones. In Fig. 5 (Hatten et al., 1984a), two populations of stained astroglia are observed. The first, constituting -10-20% of stained cells present in the cultures, had a smaller cell body and thinner, longer processes; several unstained neuronal cells bound to the arms of these astroglia distal to the cell body. On the basis of their shape and pattern of association with neurons these cells were identified as Bergmann-like glia (Rakic, 1971) (Fig. 5a,b). A second type of AbGFpositive cells (AbGf, glial filament protein) has a slightly larger cell body and stellate, shorter process (Fig. 5c). Among the arms of these astroglia were nestled several dozen unstained neurons with very fine short processes. These cells resembled astrocytes of the cerebellar granular layer as well as those of the white matter (Palay and Chan-Palay, 1974). When cells were cultured from the neurological mutant weaver, a mouse that suffers failure of granule neuron migration in concert with abnormalities in the shape and alignment of the Bergmann glia (Rakic and Sidman, 1973; Sotelo and Changeux, 1974), three findings emerged. Very few granule cells survived and little neurite outgrowth was seen, the morphology of the astroglia was abnormal, and neuron-glia associations were mostly absent. “Mixing and matching” neurons and astroglia purified from weaver and normal mice demonstrated (Hatten and Mason, 1986) that weaver neurons fail to migrate on wild-type astroglial processes in zrilro and that they impair astroglial differentiation; in contrast, normal neurons associate with weaver astroglia, inducing their differentiation and forming tight appositions like those seen in migrating neurons in vivo. These studies suggest that the granule neuron may be a primary site of action of the weaver gene and further demonstrate that neuron-glia interactions may regulate astroglia differentiation.
FIG. 5 . Specific associations of cerebellar neurons with two different forms of astroglia. After 48 hr in Vitro, glass coverslip microcultures of cerebellar cells harvested from mouse cerebellum at postnatal day 7 were stained with AbGF. (a) Several dozen unstained cells nestled at or near branch points of the processes of stained cells with a stellate morphology (A). Other stained cells (B)extend longer (150 pm) glial processes (gp)against which several unstained, phase-bright cells are apposed; one unstained cell resembles a resting neuron (m), another a migrating neuron (mn). X 348. (b) The glial process of cell B shown in (a), at higher magnification. The thickened leading process of the cell resembling a migrating neuron (mn) is contacting the stained astroglialprocess (gp).The end foot (ef)of the astroglial cell is densely packed with stained glial filaments, is adjacent to the processes of the stellate astrocyte A, and appears to end on an unstained flat cell. X 868. (c) Stained astroglia with stellate morphology (A) that bind a number of presumed neurons (n) and constitute the major astroglial cell form present in the culture. X868. Reproduced from Hatten et al.,J. Cell Biol. 98, 193, 1984a by copyright permission of the Rockefeller University Press.
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B. INFLUENCE OF NEURONS ON GLIALCELLS 1. Growth and Dfferentiation of Glial Cells
a. Schwann Cells. Although considerable work has been focused on the influence of glial cells on neuronal growth and function, attention is also beginning to be directed to the role of neurons in the growth and differentiation of the glial cell. A large body of evidence has been derived from studies of Schwann cell-axonal interactions and has been elegantly reviewed recently by Bunge and Waksman (1985), Bunge and Bunge (1986), and Ratner et al. (1986). Thus, only a few key observations will be summarized here. Schwann cell proliferation appears to be regulated by axonal contact. This is suggested both by in metrofindings that Schwann cell proliferation is dependent on a mitogenic signal provided by growing neurons (McCarthy and Partlow, 1976a,b;Wood and Bunge, 1975), and by in vivo observations that Schwann cell number in nerve trunks is proportional to the number of nerve fibers present (Aguayo et al., 1972). Studies using pure populations of sensory neurons and Schwann cells (Wood, 1976) have demonstrated that a mitogenic signal can be provided to Schwann cells by intact bare sensory axons (Salzer and Bunge, 1980; Wood and Bunge, 1975), that cell-cell contact is required (Salzer et al., 1980a,b), and that a microsomal fraction derived from cultured neurites can substitute for the intact axon (Salzer et al., 1980a,b). It has also been reported (De Vries et al., 1982) that axolemma-enrichedfractions isolated from myelinated axons of rat and bovine CNS and PNS will stimulate a quiescent population of cultured rat Schwann cells to proliferate. The mitogen in the axolemma-enriched fraction is not lost in the extensive washing used to isolate the membrane fraction and is sensitive to heat and trypsin, suggesting that it is an integral membrane protein or glycoprotein. Lemke and Brockes (1984) have reported that cultured rat Schwann cells are stimulated to divide by a protein growth factor present in extracts of bovine brain and pituitary which they named glial growth factor (GGF). Two lines of evidence indicate that GGF activity in both brain and pituitary resides in a protein: the ability of a set of four monoclonal antibodies both to precipitate GGF activity specifically from solution and to bind for the association of mitogenic activity with this molecule. The authors also claim from their results that GGF is functionally different from purified human platelet-derived growth factor, a molecule similar to GGF. However, the relation of GGF to the cell surface mitogen reported by De Vries et al. (1982) and by Salzer and Bunge (1980) remains to be established.
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In a review Ratner et al. (1987) discussed some cellular and molecular mechanisms that may underlie the response of the Schwann cell to contact with an axon: mitogen-receptor response, mitogen internalization (i.e., phagocytosis by Schwann cells of mitogenic membrane fragments), endocytosis as a necessary step in cell division, and metabolic transfer. b. Astrocytes. Several growth factors which are mitogenic for various cell types have also been reported to stimulate the proliferation of astroglial cells in primary cultures: fibroblast growth factor (Morrison and de Vellis, 1981, 1983; Morrison et al., 1982; Pruss et al., 1982), epidermal growth factor (Simpson et al., 1982; Westermark, 1976), and human platelet-derived growth factor (Heldin et al., 1977). Lim and associates have isolated a glia maturation factor (GMF), an acidic protein, from adult bovine brains. This protein stimulates the proliferation, phenotypic expression, and chemical differentiation of cultured brain astroblasts (Bosch et al., 1987; Lim, 1985; Lirn and Miller, 1984; Lim and Mitsumobu, 1974; Lim et al., 1973, 1976, 1977a,b) and triggers the release of interleukin-1 and prostaglandins from astrocytes (Fontana et al., 1983). Lim and associates have also reported that GMF stimulated the proliferation of type 1 astrocytes (described by Raff et al., 1983a,b), but GMF did not convert type 1 to type 2 astrocytes in either optic nerve- or brain-derived glia cell cultures. In addition, GMF stimulation of type 1 astrocytes derived from secondary fetal brain cell cultures resulted in an increase in A2B5 -positive progenitor cells (described by Raff and colleagues, see Miller and Raff, 1984; Miller et al., 1985; Raff et al., 1983a,b, 1984a,b). Since GMF is ubiquitous in brain tissue, it seems likely from these findings that GMF may play an important role in glial development. An astroglia growth factor (AGF) has been partially purified from bovine brain (Pettman et al., 1980, 1983). The addition of AGF to rat astroblasts cultured in a serum-containing medium or in a defined medium stimulates the proliferation of the cells (Pettman et al., 1982, 1983; Sensenbrenner et al., 1982a,b; Weibel et al., 1983). The AGF also induces a morphological change: the flat polygonal-shapedcells develop cytoplasmic processes and acquire a starlike appearance, resembling mature astrocytes (shown in Fig. 6c). In serum-free defined medium, this modification is even more pronounced and most cells have a very fibrous appearance (shown in Fig. 6d). We have been examining the influence neuronal constituents may exert on the growth of nonneuronal elements and in particular on glial cells using various culturing systems and cell populations: cells derived from dissociated adult rat spinal cord, glia-enriched cultures from newborn rat cerebral hemispheres, neuron-enriched cultures from
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FIG. 6. Two-week-oldrat astroglial cell cultures. (a and c) Cells were cultured in Waymouth's medium plus 10% fetal calf serum (a) and AGF (50 ng/ml) ( c ) . (b and d) Cells were cultures for 4 days in serum-supplemented Waymouth's medium, then for 10 days in Waymouth's medium supplemented with insulin (5 pg/ml), bovine serum albumin (0.5 pg/ml) (b), and AGF (50 ng/ml) (d). Bar, 50 pm. (From Sensenbrenner et al., 1986.)
newborn rat cerebral hemispheres, neuron-enriched cultures from chick embryo whole brain, and glia-enriched cultures from chick embryo cerebral hemispheres (Sakellaridis et al., 1984). We used the glial biochemical marker glutamine synthetase (GS) for astrocytes (Martinez-Hernandez et al., 1977; Norenberg and Martinez-Hernandez, 1979) and 2',3 '-cyclicnucleotide 3 '-phosphohydrolase (CNP) for oligodendrocytes (Poduslo,
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1975; Poduslo and Norton, 1972). We were interested in determining whether the growth of glial cells prepared from newborn rat cerebral hemispheres would be affected by the cell population derived from adult spinal cord; whether the growth of glial cells derived from chick embryonic brain would be influenced by being plated onto a neuronenriched substratum: and whether the addition of nervous tissue-derived cell suspension plated on an established glia-enrichedculture would affect glial growth. Glial cells responded differently when the cell substratum was gliaenriched versus neuron-enriched. The activity of GS was significantly reduced in co-cultures established by plating a suspension of cells dissociated from 15-day-oldchick embryo onto neuron-enriched living cell substratum from 6-day-old chick embryo. In contrast, GS activity was enhanced in co-cultures established by plating a suspension of cells dissociated from 6-day-old chick embryo onto glia-enriched living substratum from 15-day-oldchick embryo. Similar results were obtained using cell cultures from nervous tissue. The CNP activity was enhanced in the co-cultures. These findings demonstrate that astrocytes and oligodendrocytes appear to respond differently to the co-culture conditions. The variable in these co-cultures was the constituency of the cell population in the established cell substratum. These findings support our proposal that neurons exert a glial trophic role mediated either through the microenvironment or through direct cell contacts and that this trophic effect can be generated by intact neurons or by products liberated after neuronal injury. To further examine whether factors secreted from neurons into the microenvironment could influence the growth of glial cells, established glial cell cultures from 15-day-oldchick embryo cerebral hemispheres were exposed to different concentrations of medium conditioned by a neuron-enriched cell population from 6-dayold embryos (Sakellaridis et al., 1986). In our paradigm we viewed the input of culture medium as corresponding to the input of the microenvironment in the CNS. Thus, we attempted to simulate the input of the neuronal component in the microenvironment by adding neuronconditioned medium (NCM) to the glia-enriched cultures. In addition, the responsiveness to the glial cells cultured in NCM to medium containing various concentrations of fetal calf serum (FCS), the classical growth nutrient of culture medium, was examined. Addition of NCM to the cultures retarded the maturation of the glioblasts, and immature epithelioid cells were present up to day 9, when cultures were harvested. This effect was not negated by the presence of 10% FCS and was not responsive to the final concentration of the NCM alone. In contrast to the low GS activity in the cultures with NCM alone, CNP activity not only was not decreased but, in the presence of 5% NCM, CNP was
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dramatically increased. We interpret these findings to mean that epithelioid cell glioblasts are in a “transitional” stage in these cultures and thus may contain high CNP or factors in the NCM are beneficial to the expression of oligodendrocytic properties under our experimental conditions. The latter possibility supports the view of a neuronoligodendrocyte interaction (Bologa et al., 1982a),which will be discussed in the following section. The most intriguing aspect of these findings is the differential response of the oligodendrocytesand astrocytes to NCM. This phenomenon is further indication of the pluripotentiality (pluripathology) of the glioblastic cells and suggests that the presence of neuronal factors is capable of shifting the balance from astrocytic to oligodendrocytic cell expression. This view is consistent with the findings of Choi et al. (1983) that the radial glia cells give rise to both astrocytes and oligodendrocytes and the in mlro and in mvo studies of Raff and associates of a common progenitor cell for both astrocytes and oligodendrocytes (Raff et al., 1983a,b). In contrast to our findings, Hatten (1985) has found a differentiationpromoting influence of neurons on astroglia. She examined neuronal regulation of astroglial morphology and proliferation using purified cerebellar neuronal and astroglia co-cultures from mice. She reported that in the absence of cerebellar neurons, purified astroglia lost their characteristic appearance, flattened, and proliferated. However, in the presence of neurons, the flat astroglia assumed a shape characteristic of astrocytes and interacted with granule neurons. The author further described that removing the neurons from the astroglia appeared to direct the immature, partially differentiated, early postnatal cerebellar astroglia to a more primitive flat form described by Raff et al. (1983a,b). Based on additional studies Hatten and associates (Bovolenta et al., 1984; Hatten and Liem, 1981), suggested that a discrete series of steps might be required for the expression of astroglial shape and that an interruption in the sequence, or simply a loss of cell association with neurons, leads to a reversion to immature forms. In view of our findings and those of Hatten and associates, it is evident that the responsiveness of astroglial cells in culture is dependent on the animal species and brain area from which glial cells are derived, the maturational stages of the astrocytes at the time of explantation, the in m‘tro conditions, and finally, the criteria used to define astrocyte differentiation. Future studies focusing on this specific cell interaction are warranted in order to characterize the neuron-astroglia interrelationship. c. Oligodendrocytes. As discussed in another section, oligodendrocytes play an important role in the central nervous system, where they produce the myelin which is essential for the normal function of neurons
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(Bunge et al., 1962). The question, however, of whether oligodendrocytes are able to proliferate has generated numerous studies with varying results. The conflicting reports may originate from the criteria used to identify oligodendrocytes. The recent discoveries of specific antigenic markers for oligodendrocytes have enabled investigators to begin clarifying the complexity of this cell and its regulation. Evidence derived from in vitro studies is indicating that various factors, but particularly neurons, play a key role both in the proliferation of oligodendrocytes and myelin formation. Indirect evidence supporting the notion of neuron-oligodendrocyte interaction was first derived from histological studies showing that the production of myelin by oligodendrocytes begins when axons reach a certain diameter (Fraher, 1978; Matthews and Duncan, 1971). It has also been shown that the absence of healthy axons (following Wallerian degeneration) leads to an inhibition or interruption of oligodendrocyte differentiation (Fulcrand and Privat, 1977). Bologa and associates have extensively investigated proliferation and differentiation of oligodendrocytes in culture. In cultures of embryonic mouse brain, oligodendrocytes express at an early stage both galactocerebroside (GC) and myelin basic protein (MC), two antigenic markers (Raff et al., 1978; Sternberger et al., 1978), whereas in cultures of neonatal mouse brain, oligodendrocytes differentiate only later and at low rates from GC-positive cells only into cells positive for both GC and MBP (Bologa et al., 1981). In both these culture systems, the appearance, quantity, and developmental pattern of astrocytes, which were identified with glial fibrillary acidic protein (GFAP) (Bignami and Dahl, 1977) was shown to be similar (Bologa et al., 1982a,b). In contrast, the appearance, number, and development of neurons, which were identified with neuron-specific enolase (NSE) (Marangos et al., 1975), was different in embryonic and neonatal mouse brain cell cultures. Although neonatal mouse brain cell cultures are very poor in neurons, in embryonic cultures many NSE-positive neurons are present. Moreover, it was noted that sometimes NSE-positive cells are located on the border or on top of round NSE-negative clusters (presumably oligodendrocytes), indicating a possible interrelationship between these two types of cells (Bologa et al., 1982a,b, 1983). Based on these findings Bologa and associates speculate that neurons are involved in the enhancement of the ability of the oligodendrocyte to differentiate and to express myelin-related components in culture. As discussed earlier, we (Sakellaridis et al., 1986) also found that in glial-enriched cultures from 15-day-oldchick embryo cerebral hemispheres addition of neuron-conditioned medium in the cultures enhanced
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the activity of CNP. Recently, Edgar and Pfeiffer (1985) have confirmed our findings. They have reported that both a soluble and a particulate fraction from chick neuronal cultures contain factors that stimulate the proliferation of oligodendrocytes in rat brain cultures enriched for these cells, as assessed by CNP activity. 2. Neuronal Signals and Glial Cell Metabolic Responses
Neuron-glia relationships were described by Kuffler and Potter (1964) in their early studies on the leech. Extensive evidence (discussed also in a later section) now exists that glial cells are directly involved in K+ homeostasis (Orkand et al., 1981; Pentreath, 1982; Pentreath and KaiKai, 1982; Walz, 1982; Walz and Hertz, 1982, 1983a-c; Walz and Schlue, 1982; Walz et al., 1983, 1984). Neuronal activity leads to increases in extracellular K , which depolarizes the glial cell membrane (Kuffler and Nicholls, 1966, 1976; Sykova, 1983; Varon and Somjen, 1979). The potassium is eventually restored to the neurons, but in the short term a significant proportion enters the glial cells (Gardner-Medwin, 1983a,b; Nicholson, 1980). The potassium signal stimulates energy metabolism in the glial cells, which has been recorded in a number of different ways: as increased oxygen and glucose utilization (Hertz, 1978a,b), decrease in ATP (Schousboe et al., 1970), and altered levels of a pyridine nucleotide (NADH) (Orkand et al., 1973). Energy coupling between neurons and glia was proposed by H y d h several years ago on the basis of his studies with hand-isolated cells (HydSn and Lange, 1960, 1962). If neurons are stimulated (e.g., retinal ganglion cells by light, Deiter's neurons by rotation of the animal) and single neurons and clumps of glial cells from around those same neurons are analyzed, properties not different at equilibrium now display very different behaviors in the two cell types; oxygen consumption rises and COe production declines in the stimulated neurons, whereas the opposite changes take place in the glial cells. In a recent report Pentreath et al. (1987) has presented evidence that glycogen in glial cells in the leech segmental ganglia is controlled, at least in part, by cues that are released from neurons and, moreover, that K + ions appear to be particularly important. Studying [3H]glucoseand 2-deo~y[~H]glucose incorporation into glycogen in isolated ganglia, they found that electrical stimulation increases uptake of these molecules by 120%. The major site of glycogen incorporation for both substances are the packed glial cells at the edges of the ganglia, and autoradiographic studies have also demonstrated that the increased incorporation produced by antidromic stimulation is principally within the glial cells. Marked increases in the uptake and incorporation of both +
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2-deo~y[~H]glucose and [SH]glucoseinto glycogen were produced with elevated glucose and potassium in the incubation medium. The authors suggest that the glycogen changes may be subsequent to, or represent the cost of, an active uptake of K + by glial cells, or that the changes may follow K + entry into the glial cells via a spatial buffer mechanism. These findings are consistent with the proposal that K + ions released from active neurons have a key role in the regulation of energy metabolism in the glial cells. Neurotransmitters, assumed to be released from neurons, evoke a variety of responses from glial cells and consequently are implicated in glial metabolic function. The following astroglial functions have been reported to be influenced by neurotransmitter substances: (1) enzyme activities and/or levels (Cummins et al., 1983; Kumar et al., 1979; Narumi et al., 1978; Vernadakis and Nidess, 1976), (2) membrane potential (Bowman and Kimelberg, 1984; Hamprecht et al., 1976; Hirata et al., 1983; Hosli and Hosli, 1983), (3) release of macromolecules (Schwartz et al., 1977), (4) cyclic AMP-dependent protein phosphorylation (Browning and Sanders, 1981; McCarthy et al., 1985), (5) phosphinositide metabolism and calcium mobilization (Masters et al., 1984), and (6) cell morphology (Kimelberg and Pelton, 1983; Lim et al., 1973). As will be discussed in other sections, glial cells exhibit neurotransmitter uptake and release of neurotransmitters (reviews by Hertz and Schousboe, 1986; Hosli et al., 1986; Kimelberg, 1986; Massarelli et al., 1986) and express neurotransmitter receptors (reviews by Hamprecht, 1986; Lauder and McCarthy, 1986). For several years we have been interested in the role of steroid hormones in neural function, and have, along with others, suggested that steroid hormones may serve as intrinsic regulators of neural growth (see references in Vernadakis et a l . , 1979a; Vernadakis, 1982). Of particular importance is the influence of hormones and neurohormones on glial cells since glial cells can, as discussed in this review, modulate the microenvironment. We have used various culture systems including organ and organotypic cultures, monolayer cultures, and glioma cells (2B clone) (Vernadakis, 1971, 1981; Nidess and Vernadakis, 1979; Vernadakis and Nidess, 1976, 1979; Vernadakis et al., 1976, 1979a,b, 1980). We have found that the responsiveness of glial cells to neurohormones such as cortisol and norepinephrine is related to the stage of maturation at the time of treatment. For example, measuring protein and RNA synthesis in C-6 glial cells in culture we found that cortisol treatment decreases protein synthesis in mature astrocytes (dibutyryl CAMP-differentiated) but not in glioblastic cells, whereas RNA synthesis is increased following cortisol treatment in glioblasts and decreased in mature astrocytes. In
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another case, we found accumulation of [3H]norepinephrine is lower in mature astrocytes than in glioblasts. The roles of neurotransmitters as signals during early neuroembryogenesishave been reviewed by Lauder (1987) and Kater and Haydon (1987) and include participation in such events as neural tube formation; morphogenetic cell movements in the head ectoderm, otocyst, heart, gut, visceral arches, brain, and palate; cell proliferation and the onset of neuronal differentiation in the neural tube; postnatal germinal cell proliferation; neurogenesis and gliogenesis; and axonal growth and synaptogenesis. It has been advocated by these investigators and several others including ourselves (Vernadakis and Gibson, 1974; Vernadakis, 1981) and Buznikov (see refs. in Buznikov, 1984) that neurotransmitter substances act as signaling devices during early neuroembryogenesis before achieving their status as mediators of chemical neurotransmission and neuronal function in the nervous system. 3 . Myelination Central nervous system myelin is of major importance in the maintenance of normal brain function, and numerous recent reviews have thoroughly described various components of the myelination process (Lees and Brostoff, 1984; Pevzner, 1982; Pfeiffer, 1985; Ritchie, 1984). In this review, I will discuss only those aspects of myelination which demonstrate a neuron-glia interrelationship. In the CNS, myelin is formed as a specialized extrusion of oligodendroglial cell plasma membranes in which individual bilayers interact to form the multicellular structure of myelin. Myelin has a relatively simple composition of major proteins (reviewed by Lees and Brostoff, 1984) including the proteolipid protein (PLP), myelin basic protein (MBP), 2 ',3 '-cyclic-nucleotide3 '-phosphohydrolase (CNP), and myelinassociated glycoprotein (MAG). A number of uncharacterized, quantitatively minor proteins and glycoproteins of myelin and the oligodendrocyte plasmalemma have also been reported (Quarles et a l . , 1979; Schmelzer and Poduslo, 1986). Cell type-specific glycolipid markers for oligodendrocytes include galactocerebroside (GC) and sulfatide (Raff et al., 1978; Ranscht et al., 1982), the predominant glycosphingolipids of myelin. Several studies have been reported in which one or more of those specific markers have been used to define further a neuron-oligodendrocyte relationship. However, the nature of a neuronal signal in the myelin formation process remains unclear. As discussed earlier, Bologa and associates (review, 1985) have been extensively studying the role of neurons and astrocytes in the expression of MBP in oligodendrocytes in cultures derived either from embryonic or neonatal mouse brain. In cultures of embryonic mouse brain, oligodendrocytes differentiate very early into GC-positive, MBP-positive
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cells (Bologa et al., 1982b), whereas in cultures of neonatal mouse brain GC-positive, MBP-negative oligodendrocytes differentiate into GC-positive, MBP-positive cells later and in only a small percentage of cases (Bologa et al., 1981). GFAP-positive astrocytes are similar in both culture systems (Bologa et al., 1982a), but the appearance, number, and development of NSE-positive neurons are different in embryonic and neonatal mouse brain cell cultures (Bologa et al., 1982a, 1983). Moreover, neonatal mouse brain cell cultures are very poor in neurons, whereas in embryonic cultures many NSE-positive neurons are found. Based on these findings, Bologa and associates speculate that neurons are involved in the enhancement of the ability of oligodendrocytes to differentiate and to express myelin-related components in culture. Our studies on the influence of neuron-conditioned medium (NCM) on CNP activity support the idea of a neuron-oligodendrocyte relationship (Sakellaridis et al., 1986). We found that CNP activity, a marker for oligodendrocytes (discussed in an earlier section), increases in cultures derived from 15-day-old chick embryos and grown in the presence of 5% NCM. Since in these cultures the activity of GS, a marker for astrocytes, was decreased, we have hypothesized a shift in glial cell differentiation in the presence of neuron-conditioned medium that favors oligodendrocyte development. Fan and McAlister (1985), using organotypic cultures of newborn rat brains, found that functionally intact neurons were required for the enhancement of the myelination-relatedand glia-predominant enzymatic activities CNP and cGalt (UDPgalactose-ceramide glactosyltransterase). Bradel and Prince (1983) found that in oligodendrocyte-enriched cell cultures the most noticeable feature was the quantity of intercellular membranes, which were shown to be continuous with oligodendrocyte processes. These membranes often exhibited the appearance of “loose myelin” and were therefore not normally compacted. Layers of membrane with the morphological appearance of compact myelin were observed on an occasional oligodendrocyte perikaryon or process. The authors argue that their findings necessitate a reevaluation of the widely held view that oligodendrocytes are not able to elaborate myelin in the absence of neurons. Moreover, these findings are consistent with earlier reports on myelination of numerous nonaxonal structures. For example, myelinated granule cell perikarya have been reported in the normal cerebellum (Rosenbluth, 1966), and myelinated oligodendrocytes have been observed during normal development of the CNS (Hildebrand, 1971; Raine and Bornstein, 1974). Myelinated astrocyte processes (Bignami and Ralston, 1968) and oligodendrocyte perikarya have been described in a variety of pathological conditions (Fulcrand and Privat, 1977).
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Zeller et al. (1984, 1985) using molecular tools have further examined the role of the neurons in myelin protein synthesis. They have recently isolated a cDNA clone of mouse MBP, using a family of synthetic oligonucleotides as a primer for cDNA synthesis (Zeller et al., 1984). The sequence of this clone (NZ-112)revealed that it corresponds to a region of mRNA coding for an amino acid sequence present in all forms of MBP in the mouse. The nucleotide sequence of the clone shows 95% homology with that published for the rat MBP mRNA (Roach et al., 1983) and, therefore, can be used to detect the MBP mRNA species synthesized in either mouse or rat oligodendrocytes by in situ hybridization techniques. This method is ideal for the study of oligodendrocyte differentiation since it allows one to measure the expression of a particular gene at the level of transcription and to correlate it with the immunofluorescence detection of the protein. Zeller et al. (1985) studied the emergence of MBP mRNA in cultured rat oligodendrocytes. Whether the primary cultures were started from the cerebral hemispheres of 16-day-old fetuses or 0-to 3-day-old newborns, the first MBP-positive cells were detected between days 6 and 9 (postnatal age). This time of emergence corresponded to the time at which MBP was first seen in the brain of normal rats (Sternberger et al., 1978). Using double immunolabeling, they found that MBP staining of the oligodendrocyte processes increased with time, and at 10-13 days postnatal, all GC-positive cells were MBP-positive. These findings agree with reports by others (Mirsky et al., 1980; Pruss et al., 1982; Bologa, 1985). However, MBP-specific RNA could be detected 2 days before the appearance of MBP immunofluorescence when parallel coverslips of primary cultures were analyzed by in situ hybridization and by immunofluorescence. It appears from these findings that MBP, as well as GC, does not appear to depend on continuous signals from neurons. Waxman and associates have extensively examined the development and molecular organization of axonal membrane and its possible relation to glial contact (see references in review by Waxman, 1986). The freeze-fracture method has provided a powerful technique for electron microscopic examination and permits a high degree of spatial resolution of the macromolecular structure of the cell membrane. When viewed by freeze-fracture, the axon membrane displays a dramatic degree of structural differentiation, most evident at the node of Ranvier. These authors have provided evidence that axon membrane structure is modulated locally at the node by paranodal axoglial interactions with the oligodendrocytes. However, in addition to the axon and the myelinforming oligodendrocyte, differentiation of the CNS node of Ranvier appears to involve the astrocyte as well. These authors suggest that dif-
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ferentiation of the axolemma into nodal and internodal domains provides a signal demarcating areas to be covered by myelin-forming cells. Waxman (1986) further proposes the existence of recognition molecules for axoglial association, present perhaps in the axon membrane and possibly in the glial membrane. The recent report by Knapp et al. (1987) on expression of myelinspecific antigens in oligodendrocytesin culture has provided additional evidence that oligodendrocyte shape and membrane production are, in part, regulated from within the oligodendrocyte itself. However, despite the authors’ claim that in their cultures neurofilament-positive staining was gone by 5 days in culture, the possibility of some neuronal interaction between neurons and glia early in the culture period can not be entirely excluded.
111. Neuron-Glia Interactions and Synaptic Events
A. MORPHOLOGICAL EVIDENCE A historical background of morphological observations on the intimate relationship between neuroglia and nerve cells has been summarized by Hydtn and Pigon (1960). In that article, Hydtn and Pigon described the early observations by Chang and Hild (1959), de Robertis and Bennett (1954), Farquhar and Hartman (1957), Hess (1953, 1955, 1958), Holmgren (1899, 1901, 1904, 1914), Lumsden (1955, 1958), Lumsden and Pomerat (1957), Scheibel and Scheibel(l955, 1958), Schultz et al. (1957), and finally, Wyckoff and Young (1956, 1958). Hydtn and Pigon (1960) also described a functional relationship between oligodendroglial cells and nerve cells of the Deiter’s nucleus (see references in Hydtn and Pigon, 1960). Synaptic contacts between neuronal processes and glial cells have been demonstrated during development in uzvo as well as in neural tissue culture. Henrikson and Vaughn (1974), studying mouse spinal cord development, showed axoglial synapses during 11-14 days postnatally. Grainger et al. (1968), Grainger and James (1970), and James and Tresman (1969) described synaptic contacts between neuronal and glial cell processes in chick spinal cord cultures. In the adult nervous system, synapselike contacts between neurites and glial cells seem to be limited to those of the ependymal cells lining the ventricle (Leonhardt and Backhus-Roth, 1969; Noack and Wolff, 1970), and to tanycytes or astrocytes of certain periventricular regions such as the pituitary
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(Wittkowski, 1967), subcommissural organ (Driederen, 1970), medial vascular prechiasmatic gland (Le Beax, 1972), and median eminence (Giildner and Wolff, 1973). Palacios-Pru et al. (1979) have reported in vitro formation of neuroglial synapses in organotypic cultures of cerebellar cortex from 16-day-old chick embryo incubated for 6 days. If the incubation period was prolonged, the number of axoglial synapses significantly increased during the first 72 hr of incubation, after which the number of axoglial synapses remained stationary for a variable period of time. Later, at 96 hr, the structures deteriorated. The transient appearance of synapse formation supports the view that this cell-cell contact phenomenon reflects the plasticity of neuronal-glial interrelationships under specific conditions. The intimate morphological relationship between the motor nerve terminals of Locusta migratonu and the Schwann cell processes further implicates the functional importance of a morphological intimacy. Ultrastructural changes in these Schwann cells occur after massive motor nerve stimulation and subsequent rest (Reinecke, 1979). Immediately after stimulation of the motor nerve at 20 Hz for 7 min the terminal circumference covered by glial processes is significantly increased and more vesicles and vacuoles are present. Moreover, the number of ensheathing Schwann cell processes increase at the end of the stimulation period, and this increase is reversed after the rest. These changes in the glial cells after stimulation are interpreted to indicate enhanced endocytotic activity, possibly reflecting neurotransmitter uptake. The ultrastructural relationships between axons, glial cells, and connective tissue have been examined in the walking leg nerves of crabs (Lebidoclea grammania) (Selvin-Testa and Urbina-Vidal, 1975). Particular attention was directed to the structure of the connective tissue, its arrangement within the axon sheaths, and its association with the glial cells. The connective tissue of the neural lamella of the giant axons and the fascicles is formed by collagen fibrils and bands of mucopolysaccharides. Prolongation of the neural lamella divides the fascicles into bundles of contiguous axons, groups of loosely sheathed axons and nerve fibers wrapped by layers of glial cell processes alternating with layers of connective tissue. Glial cell processes close to the axons contain numerous microtubules, whereas glycogen granules predominate in the more peripheral processes. These findings support the view that the connective tissue and glial cell processes forming the envelopes of the axons together participate in the maintenance of the microenvironment around axons. Mugnaini has elegantly described evidence of membrane specializations in neuroglial cells and at neuron-glia contacts, and the reader is
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referred to several reviews for more details (Mugnaini, 1982; Mugnaini and Fiori, 1987; Mugnaini et al., 1987). Mugnaini proposes a provocative new neuron-glia interaction, that of competition between glial cells and afferent fibers for apposition to neuronal surfaces (Fig. 7) (Mugnaini and Fiori, 1987). Using the parasympathetic avian ciliary ganglion as a model, Mugnaini and Fiori reported that glial cells compete (over a long time course) with the afferent fibers for appositional sites on the ganglion cell and that this competition is actively carried on throughout the life of the animal. They proposed that the postsynaptic neuron, the presynaptic elements, and the satellite glia continually adjust themselves to some sort of balance: prior to hatching in the chick, the glia would begin to claim part of the ganglion cell surface after onset of sustained functional activity, and the preganglionic calyx would recede from part of its contact with the ganglion cell. In the adult and aging chicken
SATELLITE
SATELLITE GLIA
AXlN TERMINAL
\\ ’, \
\
\
\
FIG. 7. Schematic diagram of an idealized neuron with its complement of glial cells and afferent fibers. Neuron-neuron, neuron-glia, and glia-glia interactions are indicated by double arrows marked 1-5. Whereas interactions labeled 1-4 are widely recognized, interactions labeled 5 (question marks) have been given insufficient consideration and are the subject of this article. (From Mugnaini and Fiori, 1987.)
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the extent of the glial-neuronal contact would continue to increase slowly, and the ganglion cell would produce dendritic evaginations and craterlike invaginations to accommodate a number of synaptic contacts sufficient to ensure the maintenance of a secure synaptic linkage. Mugnaini and Fiori (1987) presented several examples that appear to support their hypothesis of glial-neuronal competition in the vertebrate CNS as well as in the invertebrate nervous system. Several studies have indicated that astrocytesmay play a role in regulation of synaptic density. In 1968 Herndon reported that after destruction of cerebellar granule cells in mature animals, Purkinje cell synaptic spines were invested by astrocytic processes. In addition, these spines maintained a morphologically complete postsynaptic apparatus, including the cleft material. Subsequent studies showed that dendritic spines could develop in the absence of afferent connections (Herndon et al., 1971; Hirano and Jones, 1972; Jones and Gardner, 1976); the postsynaptic apparatus would develop only if astrocytic processes were present to replace the missing granule cell terminals on the spines (Baloyannis and Kim, 1979; Blank et al., 1982). More recently, Meshul et al. (1987) have further examined the role of astrocytes in the regulation of synaptic density. Neonatal mouse cerebellar implants exposed to cytosine arabinoside for 5 days and subsequently maintained in normal nutrient medium were transplanted with 7-day-old mouse optic nerve at 13 days in culture and maintained in normal medium for 22-35 days in culture. These authors had previously observed (Blank et al., 1982; Seil et al., 1980) that cytosine arabinoside treatment for the first 5 days in vitro destroyed granule cells (granuloprival explants) and arrested surviving glia in an early stage of maturation. Also, in the cytosine arabinoside-treated cultures a markedly increased density of synapses on the Purkinje cell somata, probably deriving from Purkinje cell recurrent axon collaterals, was noted. Unlike normal explants, in these cultures there were few astrocytic processes directly in contact with the Purkinje cells. After the addition of optic nerve to the granuloprival explants, astrocytic processes could be seen in contact with the Purkinje cell somata; concomitantly, there was a decrease in the number of synapses on the Purkinje cell somata. Meshul et al. (1987) suggested that astroglia ensheathment, which may be neuronally directed, could be the physical element provoking the reduction in the number of synapses; control of the ensheathment process may be the means of indirectly regulating the synaptic density around the Purkinje cell somata. In the following sections further evidence will be discussed concerning the responsiveness of astrocytes. The astrocytes appear to respond
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differently to the type of signals emanating from neurons (i.e., an early differentiating neuron, an injured neuron, and an aging one will communicate different kinds of cues to the microenvironment). Moreover, the variability in the responsivenessof astrocytes reflects the pluriopotentiality of these cells, which has not yet been fully evaluated.
B. IONICREGULATION The roles of glial cells in the regulation of extracellular potassium concentration, extracellular pH, and extracellular space volume have recently been reviewed by Ransom and Carlini (1986), Walz and Hertz (1984), and White and Woodbury (1987). As discussed earlier, changes in brain extracellular potassium influence transmitter release (King and Somjen, 1981), extracellular space (Dietzel et al., 1982; Ransom et al., 1985a,b;Walz and Hi&, 1985), glucose metabolism (Salem et al., 1975), and neuronal activity (Baylor and Nicholls, 1969a,b; Sykova and Orkand, 1980; Yarom and Spira, 1982). Efficient mechanisms appear to limit evoked increases of this ion to a maximum level, the so-called ceiling level, and to expedite its removal at the conclusion of stimulation. Three major mechanisms have been proposed for glia participation in K + removal: (1) K + activation of glial Na+ ,K+-ATPase,(2) K + activation of KC1 cotransport, and (3) K + activation of a spatial buffering mechanism for K + . The participation of glial Na+ pumps remains unclear despite numerous studies (Ballanyi et al., 1984; Franck et a l . , 1983; Kimelberg et al., 1978; Tang et al., 1980; Walz and Hinks, 1985). The ability of astrocytes to accumulate net amounts of K + in response to increases in Ko+ concentration has been demonstrated in a variety of preparations (Coles and Tsacopoulos, 1979; Hertz, 1978a,b; Kettenmann et al., 1983; Schlue and Wuttke, 1983; Walz and Hertz, 1983; Walz and Hinks, 1985). Anion-coupled transport systems are present in a variety of cells (Kimelberg and Bourke, 1982) and have been well characterized in astrocytes (Kimelberg and Bourke, 1982; Walz and Hinks, 1985; Wolpaw and Martin, 1984). The concept of spatial buffering of focal increases in K$ concentration was introduced by Kuffler and his colleagues in their early studies on Necturus glial cells (Orkand et d.,1966). The extent to which a spatial buffering mechanism would be capable of dispersing accumulated increases in KO+ concentration has been recently reviewed by Gardner-Medwin (1981, 1983a,b) and GardnerMedwin and Nicholson (1983). Several studies suggest that despite the large driving force on Na+ to enter the cell, the permeability of the astrocytic membrane to Na+
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is negligible (Hertz, 1977, 1978a,b; Moonen et al., 1980; Walz et al., 1984), and that Na does not contribute significantly to maintenance of the resting membrane potential (Kimelberg et al., 1979; Moonen et al., 1980; Walz et al., 1984). However, Bowman and Kimelberg (1984) have reported depolarization of the astrocyte membrane when Nao+ concentration was reduced. There is clear evidence that astrocytes actively transport C1- into the cell against a large electrochemical gradient (Kimelberg et al., 1979; Kimelberg, 1983; Walz and Hertz, 1983a-c, 1984; White et al., 1986). Astrocytes in culture have a C1- exchange system similar to that described for erythrocytes (Dunham et al., 1980),glial cell lines (Wolpaw and Martin, 1984), in vilro brain slice preparations (Bourke, 1969), and cerebral cortex in vivo (Bourke and Nelson, 1972). The various processes that may be responsible for maintaining the intracellular concentration of C1- and its integral partner bicarbonate have been recently reviewed by White et al. (1986) and White and Woodbury (1987). The HCOs--C1- exchange system has been shown to be important in the regulation of acid-base homeostasis and is dependent in part on the glia-specific enzyme carbonic anhydrase for its substrate (see reviews in White and Woodbury, 1987; White et al., 1986). In the CNS, carbonic anhydrase, the enzyme responsible for catalyzing the conversion of C02 to HCOs- has been shown to be restricted to glia. Through hydration of C02, carbonic anhydrase provides the substrates for both HCOs--C1- and Na+-H+ exchange and is thus directly involved in acid-base homeostasis (see review in White et al., 1986). Although most authors agree that carbonic anhydrase is localized primarily in the oligodendrocyte (Ghandour et al., 1979; Delaunoy et al., 1980), Kimelberg et al. (1982) have provided both immunocytochemical and biochemical evidence that primary astrocyte cultures contain carbonic anhydrase. Extensive recent evidence has been provided by Woodbury and colleagues on the regulation of cellular pH and carbonic anhydrase in primary astrocyte cultures (review in White et al., 1986). They found that carbonic anhydrase is highly sensitive to inhibition (Ki= 2 X 10-lOM) by the carbonic anhydrase inhibitor acetazolamite. Acetazolamide significantly reduces intracellular pH (pHi) as determined from dual-labeling experiments with 5, 5-dimethyl[2-14C]oxazolidine2,4-dione (DMO) and [3H]antipyrine (Chow et al., 1983, 1984, 1985) and also by the acridine orange fluorescence method, which detects qualitative shifts in pHi (Anderson et al., 1985; Berglindh et al., Dell’ Antone et al., 1972). The role of astrocytes in the anticonvulsant actions of acetazolamide has been well demonstrated by Woodbury and colleagues (see reviews in White and Woodbury, 1987; White et al., 1986). +
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C. NEUROTRANSMISSION PROCESSES
Evidence that astrocytes may be intimately involved in neurotransmission processes has been provided from studies on electrophysiological properties of astrocytes (reviewed by Ransom and Carlini, 1986), catecholamine and serotonin uptake (reviewed by Kimelberg, 1986), amino acid uptake (reviewed by Hertz and Schousboe, 1986; Hosli et al., 1986), choline uptake (reviewed by Massarelli et al., 1986), and receptors (reviewed by Hamprecht, 1986; Lauder and McCarthy, 1986). The reader, therefore, is referred to these reviews for details and relevant references. In this review I will summarize some key findings which either demonstrate or strongly indicate a neuron-glia interaction in neurotransmission processes. 1. Neurotransmitter uptake
a. Monoamines. Intercommunication between neurons involves neurotransmitter substances that: (1) are synthesized and stored in the presynaptic neuron, (2) are released by the arrival of the impulse, and (3) act on the postsynaptic neurons. Figure 8 is a schematic representation
Glial cell
u
\
Presynaptic nerve ending
A
Postsynaptic neuron
FIG. 8. A diagrammatic representation of the fate of norepinephrine (NE) in the central nervous system. (1) Release upon stimulation. (2) Action on receptor. (3) Neuronal reuptake. (4) Degradation by catechol-0-methyltransferase(COMT). (5) Uptake by glial cell. MAO, monoamine oxidase. (From Vernadakis, 1974a,b).
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of the fate of norepinephrine in the central nervous system (from Vernadakis, 1974a,b). High-affinity, Na -dependent uptake of catecholamines by neural tissue is thought to represent reuptake by nerve endings and to be a major means of termination of their action (Snyder, 1970; Iversen, 1974; Fonnum et al., 1980), and the uptake of these transmitters by astrocytes has been shown in situ using autoradiography (Fonnum et al., 1980). Uptake of catecholamines by glial cells in culture was first reported by Pfister and Goworek (1977), who showed by histofluorescence in explant cultures from neonatal rat cerebral cortices localization of norepinephrine (NE) and dopamine (DA). More recently, Hansson (1983) reported “weak’ accumulation of both DA and NE in nialamide-treated rat primary astrocyte cultures, in comparison to “strong” accumulation of GABA and “intense” accumulation of aspartate and glutamate. Kimelberg and associates (see review Kimelberg, 1986) have found highaffinity uptake ( K , = M> of norepinephrine in primary astrocyte cultures which was Na+ -dependent and sensitive to inhibition by the tricyclic antidepressants desipramine (DMI) and amitriptyline (AMT). Thus, the uptake system in astrocytes behaves pharmacologically like the uptake system in various brain preparations. The most likely fate of the transmitter monoamines taken up into astrocytes would be metabolism and removal rather than storage in presynaptic vesicles for rerelease as is likely in nerve endings. Figure 9 is a diagram from Kimelberg (1986) that relates the probable anatomical relationships of an astrocytic process to the varicosity which represents the presynaptic specialization of many catecholaminergic or serotonergic axons in the mammalian CNS facing a receptive neuronal dendrite or cell body. For detailed discussion, see the review by Kimelberg (1986). In addition to the high-affinity NE uptake in primary astrocyte cultures, using cerebellum from 16-day-oldchick embryos maintained as an organotypic culture, we found a low-affinity ( K , = M) NE uptake which could be inhibited by cortisol, when present at a concentration of 2.76 X M in the culture medium for 24 hr (Vernadakis, 197413). In 1970, Iversen and Salt reported that certain steroids are particularly powerful inhibitors of extraneuronal uptake (Uptakez). Based on this evidence, we proposed that the low-affinity NE uptake in our cultures was reflecting glial uptake (Fig. 8 ) . The importance of extraneuronal (i.e., glial) uptake of NE in the CNS can be hypothesized. If the role of glial cells in neurotransmission is to provide a safety ratio, that is, to limit the possible buildup of neurotransmitter substances extracellularly, then inhibition of glial cell uptake could lead to an intracellular-extracellularimbalance and result in deleterious cellular +
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ASTROCYTE PRESYNAPTIC VARICOSITY
POSTSYNAPTIC
FIG. 9. Diagram of routes of release and uptake of norepinephrine (NE), dopamine (DA), or 5-hydroxytryptamine (5HT) at a monoaminergic synapse. (1 and 3), Highaffinity uptake sites on astrocytic processes and presynaptic varicosity, respectively, involving cotransport with Na+ . (2) Na+-independent, low-affinity uptake site on astrocyte. (4) Fusion of synaptic vesicle with surface membrane. ( 5 ) (Na+, K + )pump, shown only for astrocyte. Monoamine oxidase (MAO) is shown localized to mitochondria in both astrocyte and synaptic varicosity. Catechol-0-methyltransferase (COMT) present only in astrocyte. (From Kimelberg, 1986.)
effects. For example, excessive amounts of NE in the synaptic cleft would make more NE available to stimulate the CNS and would result in CNS hyperexcitability, which is known to occur with cortisol treatment (Vernadakis and Woodbury, 1963). Moreover, since the uptake system in astrocytes is also sensitive to clinically effective antidepressants, as shown by Kimelberg and associates (see review by Kimelberg, 1986), the therapeutic effects of such agents may be partially mediated by their action on astrocytes.
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Glial uptake of serotonin and dopamine has also been studied in primary cultures of the median raphe nucleus and cerebellum by using consecutive demonstrations of monoamine fluorescence and glial fibrillary acidic protein immmunofluorescence (Liesi et al., 1981). Most of the glial cells taking up monoamines have been shown to be GFAPpositive. As also supported by the reports of Hosli et al. (1975), Hosli and Hosli (1976), and Yamamoto et al. (1981), Liesi et al. (1981) found that with dopamine concentrations of l o p 5 M or less, no uptake into glial cells could be demonstrated. Liesi et al. proposed that it might be possible that the cell membranes of the astrocytes, which have a high GFAP content, could be more resistant to monoamines than those of glial cells exhibiting less intense or no GFAP immunoreactivity. It might be possible, however, that the nonexistence of monoamine fluorescence in glial cells exhibiting intense immunofluorescence could be due to high monoamine oxidase activity in these cells. Of interest in the studies of Liesi et al. (1981) are differences in glial uptake of serotonin between cultures from the median raphe nucleus and cerebellum, whereas no differences in dopamine uptake between these regions could be demonstrated. Using C-6 glioma cells, Suddith et al. (1978) have found that glial cells possess a double-affinity membrane transport system for the accumulation of serotonin. Kinetic analysis has shown a high-affinity component of 1-2 p M , which was similar to that previously described by the same authors for serotonin transport by the RN-22 neurinoma cells (Haber and Hutchison, 1976). In addition to the high-affinity system, C-6 cells also possess a second serotonin uptake mechanism of lower affinity, with a K , value of 1.4 mM. Serotonin is also bound to highaffinity binding sites on intact astrocytes in primary cultures (Hertz et al., 1979), and the astrocytic binding has been confirmed in studies using astrocytes prepared by gradient centrifugation (Fillion et al., 1980). Hertz and Tamir (1981) have reported an astrocytic protein fraction that binds serotonin and differs in some of its physical, chemical, and pharmacological properties from neuronal serotonin-binding protein. The functional rate of the astrocytic binding protein is not known. Kimelberg and Katz (1985) and Katz and Kimelberg (1985) have also observed significant uptake of serotonin by primary astrocyte cultures. The Na+ -sensitive component of this uptake showed high affinity for tritiated 5-HT (K, = 0.40 p M ) , and uptake was also very sensitive to specific inhibitors, such as various antidepressants. Hansson (1983) has reported “weak’ autoradiographic grain localization in primary astrocyte cultures exposed to tritiated 5-HT. In contrast, Kimelberg and Katz (1985) found that virtually all GFAP-positiveastrocytes had a grain density that
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was above the background level after uptake of tritiated 5-HT with Na present, and that this uptake was reduced to close to background levels when Na+ was omitted from the medium. b. Choline. Acetylcholine (ACh) differs from other neurotransmitters in that it is inactivated by extracellular hydrolysis, rather than by reuptake into presynaptic nerve endings. Furthermore, the central nervous system is capable of de novo synthesis of choline (Birks and MacIntosh, 1961; Browning and Schulman, 1968). It has been shown that both brain slices and synaptosomes accumulate choline via a high-affinity,saturable mechanism and by a second, less well-characterizedcomponent of lower affinity. It has been suggested that at cholinergic synapses, the reuptake of choline by the high-affinity system is tightly coupled to the synthesis of ACh (Haga and Noda, 1973; Kuhar et al., 1973). The transport of choline has been studied extensively using clonal cell lines of neuronal (NB41 and N2-AG) and glial (C-6 and RN-22) origin. The presence of a high-affinity transport system for choline has been studied and characterized by Haber et al. (1973) and Hutchison et al. (1976), who found that glial and neuronal cell lines responded in opposite directions to removal of sodium from the incubation medium. They found that both neuronal and glial cells possess high affinity transport systems for exogenous choline, with K,,, values in the 10-15 fM range, plus a second low-affinity system that may represent diffusion. The stimulation of the glial cell high-affinity choline transport system in the absence of sodium is a striking finding. It is not, however, an exclusive glial trait, because such diverse cell types as hepatoma and melanoma cell lines exhibit a similar response to low sodium. The significance of the differential Na modulation of choline transport in glial and neuronal cells can be speculated on in the general context of the proposed role of glial cells in regulating the ionic microenvironment. As discussed earlier, ionic fluxes generated in the process of neurotransmission may be a mechanism whereby neurons can communicate with glia. It has been shown that Na+ influx during depolarization of presynaptic terminals results in a transitory decrease in extracellular Na+ and an increase in K + . Such transitory changes in extracellular monovalent cations might trigger the influx of choline into glial cells, whereas during repolarization the external Na would increase and thus promote neuronal reuptake of choline for the resynthesis of ACh (Hutchison et al., 1976). Recently, extensive work on cholinergic enzymes and choline uptake in neuronal and glial cultures has been reported by Massarelli et al. (recent review, Massarelli et al., 1986). In glial cell cultures dissociated from chick embryo cerebral hemispheres, only a low-affinity uptake +
+
+
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ANTONIA VERNADAKIS
mechanism has been detected. The discrepancy between these results and those obtained with C-6 and NN glial cell lines may be related to the degree of differentiation of the cultures. The apparent low-affinity (Km= 10- 5 M) choline uptake is inhibited by cold and metabolic poisons and is dependent on the ionic balance of the incubating medium. Preincubation of the cultures with various inhibitors of acetylcholinesterase showed no direct parallels between inhibition of choline high-affinity uptake (apparent Km = M) and inhibition of ecto-acetylcholinesterase. We have also been using the C-6 glial cell as a model to study glial cell properties. We have found that these cells have both acetylcholinesterase and butyrylcholinesterase, both enzymes increasing when cells are exposed to dibutyryl CAMP (Vernadakis and Nidess, 1976). Moreover, the activity of the ecto-enzymes decreases with increasing cell density, reflecting cell-cell contact regulation. These findings provide further evidence that glial cells may be intimately involved in acetylcholine metabolism. In an elaborate scheme (Figs. 10 and 11), Massarelli et al. (1986)summarized choline metabolism and interactions between neurons, glia, and capillaries in two situations: steady-stateconditions and depolarizing conditions. The proposed processes are well described in the figure legends and I will not reiterate them. It is evident, however, that glial cells, in this as in other neurotransmission processes, can be active components and not “impersonal cellular entities” involved in choline uptake and metabolism (Massarelli et a l . , 1986). Of interest regarding the role of glial cells in choline accumulation are the findings of Wong et al. (1982) on the efflux of choline from neurons and glial cells of dissociated chick embryonic brain in culture. They report that spontaneous efflux of [14C]cholinewas greater in cells preincubated with higher concentrations of [*4C]cholineand the efflux was increased with increasing concentrations of “chasing” unlabeled choline. Cs+ and K + greatly stimulated the efflux both from glial cells and neurons. However, much more K + was needed to stimulate efflux of choline from neurons in comparison to glia. These data are interpreted by the authors to mean that following depolarization of the neuron, the amount of K + exiting from the neuronal membrane (or from the nerve terminal) might be sufficient to stimulate the efflux of choline from an adjacent glial cell, and this choline might in turn be taken up in the nerve terminals for the synthesis of acetylcholine. c. Amino Acids: GABA, Glutamate, and Aspartate. y-Aminobutyric acid (GABA) has been accepted as a major inhibitory transmitter in the central nervous system. It has been shown by various investigators that GABA is released from CNS tissue. More specifically, it is released from the brain in vivo and in vi2ro following electrical stimulation or exposure to veratridine or to high potassium concentrations (Jasper and
NEURON-GLIA INTERRELATIONS
189
FIG. 10. An oversimplified view of choline metabolism in the brain under nerveresting conditions. The schema includes an arterial blood vessel (ABV),glial cells (GLIA), neuronal presynaptic (Pre) and postsynaptic (Post) terminals, and a venous blood vessel (VBV). Choline (Ch) enters the brain via the end-feet (EF) processes of astrocytes or directly into the extracellular space (ECS). From ECS, Ch may be taken up into glial cells by a sodium ion gradient (Na +)-drivenprocess and be metabolized rapidly into phosphocholine (PhCh) and eventually into phosphatidylcholine (PC). Ch may also be produced by the stepwise methylation of ethanolamine (Ea)-containing compounds or by the activity of glycerophosphocholine (GPhCh) choline phosphodiesterase (EC 3.1.4.38),which is detected primarily in glial cells. Ch may exit the cells by a potassium ionic gradient ( K + ) and be taken up by nerve terminals by a similar Na -driven process. Inside the terminals, Ch follows the same metabolic steps present in glia, with the notable exception that it produces acetylcholine(ACh). The spontaneous release of ACh is terminated by the hydrolysis of the neurotransmitter into acetate (Ac-) and Ch. At present, most articles concerning the central nervous system suggest the Ac- “diffuses away” the synaptic cleft, suggesting perhaps an uptake into glial cells. Ch exits neurons also by a K + gradient. Finally, Ch exits from glial cells into venous blood vessels. (From Massarelli et al., 1986.) +
Koyama, 1969; Katz et al., 1969, Maghiyama et al., 1970; Arnfred and Hertz, 1971; De Belleroche and Bradford, 1972; Cotman et al., 1976). In brain slices it has been shown that the process is calcium-dependent (Srinivasan et al., 1969; Katz et al., 1969; Okada and Hassler, 1973). In addition, there is a calcium-independentrelease of GABA. Szerb (1979) has shown that the calcium-dependent and the calcium-independent
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ANTONIA VERNADAKIS
FIG. 11. Choline metabolism under depolarizing conditions. The schema is similar to the one shown in Fig. 10. The local inversion of Na+ and K+ gradients due to depolarizing conditions produces a stimulation of choline efflux from glial cells and the release of acetycholine (ACh) from neurons. This will result in a drastic increase in the concentrations of choline in the extracellular space. A further stimulation is obtained by a series of enzymatic activations that start with phospholipases (PLA) A, and A2 (EC 3.1.1.32 and 3.1.1.4, respectively), leading to the production of diacylglycerol (DAG), which may perhaps be considered as a second messenger, and of fatty acids (especially oleate, Ol), which may stimulate the activity of phospholipase D (PLD) (EC 3.1.4.4). Free choline (Ch) thus produced may be used for ACh synthesis (Hattori and Kanfer, 1984). (From Massarelli et al., 1986.)
release occur from two different pools. It has been postulated by Hertz and Schousboe (1982, see also 1987) that one of the two pools could conceivably be located on astrocytes, but it is disputed whether high concentrations of potassium are able to release GABA from glial cells. Hertz and Schousboe did not observe GABA release from astrocytes in primary cultures, and no GABA release was observed in C-6 glioma cells (Schrier, 1977). However, a high triggered release of GABA has been reported to occur in peripheral ganglia by Minchin and Iversen (1974). Under the conditions they used, [3H]GABAis taken up exclusively by the satellite glial cells. They have also shown a GABA efflux after exposing
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the cells to depolarizing concentrations of potassium. Qualitatively, the satellite glial GABA release is significantly lower than that of neuronal preparations. According to Hertz and Schousboe (1982, see also 1987) the released GABA molecules may be reaccumulated by the nerve ending, accumulated and metabolized by some synaptosomal parts of the neurons, or accumulated and metabolized by astrocytes. The only enzyme capable of metabolizing GABA, GABA transaminase, is located intracellularly. Thus, for GABA to be metabolized it has to be taken up from the extracellular space. Neuronal elements have been shown to have a high-affinity GABA uptake system (Henn and Hamberger, 1971; Levi and Raiteri, 1973; Martin, 1973; Sellstrom and Hamberger, 1975). This has been also shown in cells in culture (Buny and Lasher, 1975, 1978a,b; Hosli et al., 1972; Hosli and Hosli, 1978; Lasher, 1974, 1975). High-affinity GABA uptake has been also demonstrated in nonneuronal constituents of the nervous system. Henn and Hamberger (1971) have shown GABA uptake in astrocytes; it has also been shown for nonneuronal cells in peripheral ganglia (Bowery and Brown, 1972; Roberts, 1976; Schon and Kelly, 1974), Muller cells (Neal and Iversen, 1972), primary cultures of astrocytes from the cerebellum (Burry and Lasher, 1978a,b; Hosli and Hosli, 1978; Lasher, 1974, 1975), and primary cultures from the cerebral hemispheres (see also reviews by Hertz, 1979; Hertz et al., 1978a; Hertz and Schousboe, 1986, 1987; Schousboe, 1981, 1982; Schousboe and Hertz, 1987; Schousboe et al., 1977a,b). Regional differences in astrocyte GABA uptake appear to exist (as is also the case for glutamate, see below), since the uptake capacity for GABA into cortical astrocytes has been shown to be considerably higher than into cerebellar astrocytes (Drejer et al., 1983a,b; Larsson et al., 1985). Moreover, it has been shown that different types of astrocytes in cerebellar cultures take up GABA with different efficiencies (Wilkin et al., 1983). As discussed in another context in this review, treatment of cells with conditioned media from cerebellar granule cells (Drejer et al., 1983a,b), which are characterized by a dense population of GABA receptors (Meier and Schousboe, 1982), selectively stimulated astrocyte GABA uptake. It is now abundantly demonstrated that glutamate is taken up much more intensely into astrocytes than into neurons (see review Hertz, 1979; Schousboe, 1981, 1982; and Hertz and Schousboe, 1986, 1987). At a concentration of 50 of extracellular glutamate, the uptake into astrocytes is much higher than the uptake into GABAergic neurons and twice as high as the uptake into glutamatergic neurons (see Fig. 12) (Hertz and Schousboe, 1987).
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ANTONIA VERNADAKIS
Astrocyte
-f
GABAergic Neuron
Glutamatergic Neuron
f FIG. 12. Schematic drawing of evoked release and uptake of glutamate (Glu) and GABA in GABAergic or glutamatergicneurons and in astrocytes. The sizes of the arrows give an estimate of the relative magnitudes of the respective fluxes. It can be seen that neuronally released glutamate primarily accumulates in astrocytes, whereas most of the released GABA reaccumulates in neurons. (From Hertz and Schousboe, 1987.)
Culturing astrocytes from different brain regions, Schousboe and Divac (1979) have found that the capacity for high-affinity glutamate uptake in the different astrocytes correlated with glutamatergic activity in these brain regions. In a later study Drejer et al. (1982) confirmed that astrocytes originating from brain regions with a high density of glutamatergic innervation had higher V , values for glutamate uptake than those originating from regions with less glutamatergic activity. Similar findings have been reported from autoradiographic studies (Hansson, 1983). Aspartate is known to be released in vivo (Clark and Collins, 1976) and in preparations of synaptosomes (De Belleroche and Bradford, 1972; Osborne et al., 1973). Drejer et al. (1983a,b) reported the characterization of the uptake and release processes for D- and L-aspartate and compared it to that of L-glutamate in primary astrocyte cultures and cerebellar granule cells. A high-affinity uptake system for L- and Daspartate was found in both cell types. When the uptake kinetics of each one of the three amino acids was studied in the presence of the two other amino acids, no essential differences among the uptake characteristics of the amino acids were found. In addition, a Ca2+-dependent, K + -induced release from granule cells was found for both L-glutamate and D-aspartate. The question whether aspartate is a transmitter or not (i.e., whether there is a population of asparatergic neurons), remains open to further investigation. 2. Neurotransmitter and Other Related Receptors Numerous studies have recently shown that both glial cell lines and primary cultures of astroglia exhibit a variety of neurotransmitter receptor
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systems. For example, astroglia have 0-and a-adrenergic receptors (McCarthy and de Vellis, 1978); benzodiazepine receptors (Bender and Hertz, 1987; McCarthy and Harden, 1981); muscarinic cholinergic receptors (Fkpke and Maderspach, 1982); and receptors for vasoactive intestinal peptides (VIP) (Evans et al., 1984; van Calker et al., 1980); somatostatin, adrenocorticotropichormone, and melanocyte-stimulating hormone (van Calker et al., 1983); and histamine (van Calker et al., 1979; Magistretti et al., 1983). The presence of receptors in astrocytes has primarily been shown by an analysis of receptor-mediated changes in second messenger systems (i.e., CAMP)(see review by Hamprecht, 1986) or by radioligand binding to astroglia membrane preparations (McCarthy, 1983). McCarthy (1983) has reported a new technique which combines receptor autoradiography and immuncytochemistryand thus has permitted researchers to examine quantitatively the expression of receptors by single cells of known identity in a heterogeneous culture preparation. He reports that there are two distinct populations of astroglia in culture which can be distinguished on the basis of their morphology and expression of 0-adrenergic receptors (Fig. 13). Polygonal astroglia appear to exhibit far more 0-adrenergic receptors than any other cell type present. Moreover, this astroglial subtype appears to be the same as that influenced by neuronal activity in adult cortical tissue (Wolff et al., 1978). A number of astroglial functions have been reported to be influenced
FIG.13. fi-Adrenergic receptor expression by flat, polygonal (A) and process-bearing (B) GFAP-positiveastroglia. Astroglia were cultured, stained for GFAP, and labeled with an iodinated fi-adrenergic receptor antagonist. Note the high density of silver grains associated with flat, polygonal astroglia and their virtual absence over process-bearing astroglia. Similar results are obtained regardless of the age of the animals used to prepare cultures or the duration of the culture growth period. X159. (From Lauder and McCarthy, 1986.)
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by receptor stimulation including glucose uptake (Newburgh and Rosenberg, 1972), glycogen levels (Cummins et al., 1983), and lactate dehydrogenase activity (McCarthy and de Vellis, 1980). Extensive evidence reviewed recently by Hamprecht (1986) has demonstrated that various neurohormones including various transmitters, substance P, somatostatin, secretin, glucagon, adrenocorticotropin, and others (see Hamprecht, 1986) regulate the intracellular concentration of CAMP in astrogliaenriched cultures derived from murine brain. Using adenylate cyclase as one effector system, it has been clearly shown that astroglia cells are highly susceptible to several peptides and thus have to be considered as delicately regulated by neurohormones. In the context of neuron-glia interactions the responsiveness of glial cells to neurohormones and neurotransmitter substances reflects the role of neuronal signaling in providing a link between neurons and glial cells.
IV. Regeneration
As first observed by Cajal(1928), environmental influences may have considerable relevance to the regenerative capacity of severed axons and several components of the neural environment have been described: Schwann cells, fibroblasts, mast cells, basal lamina, and collagen in the peripheral nervous system; and oligodendrocytes, astrocytes, and microglia in the central nervous system. The present discussion will primarily be concerned with the possible glial role in axonal regeneration, using a few examples from in vivo and in mlro studies of vertebrates and invertebrates. The topic of the response of glial cells to injury in the CNS, the characteristic glia scar formation or reactive gliosis has been elegantly reviewed by Lindsay (1986) and Reier (1986). Gliosis represents a major effect of injury to the CNS and has been frequently viewed as being an important determinant of the extent and quality of neural repair in mammals. Because of the apparent limitation it imposes upon regeneration, glial reactivity has been traditionally viewed in a negative vein. However, a number of studies provide evidence against the notion of glial barriers to regeneration, and a beneficial role of glial cells in regeneration has been given favorable consideration. The studies of Aguayo and associates (Aguayo et al., 1978, 1981, 1982, 1983; Benfey and Aguayo, 1982; David and Aguayo, 1981; Richardson et al., 1984)have greatly contributed to our understanding of differences in axonal regeneration in CNS and PNS. In 1981, David and Aguayo
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demonstrated that adult rat spinal and medullary neurons elongate axons for distances of several centimeters through peripheral nerve grafts used as “bridges” to connect the medulla oblongata and the lower cervical spinal cord. The neurons giving rise to these axons include cells that normally project to peripheral nerves (e.g., motor neurons), but also include intrinsic neurons whose somata and projections are normally confined to the CNS. Successful elongation of axons from intrinsic CNS neurons has also been documented after the insertion of peripheral nerve segments into several other regions of the adult rat brain and spinal cord (Aguayo et al., 1983; Richardson et al., 1984). It is possible that the central axons growing into such peripheral nerve grafts may arise from either injured cells or from collaterals of uninjured neurons. However, in another study in which peripheral nerve grafts were introduced into the rat olfactory bulb and retina, the majority of the axons innervating the graft were derived from axotomized cells. Aguayo and associates (David and Aguayo, 1985) concluded that severed axons can regrow when they can interact with nonneuronal components of the PNS. Several factors may be involved in these regenerative processes including changes in the neuronal perikaryon (Grafstein and McQuarrie, 1978; Richardson and Issa, 1984; Skene and Willard, 1981), perhaps triggered by signals received through growth cones in the damaged tip of axons (Bray and Bunge, 1973; Gundersen and Barrett, 1980), from molecular cues secreted by the ensheathing cells (Richardson and Ebendal, 1982; Skene and Shooter, 1983; Varon et al., 198l), or present on cell surfaces and/or the extracellular matrix (Carbonetto et al., 1982). Smith et al. (1986) in an elegant study described the changing role of astrocytes during development, regenerative failure, and induced regeneration upon transplantation. When the cerebral midline is lesioned in the mouse embryo or neonate, the would-be callosal axons form neuronomas. An implanted Millipore bridge inserted between the neuronomas in young acallosal animals can support the migration of immature astrocytes that, in turn, support the de novo growth of commissural axons between the hemispheres. Moreover, there is a critical period for the glial to promote reconstruction of malformed axon pathways. In acallosal postnatal mice given Millipore implants on or prior to postnatal day 8, GFAP-positive, stellate-shaped astrocytes migrated and attached to the implant by inserting foot processes into the pores of the filter; this form of gliotic response is established on axon growth-promoting substratum within 24-48 hr after implantation. Of interest is the observation that during this age period there was no evidence of scar formation or necrosis at or around the implant surface. However, when acallosal mice were given implants on or later than
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postnatal day 14, extensive tissue degeneration occurred, and a mixed population of astrocytes and fibroblasts invaded the surface of the filter, producing a dense scar. Reactive cells within the scar did not promote axonal outgrowth. Furthermore, when filters coated with glia from 8-dayold mouse forebrains were transplanted into the brains of 14-day-old acallosal animals, glial scarring in the host was reduced and axonal regeneration was enhanced. Thus, axonal regeneration in the CNS at postcritical periods may be stimulated by reintroducing an immature glial environment at the lesion site. These findings are complimentary to those of Kromer et al. (1981), who have shown that transplants of intact pieces of embryonic tissue placed into gaps between the septum and hippocampus can promote the long-distance regeneration of axons. It appears from these findings that the immature glial environment within such embryonic grafts supplies essential growth and guidance cues to the regrowing axons. The authors go on to suggest that in contrast to adult reactive gliosis, the gliotic response in neonatal animals is an active rather than reactive phenomenon; activated gliosis can be considered a beneficial and constructive process. Phenomenologically similar activated astrocytes have been described in the developing (Silver and Sapiro, 1983) or regenerating optic nerve of Xenopus laevis (Reier and Webster, 1974; Bohn and Reier, 1985; Bohn et a l . , 1982) and along the regenerating olfactory nerve in adult rats (Liesi, 1985; Raisman, 1985). Smith et al. (1986) attributed the failure to regenerate after a critical period to at least two factors: the presence of ectopic basal lamina and connective tissue that form a mechanical impediment to axons, and the physical state of glial cells that migrate onto the implant. That astrocytes have different functional states is a concept supported by various investigators (for several reviews, see Vol. 2 of “Astrocytes,”Fedoroff and Vernadakis, eds., 1986). The glial role in neural repair has recently been explored in invertebrate animal models by Smith, Howes, Treherne and their associates (Howes et al., 1987; Smith and Howes, 1984, 1987; Smith et al., 1984, 1986; Treherne et al., 1984, 1986a,b, 1987). In 1984 Treherne et al. and Smith et al. reported glial repair in the CNS of the cockroach (Perz’planetaamericana L.). It appears that due to its morphology, the insect CNS offers a number of distinct experimental advantages over its vertebrate counterpart. The connectives of the abdominal ganglionic chain contain only axons and two distinct classes of nonneuronal cells, the superficial perineurial glia and the underlying subperineurial glia, which surround the axons. The perineurial cells constitute a distinct and well-defined blood-brain barrier (Schofield and Treherne, 1984),
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the integrity of which can be easily tested during repair. Because there are no neuronal cell bodies within the connective tissues, application of a glial toxin, ethidium bromide, leaves the axons intact but causes the destruction of the glial barrier. This is followed by a rapid functional repair of the perineurial barrier to exclude small cations, such as potassium (Smith et al., 1984). These early observations have suggested that undamaged axons and/or the extracellular matrix exert a profound influence on the mechanisms of glial repair. In subsequent studies Howes et al. (1987) and Smith and Howes (1987) reported that insect glial cells are capable of division and repair in organ culture after selective damage with ethidium bromide. The repair of the insect central nervous system can be divided into three phases, which show striking similarities to vertebrate repair sequences. These include the stages of initial invasion of the lesion by exogenous cells, subsequent proliferation of glial cells, and the longer-term flux of cell numbers, as the distribution of the phases and the time scale of these events. The authors suggest that the insect CNS provides an alternative system in which to study some of the cellular processes involved in CNS repair. Grafting of neural tissue has reemerged in the past decade as a powerful tool to study both basic neurobiological and clinical questions regarding the regenerative capacity of the adult mammalian CNS. Studies using fetal CNS implants into adult CNS tissue have been providing some insight into neuron-glia interrelationships in growth and regeneration processes. For in-depth information the reader should consult reviews by Bjorklund and Stenevi (1984), Gage (1987), Gage et al. (1983), and Lund (1980). The general ability of neurons within CNS implants to survive in the host brain environment appears to depend largely on the age of the tissue donor: the younger the donor, the better the chances for neuronal survival in the graft. This general rule appears to hold (1) regardless of the region used as donor graft, (2) whether solid grafts or dissociated cells (neurons plus glial cells) are implanted, (3) whether the tissue is grafted into adult CNS tissue, or into the anterior eye chamber, and (4) regardless of age of the host, CNS tissue, or the brain region where the graft is placed. These studies are still preliminary for any conclusions; however, the studies of Bjorklund and colleagues (Bjorklund and Olson, 1983; Bjorklund et al., 1983) suggest that soluble signals present in the host environment tend to increase or decrease (depending on the tissue used as host) the number of GFAP-positive astrocytes in the fetal cell-cell contact and/or cortical grafts. The role of glial cells in providing cell producing trophic factors and thus influencing the growth of grafted neurons remains to be evaluated.
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V. Neuron-Glia Interactions in Aging Processes
For many years it was believed that memory and cognitive decline of the aged was simply the result of attrition of nerve cells. However, although neuronal loss must play a role in the memory and cognitive decline of aging, it is now clear that this is not the complete explanation. As in early development, also in aging neuron-glia interactions play a key role in the neuronal function of the aged brain. It is the proposal of this author that the neuron is, in part, a “victim” of its microenvironment and that shifts in cellular homeostatic mechanisms are key epigenetic factors responsible for neuronal aging (Fig. 14). The role of glial cells in neuronal growth and homeostasis was discussed earlier. Changes, therefore, in the glial cells with aging will interrupt intercellular relationships and ultimately affect neuronal function. Detailed reviews of neuronal and glial changes in aging have been published (Vernadakis, 1985, 1986; Vernadakis et al., 1987). However, a review of neuron-glia interactions would not be complete without a brief discussion on possible changes on the neuron-glia unit with aging. Primary emphasis will be given to the role of the glial cell.
A. CHANGESIN GLIALCELLS WITH AGING Studies using Cajal’s gold stain for astrocytes and light microscopic analysis have revealed that major astrocytic reactivity (hypertrophy and increased numbers of thickened processes) occurs in the hippocampus, caudate nucleus, a number of major myelinated fiber tracts, and various other brain regions during aging (Landfield et al., 1977, 1978). This reactivity appears to be most pronounced in the hippocampus, a finding in agreement with early ultrastructural observationsby Vaughn and Peters (1974) showing little change in neocortical astrocytes during aging. Electron microscopic studies by Landfield (1978) confirmed an increase in the volume of hippocampus occupied by astroglial profiles (with no increase in astrocyte numbers) during aging. In another study Lindsey et al. (1979) examined in more detail the onset and topographic distribution of hypertrophied astrocytes in the hippocampus of aging rats. They reported that the density of astrocytes exhibiting hypertrophy in the hippocampus increased dramatically and progressively in rats of three ages (4 months, 15 months, and 25 months) and was significantly elevated even in middle-aged (15-month-old)animals. The total population of astrocytes, however, was not significantly elevated. Also, the relative
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Connective Tissue Cells (Fibroblasts, Mesenchymal C e l l s )
and Endothelial Cells
FIG. 14. Schematic representation of cell-cell interaction in the central nervous system. (From Vernadakis and Sakellaridis, 1985.)
distribution of hypertrophied glial cells and total number of glial cells remained constant at the three ages studied. The following conclusions were drawn by the authors: (1) hypertrophy of astrocytes appear to occur “in place,” and migration of hippocampal astrocytes does not appear to be a major factor; (2) glial hypertrophy does not occur uniformly throughout the hippocampus with age, and it appears more pronounced in synaptic terminal fields; and (3) glial hypertrophy appears to have its onset in middle age. Several studies using cell cultures as models have explored cell changes with aging. More specifically, somatic cells have been used as a model, and changes with cell passage in a culture have been interpreted to reflect changes with aging (Strehler, 1977). We have used early “young” passage (20-25) and late “old’ passage (50-90) C-6 glial cells (2B clone) as a model to study changes in glial cells with aging in culture (Parker et al., 1980). We compared the doubling time in young (25 passages) and old (54 passages) C-6 glial cells plated at low density (1-1.5 X cells/flask). Young cells/flask, 25 cmz) or high density (1-1.5 X passage cells plated at low cell density appeared to proliferate at a similar rate to that of old passage cells. However, when cells were plated at a high cell density, old passage cells had a doubling time of 12-16 hr, whereas young passage cells showed a doubling time of over 24 days. The rapid proliferation of the old passage cells was reflected in the higher protein and RNA content per cell in general. This biochemical difference between young and old passage cells was also reflected in the cell size, old passage cells being larger. Using the enzyme markers CNP for
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oligodendrocytes and GS for astrocytes (discussed earlier), we have examined changes in the activity of these enzymes in C-6 glial cells with cell passage (Parker et al., 1980). The activity of CNP was markedly higher in cells of young passage (21 passages) as compared to those of old passage (82 passages) at confluency (10 days in culture); enzyme activity was similar in both cell passages during logarithmic growth. Moreover, CNP activity increased in the young passage cells with days in culture, perhaps reflecting the increase in synthesis of this enzyme protein. The CNP activity did not change and, in fact, decreased in the old passage cells with days in culture, suggesting a basic cellular change occurring in these cells with cell passage. That this is the case was substantiated by the results in GS activity. In contrast to the CNP activity, GS activity was markedly higher in the old passage cells and low in the young passage cells. Moreover, the change in GS activity with days in culture was more marked in the old passage cells. Further examination using GS immunocytochemicallyas an astrocytic marker revealed that late passage cells (84 passages) have more GS than early passage cells. Recently, Raju et al. (1980) reported that aging in culture included a dramatic increase in glial fibrillary acidic protein, another astrocytic marker discussed earlier. Thus, young passage cells express predominantly oligodendrocytic properties, and old passage cells are predominatly astrocytic. Light microscopy showed that almost all cells of early passages possessed small, dark nuclei, scanty perikarya, and relatively short, thin cytoplasmic processes. In contrast, cells of late passages had significantly larger and paler nuclei, frequently abundant perikarya, and considerably longer cytoplasmic processes. This type of cell was never seen in early passage cultures. Based on these biochemical and morphological findings, we have proposed that a “transdifferentiation”of cells from oligodendrocytelike to astrocyte-likeproperties may occur in culture. Such shifts in cell types in culture has been reported by Raff and associates in their studies of cell lineages (Raff et al., 1983a,b). However, those shifts in cell types may also occur with cell aging and should be considered in future studies. Recently, we have attempted to examine properties characterizing the aging of glial cells by using primary glial cell cultures derived from newborn and adult-aged mice (Vernadakis et al., 1984, 1986). Again, as in the studies with glioma cells, we compared changes occurring with cell passage. As also reported by others, dispersed cells obtained from newborn mouse cerebral hemispheres adhere to the dish within 3-4 days, and the various populations become apparent within 10-15 days (Fig. 15). However, in the cultures derived from aged mouse brain, the dispersed cells do not adhere to the dish for at least 9-10 days. Then small patches, or “islands,”of large flat cells begin to appear sporadically on the dish
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FIG. 15. (a) Glial cell cultures derived from dissociated newborn mouse cerebral hemispheres: A 15-day primary culture (Po) under phase-contrast. Dark processbearing cells (oligodendrocytes) are seen on top of the flat background cells (astrocytes and connective tissue cells). X 362. (b) “Flat” cell cultures derived from 18-month-old mouse cerebral hemisphere: 9-day culture under phase-contrast. X 362. (c) Glial cell cultures derived from 18-month-oldmouse cerebral hemisphere: 14-dayprimary culture (Po) under phase-contrast. Primarily process-bearing cells and flat background cells are seen. X362. (From Vernadakis et al., 1984.)
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(Fig. 15). By 2-3 weeks in culture, an almost confluent monolayer of flat cells forms on top of which a smaller number of round dark processbearing cells (oligodendrocytes) can be seen. Glial fibrillary acid protein-positive and glycerol phosphate dehydrogenase (GPDH)-rhodamine-positivecells were present in both types of cell cultures, newborn and aged mouse, up to passage 4 (Fig. 16). However, with progressive cell passage, the number of GFA-positive cells declined significantly, as did the intensity and pattern of immunoreactivity (Fig. 17). The decline in GFA-positive cells with cell passage is of interest and warrants further exploration. The question that should be considered is whether there is a decline in GFA astrocytes or whether there is a cellular change in the GFA molecule so that the antibody no longer recognizes it, thus causing the decline in GFA-positive immunoreactivity. In a recent study, Lindsay et al. (1982)characterized astrocyte cultures derived from corpus callosum of adult rat after surgical lesion and a period of postoperative “priming” in vivo. They describe two distinct morphological variants in early cultures: process-bearing cells with small rounded perikarya, and very large flattened cells with readily discernible nuclei. By 3 weeks in culture, 90% of the GFAP-stained cells adopted a flattened morphology and, after 4 or more weeks in culture, process-bearing cells were virtually absent. In our cultures, flat epithelioid cells as described by Lindsay et al. (1982)were observed on the plastic floor of the culture dish. Meller and Waelsch (1984)have recently reported cyclic morphological changes of glial cells in long-term cultures of rat brain. After four subcultures and 8-10 weeks of cultivation, the following cell types could be distinguished: (1) flat epithelioid cells proposed to be precursors of (2) GFA-positive astroglial cells and (5)oligodendroglia cells. After 6 weeks, the most prominent cells are the flat epithelioid cells. Our findings also show that with cell passage the appearance of flat epithelioid cells increases. Based on these findings, we have proposed that these changes with cell passage represent cellular senescence. Changes in cell membrane that ultimately would be reflected in the size and shape of the cell have been reported to occur in various somatic cells with senescence in vitro and include changes in cell size (Simons and Swim, 1967;Guillouzo et al., 1972); sensitivity to concanavalin A-mediated agglutination (Yamamoto et al. , 1977); surface topography (Dowman and Daniel, 1975); functional specialization (Johnson et al., 1974); carbohydrate content (Courtois and Hughes, 1976;Azencott et al., 1975; Spataro et al., 1979); cell adhesion (Azencott et al., 1975); and lectin RCAI-binding sites (Aubery et al., 1980). An increasing number of the polyhedral cells were multinucleated (Fig. 18) at the late passages, especially in the cultures derived from aged
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FIG. 16. (a) GFA-positive cells from dissociated newborn mouse cerebral hemispheres, passage 3, 22 days in culture. X1432. (b) GFA-positivecells from dissociated aged mouse (18-month-old)cerebral hemispheres, passage 2, 11 days in culture. X1463. (c) GPDH-rhodamine-positive cells from dissociated newborn mouse cerebral hemispheres, passage 4, 8 days in culture. X 2079. (d) GPDH-rhodamine-positive cells from dissociated, aged (18-month-old)mouse cerebral hemispheres, passage 5, 25 days in culture. X1463. (From Vernadakis et al., 1984.)
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mice; however, similar cells were also observed in the cultures of late passage initially prepared from newborn mouse brain. Somatic polyploidy occurs widely among both plants and animals, but its function is unclear. The phenomenon has been associated and/or correlated with such diverse factors as age, hormonal action, cell enlargement, diet, and resistance to mutagenesis (D’Amato, 1977; Epstein, 1967; Evans, 1976; Gerzeli and Barni, 1976; Mysliwski et al., 1977; Nagl, 1978). Extensive evidence from studies of fibroblasts during in Vitro aging supports the view that polyploidy increases during in Vitro aging (Kaji and Matsuo, 1981; Matsuo et al., 1982). Biochemical characterization of these cultures derived from newborn or aged mouse brains, using GS and CNP activities as markers, provided complimentary data to the morphological findings. In cultures derived from newborn mouse cerebral hemispheres, CNP activity increased and remained rather constant with cell passage. In contrast, in cultures derived from aged mouse cerebral hemispheres, CNP increased at passage 2 (P2), decreased at P4, and remained at that level at all subsequent cell passages. Thus, the responsiveness of CNPcontaining glia, presumptively oligodendrocytes, to cell passage remains more or less constant. In contrast, the changes in GS activity with cell passage were dramatic. Whereas GS activity did not change with cell passage in cultures derived from newborn mouse brain, GS markedly increased in cultures derived from aged mouse brain, reaching a 20-fold level by passage 11. This marked GS activity coincides temporally with the appearance of large polyhedral multinucleated cells (see Fig. 18). Lapham (1962) has reported that in reactive protoplasmic astrocytosis, there occurs an increased number of nuclei without increased proliferation, suggesting that nuclear division in reactive protoplasmic astrocytes may have a special adaptive significance with greater functional capacity. The possibility that in our study multinucleated cells are GS-containing glial cells remains to be evaluated. In accordance with our observations in C-6 glioma cells discussed earlier, we have proposed that in culture there is a shift in the types of astrocyte population, with a decline in GFA-positive cells and an increase in GS-containing cells. Recently, Hallermayer and Hamprecht (1984) have reported colocalization of GS and GFA in primary cultures of dissociated cells from newborn mice. FIG.17. Phase-contrast photomicrographs of cultures prepared from mouse cerebral hemispheres. (A and B) Cultures from newborn mouse, cell passage 10, 24 days in culture. (C and D) Cultures from aged mouse (Wmonths-old), cell passage 11, 24 days in culture. Arrows demonstrate GFA-positive immunoreactivity.(From Vernadakis et a l . , 1986.)
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FIG. 18. Phase-contrast photomicrographs of cultures prepared from mouse cerebral hemispheres. Cells were from late passages (P10 or P11) and were grown on the floors of plastic culture petri dishes. (A) Cultures from newborn mouse, cell passage 10, 24 days in culture. (B) Cultures from aged mouse (18 months old), cell passage 11, 24 days in culture. Arrow indicates a multinucleated cell. (From Vernadakis et al., 1986.)
Extrapolating from these observations to ours, it is conceivable that the decline in GFA-positive glial cells (see Fig. 17) and the increase in GS activity represents a shift toward GS-containing glia. This shift in cell types is also evident in the decline of oligodendrocytes and rise in astrocytes with cell passage. Whether these cell phenomena are a result of in vitro conditions or indeed reflect cell plasticity and adaptability warrants consideration in order to begin to understand cell aging. In the context of neuron-glia interactions, the marked activity in GS-containingastrocytes is of importance. As discussed in another section, astrocytes are intimately involved in the compartmentation of glutamate-glutamine-GABA (see also Fig. 12). Increases in GS activity would be expected to lead to increases in both cellular and extracellular glutamine released from astrocytes. Glutamine is a precursor for GABA synthesis and also has been shown to serve as a precursor to glutamate release (see refs. in review by Hertz and Schousboe, 1987). Thus, changes in GS activity in GS-containing astrocytes will be reflected in both
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GABAergic and glutamatergic neuronal activity. The consequences of such shifts in cellular activity during aging of the CNS have only very recently been considered.
B. SYNAPSE FUNCTION There is now evidence that synaptic loss and decline in synaptic function occur in the senescent brain and may play a role in the age-related decline in brain function (for review, see Vernadakis, 1985). Recent studies have attempted to explore the possibility of compensation for neuronal loss by synapse growth in the aged brain (Cotman and Scheff, 1979). Studies in young and mature animals have shown that when neurons die or are destroyed, they leave their target cells with fewer synapses. In some cases, however, the remaining nearby neurons will make new synaptic contacts to replace those lost. This process is referred to as reactive synaptogenesisor axon sprouting (Cotman, 1978; Cotman and Lynch, 1976; Cotman and Nadler, 1978). Cotman and Scheff (1979), using the hippocampus as a model, compared reactive synaptogenesisin the mature and aged brain. In these experiments, the entorhinal cortex was unilaterally removed and the denervated area of the dentate gyrus ipsilateral to the lesion was examined. In this procedure, the contralateral side of the brain serves as the control. The reactive synaptogenesis response of 24-month-old rats to brain lesions was considerably slower than in the younger animals and not as extensive. The series of events occurring in reactive synaptogenesis as proposed by Cotman and Scheff (1979) are illustrated in Fig. 19. In this complex series of events, the glial component is proposed to be involved in the initiation of growth and axon and/or dendritic growth. The role of glial cells in synaptic remodeling and synaptic turnover has been further considered in a recent study by Adam and Jones (1982). Ultrastructural observation of the molecular layer of the parietal cortex of rats aged 3, 6, 10, and 17 months revealed various atypical synaptic profiles, as well as typical synapses. The atypical synapses were frequently in the vicinity of hypertrophied astrocytic profiles and were sometimes completely surrounded by astrocytic processes. The total surface area of astrocytic profiles and the numbers of atypical synapses increased significantly between 3 and 10 months. Moreover, astrocytic acquisition of degenerating terminals was repeatedly observed over this period; however, the most significant increase in astrocytic surface and density of atypical synapses was found by 10 months of age, with no further increase at 17 months. The authors propose that the removal of
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I Degeneration S,
Hormones
Initiation
I Axon and/or Dendritic Growth
FIG. 19. Model for reactive synaptogenesis. Partial denervation upsets normal trophic relationships between input and target neurons and induces a reactive growth response. The initiation of growth is dependent on trophic signals, hormones, and other growth-regulating factors. The glial cells prepare the neuropil for the growth of new synapses and may serve their own regulatory function. Once the growing fibers reach their targets, synapses rapidly form. Reactive synaptogenesis is slower in aged animals, presumably because of a less robust set of events in the initiation phase. Both hormonal and glial influences may act to suppress growth. (From Cotman and Scheff, 1979.)
degenerative debris by astrocytes may be an integral aspect of synaptic turnover. In the aged brain, synaptic replacement and astrocytic activity may be less than optimal, as reflected by the decrease in synaptic numbers at 17 months and no further significant increases of hypertrophied astrocytic profiles at this age. The intimate astrocytic-synaptic relationship appears to be a key factor in synaptic degeneration and remodeling. An imbalance in this relationship would impede synaptic turnover and consequently affect brain plasticity.
VI. Summary
Considerable progress in our understanding of neuron and glial cell interrelationships has emerged during the last decade from in vitro and in vivo studies. Neural culture systems have provided powerful tools to delineate cellular and molecular events. Moreover, the advances in development of immunocytochemical and biochemical specific cell markers has made possible the characterization of complex cell behaviors. Glial cells actively participate in several aspects of neuronal growth and differentiation both by providing cell-cell contact interactions and by secreting neuronal growth-promoting factors. In turn, neurons influence the cellular behavior of both astrocytes and oligodendrocytes, primarily by secreting substances into the microenvironment. Such
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substances as neurohormones and neurotransmitters have been shown to affect several glial functions including electrophysiological responses, energy metabolism, and ionic homeostasis. In several instances these effects appear to be mediated through receptors on glial cells. Astrocytes actively participate in the regulation of the ionic environment. They take up and release several neurotransmitter substances and can modulate the concentration of a neurotransmitter substance at the synaptic cleft and thus monitor neuronal activity. The evidence of neuron-astroglia synaptic contacts supports the view that such contacts are present during early neuroembryogenesisand thus may provide contact signals for neuronal growth. The process of myelination in the CNS appears to be regulated by both neuronal signals to the oligodendrocyte and also intrinsic programming in the oligodendrocytes to produce myelin components. The prevailing view that astrocytes impede regeneration appears to be shifting towards a more favorable notion of the role of these cells in promoting this process. Of interest is the concept that there is a critical period in the ability of astrocytes either to enhance regeneration or to form a gliotic scar and impede this process. The role of glial cells in the aging process of the neuron is only beginning to be appreciated. If glial cells are actively involved in the regulation of the microenvironment, then it follows that any changes in the behavior of glial cells with aging will ultimately affect neuronal function. It is abundantly clear from in vitro studies that glial cells are pluripotential cells with several functional capabilities. Their responsiveness to an environment in which neurons are maturing as compared to an environment where neurons are injured or aging clearly portrays the multifunctional role of the astrocyte. Despite the optimism derived from contemplation of the studies reviewed here, the question of the degree to which neuron-glia interactions are a reflection of in vitro phenomena will continue to be of concern. It is this concern that has brought about the recent “boom” in brain tissue and cell transplantation research. Whether the transplanted neuron will carry on its programmed behavior or express new functions may well be regulated by the neuron-glia unit. Thus, more research focusing on this cell-cell interrelationship should continue to be pursued.
Acknowledgment
The work by the author’slaboratory reported in this review was partially supported by research Grant HD 18894 from NICHD and a grant from the Developmental
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Psychobiology Research Endowment Fund. The able assistance of Ms. Jackie Jones in library research is gratefully acknowledged.
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Vernadakis, A., and Gibson, D. A. (1974). In “Perinatal Pharmacology: Problems and Priorities” 0. Darcis and J. C. Hwang, eds.), pp. 65-77. Raven, New York. Vernadakis, A., and Nidess, R. (1976). Neurochem. Res. 1, 385-402. Vernadakis, A., and Nidess, R. (1979). In “Hormones i n Development” (L. Macho and V. Strbak, eds.), pp. 153-178. VEDA Publ., Bratislava. Vernadakis, A . , and Sakellaridis, N. (1985). In “Progress in Neuroendocrinology” (H. Parvez, S. Parvez, and D. Gupta, eds.), pp. 17-44. VNU Science Press, The Netherlands. Vernadakis, A., and Woodbury, D. M. (1963).J Phurmucol. Ex$. Ther. 139, 110-113. Vernadakis, A., Nidess, R.,Timiras, M. L., and Schlesinger, R. (1976). Exp. Cell Res. 97, 453-457. Vernadakis, A., Nidess, R., and Arnold, B. E. (1979a). In “Neural Growth and Differentiation” (E. Meisami and M. A. B. Brazier, eds.), pp. 27-38. Raven, New York. Vernadakis, A., Nidess, R.,Culver, B., and Arnold, B. E. (1979b). Mech. Ageing Dev. 9 , 553-566. Vernadakis, A., Parker, K., and Norenberg, M. (1980). In “Tissue Culture in Neurobiology” (E. Giacobini, A. Vernadakis, and A. Shahar, eds.), pp. 411-426. Raven, New York. Vernadakis, A., Mangoura, D., Sakellaridis, N., and Linderholm, H. (1984).J. Neurosci. 11, 253-262. Vernadakis, A,, Sakellaridis, N., and Mangoura, D. (1986).J. Neurosci. Res. 16, 397-407. Vernadakis, A., Sakellaridis, N., Mangoura, D., and Davis, D. (1987). In “Model Systems of Development and Aging of the Nervous System” (A. Vernadakis, A. Privat, J. M. Lauder, P. S. Timiras, and E. Giacobini, eds.), pp. 475-490. Martinus Nijhoff, Boston. Virchow, R. (1846). Allg. 2. Psychiutz 3, 424-450. Walz, W. (1982).J. Neurosci. Res. 7, 71-79. Walz, W., and Hertz, L. (1982). J. Neurochem. 39, 70-77. Walz, W., and Hertz, L. (1983a). Bruin Res. 277, 321-328. Walz, W., and Hertz, L. (1983b). J. Neurosci. Res. 10, 411-423. Walz, W., and Hertz, L. (1983~).Prog. Neurobiol. 20, 133-183. Walz, W., and Hertz, L. (1984). J. Neurosci. Res. 11, 231-239. Walz, W., and Hinks, E. C. (1985). Bruin Res. 343, 44-51. Walz, W., and Schlue, W. R. (1982). Bruin Res. 239, 119-138. Walz, W., Wuttke, W., and Schlue, W. R. (1983). Bruin Res. 267, 93-100. Walz, W., Wuttke, W., and Hertz, L. (1984). Bruin Res. 292, 367-374. Watson, W. E. (1974). Physiol. Rev. 54, 245-271. Waxman, S. G. (1986). In “Glial-Neuronal Communication in Development and Regeneration” (H. H. Althaus and W. Seifert, eds.), NATO AS1 Series, pp. 711-736. Springer-Verlag, New York. Weibel, M., Pettman, B.,Daune, G., Labourdette, G., and Sensenbrenner, M. (1983). In “Hormonally Defined Media” (G. Fischer and R. J. Wieser, eds.), pp. 229-231. Springer-Verlag, New York. Wessels, N. K., Letourneau, F! C., Nuttall, R. P., Luduena-Anderson, K., and Geiduschek, J. M. (1980). J. Neurocytol. 9, 647-664. Westermark, B. (1976). Biochem. Biophys. Res. Commun. 69, 304-310. White, H. S . , and Woodbury, W. M. (1987). In “Model Systems of Development and Agingof the Nervous System” (A. Vernadakis, A. Privat, J. M. Lauder, P. S. Timiras, and E. Giacobini, eds.), pp. 171-191. Martinus Nijhoff, Boston. White, H. S., Honda, T., Chow, S . V., and Woodbury, P. M. (1986). In “Astrocytes” (S. Fedoroff and A. Vernadakis, eds.), Vol. 2, pp. 239-250. Academic Press, Orlando, Florida.
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Wilkin, G. P., Levi, G., Johnstonne, S. R., and Riddle, P. N. (1983). Deu. Bruin Res. 10, 265-277. Wittkowski, W. (1967). Actu Anat. 67, 338-360. Wolff, B. B., Harden, T. K., Sporn, J. R., and Molinoff, P. B. (1978). J. Pharmucol. Exp. Thez 207, 446-457. Wolpaw, E. W., and Martin, D. L. (1984). Bruin Res. 297, 317-327. Wong, T. Y., Hoffman, D., Dreyfus, H., Louis,J. C., and Massarelli, R. (1982). Neurosci Lett. 29, 293-296. Wood, P. M. (1976). Bruin Res. 115, 361-375. Wood, P. M., and Bunge, R. P. (1975). Nature (London) 256, 662-664. Yamamoto, K., Yamamoto, M., and Ooka, M. (1977). Exp. Cell Res. 108, 87-93. Yamamoto, M., Chan-Palay, V., and Palay, S. L. (1981). Anut. Embryol. 159, 137-149. Yarom, Y., and Spira, M. E. (1982). Science 216, 80-82. Zeller, N. K . , Hunkeler, M., Campagnoni, A. T., Sprague, J., and Lazzanni, R. A. (1984). PTOC.Natl. Acud. S C ~ U.S.A. . 81, 18-22. Zeller, N. K., Behar, T. N., Dubois-Dalcq, M. E., and Lazzarini, R. A. (1985). J. Neurosci. 5, 2955-2962.
CEREBRAL ACTIVITY AND BEHAVIOR: CONTROL BY CENTRAL CHOLINERGIC AND SEROTONERGIC SYSTEMS By C. H. Vanderwolf Department of Psychology The University of Western Ontario London, Ontario, Canada, N6A 5C2
I. Introduction 11. Hippocampal Activity in Relation to Behavior
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A. Slow Waves B. Unit Activity C. Place Cells D. Behavior and Mental Processes Neocortical Activity A. Genesis of the Electrocorticogram B. Neocortical Activity in Relation to Movement Cholinergic and Serotonergic Control of Cortical Activation A. Hippocampus B. Cellular Mechanisms in the Hippocampus C. Neocortex: Cholinergic Control D. Neocortex: Serotonergic Control E. Neocortex: Mechanisms of Activation F. Cingulate Cortex G. Role of the Thalamus in the Control of Neocortical Activity H. Behavioral Effects of Central Cholinergic and Serotonergic Blockade Sleep and Waking A. Is There a Sleep Center in the Basal Forebrain? B. Does Serotonin Promote Slow Wave Sleep? Interpretation and Discussion References
1. Introduction
A major long-term aim of neurobiology is to provide a comprehensive account of how the neurochemical and electrical activity of the nervous system generates behavior. A well-developedscience of behavior, capable of posing fundamental questions that neurobiology must attempt to answer, is an essential requirement of this program. However, at the present time, there appears to be very little general agreement on how INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 30
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behavior should be studied or even on the identification of the major problems in the field. Studies of animal behavior focus on extensive observation of spontaneous behavior under natural or seminatural conditions (Alcock, 1984). In contrast, psychological studies of animals rely very heavily on contrived tests of learning such as mazes or lever boxes. Human behavior is usually studied by paper and pencil tests or by having subjects press keys while watching a visual display. For many people, the central problem in this entire field is a centuriesold philosophical conundrum. How does the activity of the conscious mind, known introspectively, relate to the activity of the brain? According to time-honored concepts, the mind consists of a number of component processes. William James (1890) entitled chapters of his famous book “The Principles of Psychology” with such terms as attention, memory, sensation, imagination, reasoning, and the emotions. Much neurobiological work in this century has had as one of its aims the discovery of the neural basis of mental processes of the type that James discussed (Delafresnaye, 1954; Eccles, 1966; Popper and Eccles, 1977; Buser and Rougeul-Buser, 1978). However, it has also been the case that in this century the traditional philosophical and psychological account of the mind has been challenged by repeated demonstrations that introspection reveals little of the activity of the mind (Hebb, 1977, 1980; Lyons, 1986). It has been claimed that knowledge of the mind (one’s own as well as others) is inferential rather than direct, and that we observe behavior and external circumstances and then infer whether a particular psychological process is present or not. In the natural sciences there is a long tradition of rigorous distinction between observation and inference. In keeping with this, it can be argued that descriptions of behavior should be made initially in terms of what can be observed, that is, in terms of overt motor activity and the correlation of such activity with the presence of possible controlling factors such as features of the physical environment. Inferences, including those concerning internal psychological states, should be made only after the observable facts have been ascertained and clearly described. This review will describe attempts to apply this simple precept to some longstanding problems in mammalian neurobiology. Much of the existing neurobiological literature that is relevant to behavior is based on traditional philosophical and psychological concepts. Twentieth century analyses of the mind and behavior have had little influence as yet. A prominent example of this is the conventional theory of the electrocorticogram and the reticular formation. It has been known for several decades that electrical stimulation of the medial
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brainstem can transform neocortical electrical activity from a pattern consisting of large slow waves associated with widespread synchronized bursts of neuronal action potentials to a pattern consisting of low-voltage fast activity (LVFA) associated with a continuous asynchronous barrage of firing in principal neocortical neurons (Moruzzi and Magoun, 1949; Moruzzi, 1972; Steriade, 1981). This effect, commonly referred to as cortical activation, is attributed to an action of the ascending reticular activating system. The discovery of this system constituted a major landmark in the history of mammalian neurobiology, appearing to provide a broad avenue for the physiological investigation of the previously inaccessible regions of the mind, of attention, motivation, emotion, and of consciousness itself (Delafresnaye, 1954). The broad outlines of the conventional view of the reticular activating system and its influence on cortical activity and behavior can be found in any comprehensive contemporary textbook in neuroscience (Mountcastle, 1980; Brodal, 1981; Kandel and Schwartz, 1985; Carlson, 1986). It is generally assumed that cortical activation is dependent on projections from the mesencephalic reticular formation to the medial thalamus, especially the intralaminar nuclei, Ascending projections from the intralaminar nuclei are thought to permit local or generalized activation of the neocortex, thus altering the pattern of the spontaneous electrocorticogram, cortical evoked potentials, and the discharge patterns of cortical neurons (Steriade, 1981). High levels of activity in this ascending pathway are assumed to provide the basis for alertness, attention, vigilance, or excitement whereas low levels of activity are assumed to be associated with relaxation, sleep, or coma. A few years ago, T.E. Robinson and I summarized the available evidence to show that the conventional view of cortical activation is wrong in several respects. Cortical activation does not correlate closely with consciousness or sleep as those words are conventionally understood. Animals, including humans, may display either cortical activation or nonactivation regardless of whether they are awake, asleep, anesthetized, or in coma following a brainstem lesion (Vanderwolf and Robinson, 1981). As this review will show, neocortical activity does, in fact, correlate very closely with behavior, but not in a way that can be related to folk psychological concepts such as attention or consciousness. New evidence will be discussed here showing that cortical activation is dependent on both cholinergic and serotonergic inputs to the neocortex. Ascending cholinergic and serotonergic pathways innervate both the thalamus and the neocortex. It may be that the thalamus and the cerebral cortex are activated in parallel and that thalamic activation is not the primary cause of neocortical activation.
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The newer view of the nature of cortical activation developed from experiments on the relation between behavior and the electrical activity of the hippocampal formation. Therefore, research on the hippocampus will be discussed, but only in a brief outline, since several recent reviews and symposia are already available (Buzsski, et al., 1983; Seifert, 1983; Buzsziki and Vandenvolf, 1985; Bland, 1986; Isaacson and Pribram, 1986). A brief review of current knowledge of the genesis of the electrocorticogram will also be presented to provide a basis for understanding the nature of cortical activation.
II. Hippocampal Activity in Relation to Behavior
A. SLOWWAVES The fact that spontaneous hippocampal activity changes from moment to moment in close correlation with concurrent motor activity was originally demonstrated by recording spontaneous behavior (using a keyboard) and hippocampal slow wave activity simultaneously on a polygraph. It was observed that a pattern of rhythmical slow activity (RSA, or 8 rhythm) is present when a rat turns its head, takes a step forward, changes posture, walks, runs, swims, climbs, jumps, or manipulates objects with its forelimbs (voluntary or Type 1 behavior, Fig. 1). A pattern of large amplitude irregular activity (LIA) is ordinarily present when a rat stands motionless or engages in a variety of more reflexive activities such as licking, gnawing, face washing, or shivering (automatic or Type 2 behavior). During a mixed sequence of Type 1 and Type 2 behavior, the pattern of activity observed in the hippocampus varies in close temporal correspondence with the concurrent motor activity. For example, a rat may walk to a water dish (RSA present); stop walking while lowering the head (RSA present); lick water (LIA present); raise the head and sit up (RSA present) and begin washing its face (LIA present); cease face washing, with no change of posture, and remain motionless for several seconds in response to an unfamiliar sound (LIA present) (Vandenvolf, 1969; Vanderwolf et al., 1973, 1975). If Type 1 and Type 2 behaviors are performed simultaneously, RSA is always present. For example, sniffing in rats is usually associated with head movements and locomotion and is then accompanied by RSA. If rats sniff while remaining immobile, LIA is present in the hippocampus. Thus, it appears that RSA is related to head movement and locomotion rather than sniffing per se.
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FIG.1. Hippocampal activity in relation to behavior in the rat. Type 1 behavior includes walking, running, jumping, swimming, rearing, digging, manipulation of objects with the forelimbs, isolated movements of the head or one limb, and shifts of posture. Type 2 behavior includes both alert immobility in any posture and licking, chewing, chattering the teeth, sneezing, startle response, vocalization, shivering, tremor, face washing, scratching the fur, pelvic thrusting, ejaculation, defecation, urination, and piloerection. (A) Walking is associated with rhythmical slow activity (RSA) whereas alert immobility is associated with large amplitude irregular activity (LIA). (B) A dentate granule cell, identified by electrophysiologicalcriteria, fires at a high rate during walking and a low rate during drinking. (C) Evoked potentials occurring in stratum pyramidale (upper trace) and stratum radiatum (lower trace) of field CA1 following stimulation of the angular bundle during walking (Type 1 behavior) or drinking (Type 2 behavior). Ten superimposed sweeps. The early in-phase potentials are volume-conducted from the dentate gyrus. Note that phase-reversed evoked potentials often occur in CA1 during drinking but never occur during walking. The traces shown were extracted from Vandenvolf et al. (1975) and Buzsiki et al. (1983).
It is interesting that phasic muscular activity such as a postural change or locomotion is consistently accompanied by RSA but that the maintenance of a static posture is accompanied by irregular activity even when considerable muscular effort is involved. Thus, RSA does not appear in a rat standing totally motionless on its hind legs or hanging motionless from the edge of a table supported by its forepaws. Such
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examples also suggest that the absence of RSA is not related to relaxation or to an absence of muscular effort. This is confirmed by other data. Heart rate correlates very closely with oxygen uptake and can be regarded as a convenient index of the metabolic requirements of various motor activities (Carlsten and Grimby, 1966). Since face washing (not associated with RSA) is accompanied by a higher heart rate in rats than walking (always associated with RSA) it is evident that the absence of RSA is not necessarily associated with muscular relaxation and inactivity (Vanderwolf and Vanderwart, 1970). The question of whether RSA is involved in the causal sequence that leads to motor activity or whether it is a consequence of sensory feedback or corollary discharge has been partially answered. First, passive movements generally do not produce RSA, especially the atropineresistant type (see Section IV,A and Vanderwolf, 1975), suggesting that RSA is not produced primarily by sensory input from moving body parts. Experiments on curarized animals point to the same conclusion. Black (1975), using a shock-avoidance procedure, trained dogs to press a lever upon presentation of one auditory signal and to refrain from pressing during the presentation of a second signal. Clear RSA occurred in response to the first signal, but not to the second, both in the normal state and following general muscular paralysis induced by gallamine triethiodide (Flaxedil) (Black and Young, 1972) (Fig. 2). More remarkably, it is possible to use shock avoidance techniques to train gallamine-treated dogs to display RSA in response to one auditory signal and to refrain from displaying RSA in response to a second auditory signal (Black et al., 1970; Dalton, 1969). When the dogs have recovered from the gallamine-induced paralysis, they respond to the first signal by struggling and attempting to escape from the apparatus (accompanied by RSA) and respond to the second signal by remaining motionless (Black, 1970; Black et al., 1970). These experiments indicate that RSA is closely related to the activity of central mechanisms that control voluntary motor activity and that sensory feedback from moving body parts does not play an important role in the generation of RSA. If this is true, then RSA might be expected to occur somewhat in advance of the motor activity that it accompanies. Anticipatory activity of this type can be readily demonstrated in rats trained to jump out of a box and up to a landing platform to avoid an electric shock delivered through the grid floor of the apparatus. In this situation the rats rear up on their hind legs several seconds before jumping, remaining almost totally motionless until the instant that the hind legs are abruptly extended. In well-trained rats, hippocampal RSA of about 6 Hz may precede the act of jumping by as much as several seconds. Beginning
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about 0.5 sec prior to the jump, the RSA frequency rises to 8-12 Hz, reaching a peak at jump initiation or immediately afterwards as the rat scrambles up on the landing platform (Vanderwolf, 1969; Whishaw and Vanderwolf, 1973; Morris and Hagan, 1983). If the rise in RSA frequency fails to occur, the rat invariably fails to jump. This can be demonstrated by fatiguing well-trained rats by forcing them to jump hundreds of times in quick succession (Vanderwolf and Cooley, 1974) (Fig. 3) or by treatment with neuroleptic drugs to produce a partial failure of avoidance response (Vanderwolf, 1975). At first glance, these data provide strong support for the idea that hippocampal RSA is related to the generation of a voluntary movement. However, on closer examination they raise an important problem. It
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becomes evident that clear RSA can be present for a considerable period during total behavioral immobility. During spontaneous behavior in normal rats this is a rare occurrence; prolonged aversive training is evidently a special case (see below). Dogs (Black, 1975; Lopes da Silva and Arnolds, 1978; Arnolds et al., 1979a,b) and Mongolian gerbils (Kramis and Routtenberg, 1969; Whishaw, 1972) rarely or never display clear-cut RSA during behavioral immobility. In rabbits (Harper, 1971; Klemm, 1971; Winson, 1972; Kramis et al., 1975), cats (Bennett, 1975; Kemp and Kaada, 1975; Arnolds et al., 1984; Sainsbury, 1985), and guinea pigs (Sainsbury, 1970), however, clear RSA can be demonstrated readily following the presentation of novel sensory stimuli even though the animals display absolutely no movement and no change in EMG activity (Kramis et al., 1975). Short bursts of RSA can also occur spontaneously during complete immobility. The presentation of other animals to which the experimental subjects have strong instinctive responses appears to be especially effective in producing RSA during immobility. Thus, immobile guinea pigs display clear RSA when presented with a snake or the recorded calls of raptorial birds such as the great horned owl (Sainsbury and Montoya, 1984). Even rats will display RSA during immobility in the presence of a cat or ferret (Sainsbury et al., 1987). Despite the complications introduced by the presence of some RSA during Type 2 behavior, an overall correlation of RSA with motor activity can be readily demonstrated in both cats (Black, 1975; Arnolds et al., 1984) and rabbits and guinea pigs (Sainsbury, 1970; Kramis et al., 1975). Clarification of the problem posed by the fact that RSA can sometimes occur during Type 2 behavior, as well as during Type 1 behavior, came with the discovery that there are actually two distinct types of RSA. Data discussed in Section IV,A indicate that a cholinergic input to the hippocampus produces RSA both during Type 1 behavior and, sometimes, during Type 2 behavior. A serotonergic input appears to produce RSA only during Type 1 behavior. The transition from a pattern of large amplitude irregular activity to one of RSA is only one aspect of a very general change in hippocampal activity. For example, a spectral analysis by Leung et al. (1982a) showed that power in the 40- to 60-Hz waveband increased significantly during the presence of RSA and Type 1 behavior. In succeeding sections data will be reviewed to show that unit activity and evoked potentials in the hippocampus also co-vary with the RSA pattern (see Sections 11,B; IV,A; and IV,B). The primate hippocampus does not appear to generate the clear RSA seen in rats, rabbits, cats, and dogs, but its activity is nonetheless correlated with motor activity. Arezzo et al. (1987) showed that a
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well-practicedself-initiated extension movement of the wrist in macaque monkeys is preceded and accompanied by a complex “motor potential” and multiple unit discharge in the contralateral hippocampus (Fig. 4). Concurrent with this, a brief period of reduced voltage and increased frequency activity is often visible in the spontaneous hippocampal slow wave activity. Possible correlations of human hippocampal activity with motor activity have not been extensively explored. Clear rhythmical waves of 5-7 Hz have been observed in the hippocampus of a 12-year-oldpatient during electrical stimulation of the posteromedial hypothalamus (Sano et al., 1970). The overall effect is somewhat similar to the waves that can be observed during stimulation of the posterior hypothalamus in rats (Bland and Vandenvolf, 1972a). Arnolds et al. (1980) reported a spectral component of 3-5 Hz in hippocampal activity that increased in frequency and rhythmicity during writing and speaking, but not
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FIG. 4. Motor potentials (MP) and multiunit activity (MUA) recorded simultaneously within the hippocampal formation and precentral neocortex of a macaque monkey performing a well-trained, self-paced extension movement of the wrist. Records are taken contralateral to the moving limb. The arrow indicates the onset of muscle contraction. N,,NZa, and NZb refer to successive negative potentials; PZ to the positive potential. EMG, electromyogram. (From Arezzo et al., 1987, by permission of Elsevier Science Publishers B.V.)
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during walking, in one epileptic patient. Halgren et al. (1978), however, reported a desynchronization during movement that is similar to the results of Arezzo et al. (1987).
B. UNITACTIVITY Research by O’Keefe and Dostrovsky (1971) and Ranck (1973) showed that two major types of cells can be identified by electrophysiological criteria in the hippocampus of the freely moving rat. These cell types, usually referred to as 8 cells and complex spike cells, differ in several ways including spike morphology and duration, firing rate and pattern, and relation to behavior. Complex spike cells sometimes discharge single action potentials, but at other times they fire in bursts of two to seven individual spikes of progressively declining amplitude and an interspike interval of 1.5-6 msec (complex spikes). Ranck defined hippocampal 8 cells as cells that fire at a high rate if, and only if, a 8 (RSA) rhythm is present in the concurrent slow wave activity. The 8 cells display shorter duration spikes than complex spike cells, often fire at considerably higher rates, and never display complex spikes. Based on anatomical location and the response to stimulation of hippocampal fiber tracts, complex spike cells in Ammon’s horn appear to be projection neurons (mainly pyramidal cells) whereas 8 cells may be interneurons (Fox and Ranck, 1981). A series of investigations has led to a general consensus that the extracellular RSA rhythm in rats and rabbits is produced by synaptically induced current flows in both CA1 pyramidal cells and dentate granule cells (Winson, 1974; Bland et al., 1975; Bland and Whishaw, 1976; Buzsaiki et al., 1983; Leung, 1984a,b; Bland, 1986). However, the mechanism by which the RSA is produced and timed remains rather obscure. The 8 cells (putative interneurons) in the hippocampus tend to fire rhythmically in phase with RSA and at a high rate during Type 1 behavior but fire more sporadically and at a lower rate during the LIA accompanying Type 2 behavior (O’Keefe and Dostrovsky, 1971; Feder and Ranck, 1973; Ranck, 1973; Buzsaiki et al., 1983). Complex spike cells (putative pyramidal cells) tend, as a class, to fire at a rather low rate when Type 1 behavior and RSA occur, but individual cells sometimes fire at relatively high rates, also with a tendency to discharge in phase with the local RSA (Buzsaiki et al., 1983; O’Keefe and Dostrovsky, 1971; Feder and Ranck, 1973; Ranck, 1973; Fox and Ranck, 1975, 1981; O’Keefe, 1976). Dentate granule cells fire in relation to RSA and Type 1 behavior in much the same way as hippocampal 8 cells do [Buzsaiki et al., 1983;
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Rose, 1983 (see Fig. l)]. Firing rates are very low during immobility and other Type 2 behavior but increase dramatically during walking, running, and head movements. This finding is of considerable importance since most of the afferent fibers to the hippocampus consist of projections from the entorhinal cortex to the dentate gyrus and Ammon’s horn (RamBn y Cajal, 1911; Blackstad, 1956, 1958; Steward, 1976; Steward and Scoville, 1976). The entorhinal-dentate projection is the first step in the well-known trispaptic pathway, which includes perforant path projections from the entorhinal cortex to the dentate gyrus, mossy fiber projections from dentate granule cells to CA3, and Schaffer collateral projections from CA3 to CA1 (RamBn y Cajal, 1911; Andersen, 1975). An interesting aspect of the correlation of hippocampal activity with concurrent motor activity is the sporadic occurrence of 30- to 120-msec sharp waves in CA1 during Type 2 behavior and the LIA pattern associated with it. These sharp waves in CA1, associated with bursts of firing in CA1 complex spike cells and theta cells, appear to be caused by prior bursts of firing in CA3 complex spike (pyramidal) cells and the Schaffer collaterals, but associated discharges also occur in the dentate gyrus and subiculum (Buzsaiki et al., 1983; Buzsaiki, 1986; Suzuki and Smith, 1985). Hippocampal sharp waves are strongly suppressed during Type 1 behavior and the associated RSA. Stimulation of the perforant path during LIA and the associated Type 2 behavior tends to produce, in CA1, large evoked potentials with a morphology and phase reversal similar to those of the spontaneously occurring sharp waves. The similarity between these evoked potentials and the spontaneous sharp waves suggests that the latter are due to spontaneous activity generated within the trisynaptic entorhinal-to-CA1pathway. During Type 1 behavior and the associated RSA, potentials evoked in CA1 by perforant path stimulation are strongly suppressed (Buzsaiki et al., 1983; Buzsaiki, 1986; Fig. l),indicating that transmission through the trisynaptic pathway is closely regulated in correlation with RSA and Type 1 behavior. The suppression of CAI sharp waves when RSA is present also suggests that intrahippocampal transmission is closely regulated by afferent input during Type 1 behavior and is relatively unregulated during Type 2 behavior. In agreement with this, it has been found that potentials evoked in CA1 by stimulation of the Schaffer collaterals ( h u n g , 1980, 1985), the interhemispheric commissural fibers, or the perforant path input to the dentate gyrus (Segal, 1978a; Buzsaiki et al., 1981) vary in form depending on whether stimulation is applied during Type 1 behavior and the accompanying RSA or during Type 2 behavior and the accompanying irregular activity. Winson (1986) and Winson and Abzug (1978), who carried out experiments similar to thoseof Buzsaiki et al. (1983), reported somewhat
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different results. Major differences were found between “still alertness” and slow wave sleep, with more variable results when RSA was present. “Still alertness” in Winson’s experiments apparently consisted of the behavioral immobility produced by startling a rat with a loud noise (Winson and Abzug, 1978, p. 719). If a rat in slow wave sleep is suddenly awakened by a noise, the resulting hippocampal slow wave activity is likely to consist of small amplitude irregular activity (SIA), which appears to be a very low-amplitude form of LIA (Whishaw and Vanderwolf, 1973; h u n g et al., 1982a). It may be that transmission through the trisynaptic circuit during SIA is suppressed in comparison with transmission during normal spontaneous LIA, thus accounting for the differing results of Winson (1986) and Buzsa’ki et al. (1983). In agreement with this hypothesis, Buzsaiki (1986) has recently shown that a startling stimulus suppresses hippocampal sharp waves regardless of whether RSA and Type 1 movement are elicited or not.
C. PLACECELLS When a rat is placed in a spatially extended environment such as an open field or maze, individual complex spike cells display a remarkable tendency to fire at high rates in specific locations (place fields) that differ from cell to cell. Such units have been called place cells by O’Keefe (O’Keefe and Dostrovsky, 1971; O’Keefe, 1976). The consensus from a number of studies of this phenomenon (O’Keefe, 1979) is that locationspecific high rates of firing are controlled by a complex of environmental stimuli rather than any specific stimulus such as local odor or a specific visual feature. Firing is also broadly independent of specific concurrent motor acts, although it is true that place fields are better defined during RSA and the associated Type 1 behavior than during LIA and Type 2 behavior (Kubie et al., 1985). O’Keefe and Nadel (1978) proposed that these empirical findings indicate that hippocampal pyramidal cells are elements in a “cognitive map” in which absolute Euclidean space is represented in the brain. According to this idea, a rat could determine its location in space by consulting, in some way, the firing rate of a number of place cells. However, recent data suggest that this interesting idea may not be entirely correct. A single place cell may have more than one field, leading to an ambiguous signal with respect to spatial location (Muller et al., 1987; Muller and Kubie, 1987). More importantly, introducing a barrier into a place field modifies it profoundly regardless of whether the barrier is opaque fiberboard or a transparent sheet of Plexiglas (Kubie et al., 1985; Muller and Kubie, 1987). A low barrier over which a rat can easily
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climb has very little effect on spatial fields. These observations may suggest that place cells fire in relation to the behavioral possibilities at a particular location (i.e., whether or not a barrier impedes movement) rather than the specific stimulus characteristics or absolute position in space. A further complication is introduced by the demonstration that 83% of a sample of complex spike cells increased their rate of firing on presentation of the conditioned stimulus (tone) in a corneal air puff and nictitating membrane conditioning task in rabbits, even though the rabbits were restrained from moving about (Berger et al., 1983). It would appear that the behavioral correlates of complex spike cell firing are, as yet, incompletely understood.
D. BEHAVIOR AND MENTALPROCESSES An important early step in the scientific study of the behavior of any species of animal is the compilation of a catalog (sometimes called an ethogram) of the behaviors, primarily the postures and movements, it displays (Tinbergen, 1951). Similarly, an early step in brain-behavior research should include a wide-ranging investigation of possible relations between the various items of behavior and the electrical activity of different brain structures. It is astonishing how seldom this simple approach has been applied. Implicit acceptance of traditional philosophical and psychological accounts of the mind appear to have diverted investigators from simple, straightforward studies of behavior. In the case of the spontaneous activity of neurons in the reticular formation of the lower brainstem, for example, the existence of clear-cut correlations between firing rate and the occurrence of specific motor patterns was recognized only after a large number of much more complicated hypotheses had been shown to be inadequate (Siegel, 1979). In the case of hippocampal slow waves, the publication of simple descriptions of the correlations between electrical activity and concurrent motor activity has been the cause of considerable controversy, as shown, for example, by the commentaries on the paper by Vanderwolf and Robinson (1981). It appears to be widely held that simple descriptions of motor activity are inadequate as a starting point for the analysis of the brain mechanisms of behavior and, further, that a realistic approach to the mammalian brain and behavior must make use of traditional philosophical and psychological concepts such as attention, emotion, motivation, cognition, and memory. In fact, many theorists have discussed hippocampal activity in precisely such terms (Black, 1975).
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This raises a general problem that is applicable to neurochemistry, neuropharmacology, and the new brain-imaging techniques as well as to electrophysiologicalstudies relating to behavior. How can one determine whether a given physical measure of brain activity is related to observable motor activity or to a hidden psychological process? If the latter, how can one identify the relevant psychological process from among the large number of possibilities? Skinner (1974, pp. 207-208) lists 68 common English terms for psychological processes; undoubtedly, more could be added. In everyday usage, folk psychological terms such as attention, fear, belief, or desire refer to rather complex situations. A classic study by Hebb (1946) showed that the attribution of emotions such as fear, rage, hate, shyness, or nervousness to chimpanzees by human observers involved a knowledge not only of the chimpanzee’s present behavior and the present environmental circumstances, but also of its past behavior and experience. Identical motor performances in two different animals might be ascribed to different emotions by observers as a result of taking a number of such factors into account. Presumably this is also true when humans attribute emotions and other psychological entities to one another (Hebb, 1946). For this reason, the ordinary terms of folk psychology are seldom descriptive of overt motor activity in any simple way. For example, someone may be described as closely attentive while sitting motionless listening to a lecture or while walking down a street listening to the talk of a friend. The term “fear” may be applied in cases of behavioral immobility (freezing behavior, “scared stiff,” paralysis of terror) or vigorous movement (rapid flight). Learning is said to occur equally in students sitting motionless in a classroom or walking on a field trip with their instructor. The foregoing examples suggest that folk psychological processes, whatever their ultimate nature may be, can occur during both immobility and movement. This, in turn, suggests a general procedure for distinguishing the brain correlates of Type 1 behavior from the brain correlates of psychological processes. Experimental situations can be created in which a given psychological process is believed to be present during both immobility and Type 1 movement and in which the process is believed to be absent during both immobility and Type 1 movement. Table I outlines the general strategy with respect to learning and memory, but similar analyses can be applied to attention or various motivational or emotional states. Systematic experimentation in this field has usually involved training animals to move (press a lever, run, etc.) in the presence of one discriminativestimulus and refrain from moving in the presence of a different discriminative stimulus. Perception, attention,
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TABLE I EXPERIMENTAL DESIGN TO DISTINGUISH BRAINCORRELATE, OF MOVEMENT FROM THOSE OF LEARNING AND MEMORY Behavior
Movement condition
Immobility condition
Specially trained
Lever pressing, running, walking, etc. to avoid shock or obtain food
Passive avoidance (i.e., learning to remain motionless to avoid shock), training with reinforcement contingent on immobility
Untrained
Running, walking, etc., spontaneously or following novel sensory stimuli
Spontaneous immobility in the alert state; “freezing” in response to novel sensory stimuli
motivation, and memory are presumably required under both circumstances but movement is required in only one of the two. The training techniques have involved avoidance of electric shock (Dalton and Black, 1968; Bland and Vanderwolf, 1972a; Black, 1975; Vanderwolf and Ossenkopp, 1982), food reward (Morris and Black, 1978), or rewarding intracranial self-stimulation (Paxinos and Bindra, 1970). In rats and dogs, without exception, clear RSA was found to occur in association with Type 1 movement but did not occur when correct responding consisted of remaining motionless. It is also the case that RSA is associated with Type 1 movement regardless of whether such movement can be assumed to be associated with low or high levels of attention, memory, and motivation (Vanderwolf et al., 1975). In sum, the experimental evidence shows quite clearly that hippocampal RSA is correlated primarily with the occurrence of Type 1 behavior and is not directly related to attention, learning, memory, or motivation in the sense in which such terms are understood in everyday speech. Previous proposals that hippocampal RSA is related to inferred psychological processes have generally failed to take into account the correlation of RSA with motor activity (Whishaw and Vanderwolf, 1973; Vanderwolf and Leung, 1983; Vanderwolf and Ossenkopp, 1982). The situation is complicated but not fundamentally altered by the existence of a distinct type of RSA that can occur at times during Type 2 behavior. In this review, the term “behavior” refers primarily to the overt postures and movements that animals display. Thus, “sleep”in a rat refers to a behavior comprising long periods of immobility with the head resting on the substrate, the eyes partly or totally closed, and the tail and trunk
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frequently in a curled-up position. “Waking immobility” refers to a behavior in which the eyes are usually open and the head is supported against gravity. A longer list of behaviors that is relevant to gross cerebral electrophysiology is given in Vanderwolf et al. (1987). This approach emphasizes simple descriptions of what can be observed with a minimum of inference. In particular, it is not assumed, on an a priori basis, that the traditional philosophical and psychological subdivisions of the mind can be applied directly to the organization of the brain (Vanderwolf, 1983).
111. Neocortical Activity
A. GENESIS OF
THE
ELECTROCORTICOGRAM
Theoretical understanding of how synaptic potentials and propagated action potentials produce extracellular field potentials is most advanced for simple cortical structures such as the olfactory bulb, prepyriform cortex (Freeman, 1975; Shepherd, 1977), cerebellar cortex (Nicholson and Llina’s, 1971; Spencer, 1977), and hippocampal formation ( h u n g , 1984b). However, in the neocortex, the variety of cell types and the complexity of their anatomical interconnections have, so far, precluded any comprehensive quantitative theoretical analysis (Elul, 1972; Mitzdorf, 1985). It has long been thought that the main source of the electrocorticogram is the occurrence of localized synaptic potentials in the dendrites and somata of extended vertically oriented cells in the neocortex (Purpura, 1959). In support of this, there is consistent evidence of the existence of vertical flows of current in the cortex during the occurrence of slow waves or spindle waves but no evidence of tangential current flows (Calvet et al., 1964a; Ball et al., 1977a,b). For this reason alone, it seems probable that the large, deeply located pyramidal cells, with apical dendrites that extend radially to the cortical surface, must play a major role in the genesis of the electrocorticogram, as was originally suggested by one of the pioneers of cerebral electrophysiology (Bremer, 1938). An important development in understanding the spontaneous electrocorticogram was the demonstration by Spencer and Brookhart (1961a,b) that spontaneous spindle bursts in cerveau isolC cats consisted of two types of waves. Type 1 waves, consisting of initially surface-positive, deepnegative potentials, were consistently associated with bursts of multiunit activity in layers III-V of the cortex. Type 2 waves, consisting of
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primarily surface-negative, deep-positive potentials, had a weaker relation to multiunit activity in layers III-V. Spencer and Brookhart suggested that these spontaneous waves resembled certain waveforms elicitable in the neocortex following electrical stimulation of various thalamic nuclei. A role of the thalamocortical pathways in the control of cortical electrical activity had been demonstrated in earlier research by Bishop, Bremer, Dempsey, Jasper, Morison, and others (Andersen and Andersson, 1968). This early work had shown that a single pulse stimulus to a specific sensory nucleus such as ventralis lateralis or ventralis posterolateralis evokes a short-latency, surface positive-negative wave sequence, the primary response, in the sensorimotor area. During repetitive stimulation at a low frequency the primary response is replaced by a longer-latency, surface positive-negative wave sequence of increased amplitude, the augmenting response. The initially surface-positive(deepnegative) component of the augmenting response is associated with increased unit discharge in the deep layers of the neocortex. Thus, augmenting waves resemble Type 1 spindle waves. This suggests that Type 1 spindles may be dependent on a repetitive thalamocortical discharge involving the classical thalamic relay nuclei. There is a great deal of evidence supporting this concept (Andersen and Andersson, 1968; Steriade and Deschenes, 1984). However, despite extensive analysis, the basis of the augmenting response has been difficult to elucidate. It has been attributed to activation of specific thalamocortical afferents (Sasaki et al., 1970), to the development of activity in intrinsic neocortical pathways (Morin and Steriade, 1981). and to the postsynaptic response of neocortical cells activated by the axonal collaterals of antidromically driven corticothalamic cells whose somata lie in layer VI (Ferster and Lindstrom, 1985a,b). If the latter suggestion is correct, it may suggest that there is a powerful activation of the layer VI corticothalamic cells during spontaneous spindle bursts. Spencer and Brookhart suggested that their Type 2 spindles were analogous to the recruiting potentials that are elicited by low-frequency repetitive stimulation of the medial thalamus, especially the intralaminar nuclei (Morison and Dempsey, 1942; Jasper, 1949). The recruiting response consists of a series of surface-negative, deep-positivepotentials (Li et al., 1956) which increase gradually in amplitude during the course of a brief train of stimulus pulses. They may or may not be associated with increased firing in cortical neurons. Augmenting responses are more reliably associated with unit discharges in the neocortex than are recruiting responses (Arduini and Whitlock, 1953; Brookhart and Zanchetti, 1956).
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Spencer and Brookhart (1961b) and later investigators (Jasper and Stefanis, 1965; Creutzfeldt et al., 1966a-c; Ball et al., 1977a) suggested that the surface-negative, deep-positive type of potential exemplified by the recruiting response and one type of spontaneous spindle wave (Spencer and Brookhart Type 2) may be due to a depolarization by nonspecific afferents from the intralaminar region of the thalamus of the apical dendrites of cortical pyramidal cells, with the cell somata and basal dendrites acting as passive sources. A recent current source density analysis of recruiting waves has confirmed this and added the finding that sinks occur simultaneously in both the superficial and middle layers of the neocortex during recruiting waves (Pellegrini et al., 1987). Intracellular recording from unidentified cells (probably mainly pyramidal cells) shows that recruiting waves and the related spindle waves usually consist of a rhythmical series of depolarizations, which may or may not be sufficient to initiate spike discharges and may (Jasper and Stefanis, 1965) or may not (Creutzfeldt et al., 1966c)be associated with intervening hyperpolarizations. In contrast to the superficial depolarizing effect of stimulating the intralaminar nuclei, stimulation of the classical sensory relay nuclei appears to produce depolarization of cell somata in the deep layers of the neocortex. Thus, during augmenting responses, and also during a Spencer and Brookhart Type 1 spindle, it appears that the apical dendrites of pyramidal cells act as passive sources, giving rise to an initially surface-positive, deep-negative response (Spencer and Brookhart, 1961a,b;Calvet and Calvet, 1965;Jasper and Stefanis, 1965; Creutzfeldt et al., 1966a-c; Sasaki et al., 1970). The region of negativity subsequently invades the superficial layers of the cortex, as well as the deep layers, suggesting a widespread depolarization. This phase in turn is commonly followed by a surface-negative, deep-positivewave associated with hyperpolarization and cessation of firing in deep pyramidal cells. However, according to the data of Creutzfeldt et al. (1966a,b) this surface-negative wave may reverse to positivity well before the hyperpolarization of the deep cells is completed. J. Calvet and his colleagues (Calvet and Calvet, 1965; Calvet et al., 1964a,b, 1973) confirmed many of the observations of Spencer and Brookhart but made the important additional observation that in unanesthetized animals there occurs a long-duration, surface-negative, deep-positive wave associated with suppression of deep multiunit activity and hyperpolarization (recorded intracellularly) of presumed pyramidal cells. On the basis of these findings, Calvet and his colleagues proposed that the electrocorticogram is based on the activity of three vertically oriented generators: A, B, and C (Fig. 5). The A generator produces
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A
B
FIG. 5 . A simple schematization of the relation between slow waves and action potentials in pyramidal cells in the neocortex. Slow wave negativity up. (A) Potentials resulting from depolarization of apical dendrites are associated with a relatively small increase in discharge rate. (B) Potentials resulting from somatodendritic depolarization are associated with a larger increase in discharge rate. (C) Potentials resulting from somatodendritic hyperpolarization are associated with suppression of unit discharges. Modified from Calvet et al. (1964a).
a negativity in the superficial layers of the cortex (from the surface to a depth of about 600 pm) attributable to depolarization of the apical dendrites of pyramidal cells. Waves due to the A generator occur during one type of spontaneous spindle burst (in both unanesthetized and lightly barbiturized animals), during recruiting responses elicited by electrical stimulation of the thalamic intralaminar nuclei, and as isolated events, often intermingled in a sequence with other wave types. Calvet and Calvet (1965) and Calvet et al. (1964a) reported no positivity in the depths of the cortex during the presence of A-wave surface negativity but other authors, using monopolar recording, have demonstrated a deep positive wave occurring simultaneously with a surface negative wave during recruiting responses (Li et al., 1956; Spencer and Brookhart, 1961a). As we have seen, activity of the A generator can elicit spike discharges in pyramidal cells, bat the level of depolarization achieved is often insufficient to do so. Calvet’s B generator gives rise to a surface-positive, deep-negative wave associated with depolarization (recorded intracellularly) and increased firing rates in neurons deep in the cortex (see Fig. 5 ) . B-waves occur during one type of spontaneous spindle activity (Spencer and
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Brookhart Type 1), during augmenting waves produced by electrical stimulation of specific thalamic nuclei (such as the ventral nuclei), as isolated events, or intermingled in a sequence with other wave types. Calvet et al. (1964a) suggested that B-waves are attributable to a somatodendritic depolarization of pyramidal cells with the apical dendrites acting as passive sources. B-waves were subdivided into (1) B1-waves, which occur during spontaneous spindle bursts and have a mean duration of 62 msec and (2) Be-waves, which occur as isolated events or during a sequence of slow waves during natural sleep and have a mean duration of 268 msec (Calvet et al., 1964a). Finally, Calvet’s C generator produces a long-duration, surfacenegative, deep-positive wave associated with hyperpolarization and suppression of spike potentials of cells located deep in the cortex (see Fig. 5 ) . Such waves were also described by Stefanis and Jasper (1965) and Ball et al. (1977b). Calvet suggested that C-waves are due to somatodendritic inhibition of pyramidal cells (Calvet et al., 1964a). Such inhibition might be mediated by interneurons that contain y-aminobutyric acid (GABA) and synapse preferentially on or near the somata of pyramidal cells (Colonnier, 1966; Ribak, 1978). Skrebitsky (Skrebitsky and Sharonova, 1976; Skrebitsky et al., 1980) has published very clear demonstrationsof surfacenegative, deep-positive gross potentials (C-waves)occurring in correlation with hyperpolarization and suppression of spike discharges in unidentified deep cells (probably pyramidal Lells) in the striate cortex of waking rabbits following a light flash. However, C-waves can occur as isolated events and are prominent components of the electrocorticogram during natural slow wave sleep or following treatment with systemic antimuscarinic drugs (Schaul et al., 1978). C-waves are depressed by anesthetics and rarely occur in acute anesthetized preparations (Calvet et al., 1973). Consequently, they have not been extensively studied by electrophysiologists. For example, the frequently cited experiments of Creutzfeldt et al. (1966b) were conducted entirely in barbiturate-anesthetized cats, in which C-waves were, presumably, poorly developed or absent. However, Calvet’s classification does not mention the occurrence of a type of C-wave that can occur in barbiturate-anesthetized animals. Surface-negative, deep-positive waves that are associated with hyperpolarization of deep pyramidal cells have been observed by several authors (Creutzfeldt et al., 1966c; Humphrey, 1968; Pollen, 1964). It may be suggested that the C-waves, like the B-waves, consist of two subtypes and that only one of these subtypes occurs in barbiturate-anesthetized animals. Surface-negative waves associated with suppression of spike discharges deep in the neocortex have durations of up to 300 msec or more in sleeping cats (Calvet et al., 1973) but the surface-negativewaves
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occurring during spindles (the only type observed during barbiturate anesthesia) are considerably briefer than this. Consequently, the C-waves occurring under these two conditions may have somewhat different origins (see Section IV,G). There are other possibilities that are not taken into account in Fig. 5 or in the original classification of Calvet et al. (1964a). One is the occurrence of hyperpolarization of the apical dendrites of pyramidal cells in the superficial layers of the cortex, occurring independently or concurrent with hyperpolarization of the deeply located cell bodies. There is good evidence for hyperpolarization and inhibition in dendrites remote from the cell body in the olfactory bulb and the hippocampus (Nicoll, 1969; Llina’s, 1975; Leung, 1978; Alger and Nicoll, 1982). Such an effect might account for surface positivity concurrent with hyperpolarization of deep pyramidal cells as observed by Creutzfeldt et al. (1966a-c). It is also possible for the deep and superficial layers of the cortex to show negativity simultaneously, perhaps as a result of activation of the A and B generators simultaneously. This occurs, for example, during one phase of the augmenting response (Spencer and Brookhart, 1961a) and during the transcallosal evoked potential (Vanderwolf et al., 1987). Finally, it must be said that a model recognizing inputs to only the apical dendrites and the basal-somatodendritic region, as in Fig. 5, is overly simplistic. Since synaptic inputs impinge on the entire somatodendritic surface, it is probable that depolarizing and hyperpolarizing inputs can occur in a number of localities on the large pyramidal cells. However, data are not yet available for the construction of more realistic models of neocortical activity. Presumably, such data will be increasingly provided by current source-density analysis (Mitzdorf, 1985). Traditional studies of polarity reversals are not infallible guides to the location of current sources and sinks. It is apparent from Fig. 5 that there can be no simple relation between the discharge of deep pyramidal cells and the polarity of gross potentials occurring at the cortical surface. Cells may fire during either surfacenegative or surface-positivewaves depending on the location of the inputs that produce excitation. Published experimental data are generally consistent with this interpretation (Whitlock et al., 1955;Jasper and Stefanis, 1965; Creutzfeldt et al., 1966b; Frost and Gol, 1966; Fromm and Bond, 1967; Frost, 1967; Ball et al., 1977a,b; Speckmann et al., 1978). It is also possible for hyperpolarization and inhibition of spike discharges in deep pyramidal cells to be associated with surface-positive gross potentials, although surface-negative potentials are more commonly observed in this situation. Furthermore, it is clear that at times gross potentials at the surface and in the deep layers of the neocortex are closely related (sometimes
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in phase but more commonly phase-reversed), although at other times activity at the surface and in the depths seems to proceed independently (Petsche et al., 1978). For example, Humphrey (1968) described a shortlatency wave, positive throughout the thickness of the cortex, following antidromic stimulation of the pyramidal tract. This wave was attributed to synchronized antidromic activity in pyramidal tract axons. It was followed by two successive negative waves (N1,No) which were very evident in the depths of the neocortex (and were correlated with antidromic discharge of pyramidal tract cells) but were not present at all in the surface records. As in this case, it appears to be generally true that gross potentials led from the deep layers of the neocortex correlate more closely with the discharges of deep pyramidal cells than potentials led from the neocortical surface. Additional examples of this are provided by Speckmann et al. (1978). The large pyramidal cells and fusiform cells (modified pyramids) that give rise to cortical efferent fibers are located mainly in layers V and VI of the neocortex (Ilorente de N6, 1949). Inputs to either the apical dendrites or the somata and basal dendrites of such cells should give rise to gross potentials of opposite polarity at the surface and in the depths of the neocortex. A difference recording from a transcortical electrode pair with one lead on the cortical surface and one lead in layer V or VI adds these opposite potentials but potentials that are in phase at the two sites or any potentials that are localized to either the surface or the depths are not added in this way. Consequently, as pointed out by Calvet et al. (1964a). a transcortical gross potential record is likely to be dominated by the activity of the deep large pyramidal cells and, therefore, correlates very closely with their activity. Gross potentials that are led monopolarly from the cortical surface are often more complex than those that are led from the depths of the neocortex and may correlate only weakly with the activity of the large deep pyramidal cells. Such surface records may be derived, in part, from the activity of the smaller, more numerous (Ramon-Moliner, 1961) cells located in the superficial cortical layers (i.e., pyramidal cells in layer 111). although the relative contributions of layer 111 cells, as compared to layers V and VI cells, have never received systematic study. In unanesthetized rats, bipolar transcortical (surface to layer V) records reveal three principal patterns of activity: (1) low-voltage fast activity (LVFA), (2) spindle bursts, and (3) large-amplitude, irregular slow waves (Fig. 6). The LVFA pattern is associated with a continuous multiunit discharge while the other two patterns are associated with a burst-suppression pattern of multiunit activity. The correlations between cell discharges and gross potentials are demonstrated more readily in multiunit records than in single unit records, presumably because the
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FIG.6. Slow waves and multiunit activity (MUA) recorded simultaneously from a a rat. Unit bipolar surfaceto-depth (layer V) electrode pair in parietal ~ o c o rint ~ activity is derived from thedeep electrode. Deep positivity (surface negativity) up. Note continuous MUA during low-voltage fast activity (WFA) (a); brief rhythmical bursts of MUA in phase with deep negative component of slow waves and longer lasting sup pression of MUA during deep positive component of slow waves during spindling (b); and long duration bums of MUA interrupted by amhythmk periods of MUA suppression (correlated with deep positive waves) during l a w amplitude i n r g S r slow activity (c). Calibration: 0.5 mV for slow waves; 0.050 mV for MUA; 200 msec.
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multiunit record is a type of “averaged”response that gives a good indication of the activity of a large population of cells. The correlation of slow wave spindle activity with rhythmical discharges in the principal neurons of the neocortex has long been known (Adrian and Moruzzi, 1939; Purpura, 1959). Cells commonly fire in brief rhythmical bursts, associated with field potential surface positivity and deep negativity (B generator) in alternation with longer periods of suppression of firing associated with surface negativity and deep positivity (C generator, see Figs. 5 and 6). During large amplitude, irregular slow waves (LISA) of the type occurring during slow wave sleep, the bursts of cell discharges are much longer than during spindle activity. At irregular intervals a surfacenegative, deep-positive wave occurs in association with a suppression of cell firing (C generator). The theory that the electrocorticogram is due to the activity of three principal generators located in the somata and dendrites of cortical pyramidal cells does little to clarify the origin of low-voltagefast activity. Calma and Arduini (1954) appear to have been the first to mention that pyramidal tract neurons fire in a continuous irregular pattern with no particular relation to the details of the electrocorticogram during the occurrence of LVFA in unanesthetized (curarized) cats. During the occurrence of spindle activity (provoked by injection of pentobarbital) a burst-suppression pattern of activity was observed. Similar phenomena were observed in freely moving cats by Hubel (1959) and by many subsequent workers (Evarts, 1964; Harper, 1973; Steriade and Hobson, 1976). Steriade et al. (1978) demonstrated that parietal neocortex neurons that can be identified as efferents by antidromic excitation from subcortical sites fire in a burst-suppression pattern during slow wave sleep and in a continuous irregular pattern at a higher rate during the presence of LVFA. These different patterns of firing, associated with large slow waves and LVFA, respectively, appear to occur throughout the neocortex (Steriade and Hobson, 1976). It would appear that the burst-suppression pattern in cortical projection cells is due to the presence of powerful inhibitory processes that occur synchronously in large numbers of neurons. During LVFA periods a more continuous pattern of firing occurs, partly perhaps as a result of a reduction in synchronized inhibition.
B. NEOCORTICAL ACTIVITY IN RELATION TO MOVEMENT A variety of different phenomena demonstrate the well-known fact that the electrical activity of the sensorimotor area of the cortex is closely related to motor activity.
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1 . Blockade of the 0 rhythm and the p, or wicket, rhythm in the human Rolandic area occurs prior to and during voluntary movement (Jasper and Penfield, 1949; Gastaut, 1952; Chatrian et al., 1959). Similarly, rhythmical waves, resembling the human wicket rhythm and occurring in the frontal and parietal cortex of waking immobile cats, are replaced by low-voltagefast activity whenever a voluntary movement occurs (Rouged et al., 1972; Cervantes et al., 1975). Spindle-shaped waveforms of 6-10 Hz (see Fig. 6) that occur in the frontal cortex of waking rats during periods of behavioral immobility (Klingberg, 1971; Vandenvolf, 1975; Semba et al., 1980), but not during voluntary or Type 1 behavior, probably represent a rodent homolog of the wicket rhythm. 2. Averaging techniques reveal that a slowly developing surface negativity (“readiness potential”) in the human Rolandic area precedes voluntary movement by as much as 0.5-2.0 sec. A more complex positivenegative wave sequence occurs 50-100 msec prior to the onset of electromyographic (EMG) activity (Kornhuber et al., 1965; Gilden et al., 1966; Vaughan et al., 1968). Related phenomena have been studied in monkeys (Sasaki et al., 1981). 3 . Single unit recording studies have revealed that pyramidal tract neurons fire prior to and during a voluntary movement (Evarts, 1966, 1981).
These findings, together with earlier work on the effects of lesions and electrical stimulation of the sensorimotor region (Fulton, 1949), justify the conclusion that this part of the cortex plays an important role in voluntary movement. However, it is not widely appreciated that electrical activity in neocortical areas outside the classical motor cortex, as well as in a number of subcortical structures, also correlates strongly with voluntary motor activity. Data demonstrating this fact will be reviewed here. Much of this work is based on the study of evoked potentials. Two main hypotheses appear to dominate experimental work on the nature of cerebral potentials evoked by natural or electrical stimulation of afferent pathways. One view holds that evoked potentials vary in form and amplitude in relation to the level of attention, consciousness, or vigilance (Bremer, 1954, 1961; Jouvet, 1967; Steriade, 1970). A second hypothesis suggests that variations in the form and amplitude of evoked potentials are a consequence of the “gating” of inputs that might influence ongoing motor activity (Rushton et al., 1981). An often mentioned illustration of this hypothesis is the suggestion that low-level flexion reflexes may be temporarily blocked by suppression of cutaneous inputs while one sets down a hot teacup.
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The classical physiological investigations of cerebral evoked potentials were carried out in anesthetized or immobilized preparations (Gauthier et al., 1956; Bremer, 1961; Steriade, 1970). Experiments of this type facilitate detailed analysis of neuronal mechanisms but are incompatible with study of the behavioral significance of whatever is discovered. Experiments in freely moving animals complement work done in immobilized preparations. An early experiment on freely moving cats by Starr (1964) indicated that responses evoked in the auditory cortex by a click stimulus, or by a single-pulseelectrical stimulus to the cochlear nucleus, were reduced in amplitude during skeletal movement as compared to periods of immobility. Similar effects were noted in subcortical structures such as the colliculi, medial geniculate nucleus, and the reticular formation. Beyer and Sawyer (1964) and Thompson and Shaw (1965) also found that nonspecific responses occurring in a variety of neocortical and subcortical structures following acoustic, somatic, or visual stimulation were generally reduced in amplitude during skeletal movement. Both Beyer and Sawyer (1964) and Starr (1964) noted that attention in a motionless animal had no effect on evoked potentials: evoked potential suppression was correlated specifically with movement. Studies by Ghez and Lenzi (1971), Ghez and Pisa (1972), and Coulter (1974) indicated that the response in the medial lemniscus to an electrical stimulus of the radial nerve is depressed, in comparison with the amplitude during immobility, up to 200 msec prior to and during a trained movement of the forelimb in cats. Similar results were obtained in records taken from the nucleus gigantocellularis, the nucleus centrum medianum, and the anterior suprasylvian cortex in cats engaged in pedalpressing behavior (Ciancia et a l . , 1980). This indicates that transmission through the dorsal column nuclei and also in extralemniscal pathways is reduced in preparation for, and during, voluntary movement. Coulter (1974) reported that passive movements, in contrast with active movements, have very little effect on potentials recorded in the medial lemniscus. This suggests that the reduction in transmission through the lemniscus is related to the production of movement and not to sensory feedback from the moving limbs. However, the force of this finding is somewhat weakened by the fact that the passive movement tests were carried out under general anesthesia. In scalp recordings in humans, the potentials evoked by electrical stimulation of the fingers are also strongly modified (different components of the response can be increased or reduced in amplitude) before and during voluntary movements of the stimulated hand (Cohen and Starr, 1987). Passive movements of the stimulated hand may (Coquery et al., 1972; Rushton et al., 1981) or may not (Lee and White, 1974) have the
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same effect as active movement. Movements of other parts of the body have a reduced effect or no effect. Hazemann et al. (1975) found that auditory as well as somatosensory evoked potentials were reduced in amplitude during a flexion movement of the hand in humans. In the foregoing studies, little attempt was made to examine the effect of different types of movement on transmission in afferent pathways. Schwartzbaum and his colleagues showed that a late component of the striate cortex response to a light flash in freely moving rats was reduced in amplitude during the presence of RSA in the hippocampus relative to periods when RSA was absent (Schwartzbaum et al., 1971; Schwartzbaum and Kreinick, 1973). This amounts to showing, indirectly, a relation to voluntary or Type 1 motor activity, since RSA is closely correlated with such activity. Similarly, Pickenhain and Klingberg (1965) showed that a multiple response (Kimura, 1962) or afterdischarge resembling a spontaneous 6 - to 10-Hz spindle burst occurs in rat striate cortex in response to a light flash during immobility and “comfort movements” (presumably grooming, which comprises a number of Type 2 behaviors) but not during “intended movements” (probably walking and other Type 1 behavior was meant). This type of work was developed further by Fleming and Bigler (1974), who noted that photically evoked afterdischarges often occur in striate cortex when RSA is absent in the hippocampus of freely moving rats but never occur when RSA is present. The nature of the potentials dealt with in these studies has been elucidated by Sumitomo and Klingberg (1972), who noted that unilateral stimulation of the lateral geniculate body often leads to a rhythmical afterdischarge bilaterally in the occipital cortex. Surgical destruction of the lateral geniculate body contralateral to the stimulated side results in a disappearance of the afterdischarge ipsilateral to the lesion. This indicates that the cortical potentials are driven by discharges from the lateral geniculate body and that the two geniculate bodies are in communication. The nature of the rhythmical potentials in the neocortex itself has been revealed by Skrebitsky and Sharonova (1976) and Skrebitsky et al. (1980). In restrained waking rabbits a light flash produced, in the striate cortex, a long-duration, surface-negative, deep-positive gross potential (C-wave) associated with equally long-lasting hyperpolarizations and spike suppression in impaled deep cortical neurons. Sometimes a series of such rhythmical inhibitory postsynaptic potentials (IPSPs), associated with a multiple response in the gross potential record, was triggered. Stimulation of the reticular formation eliminated this multiple response as well as the initial C-wave and the associated hyperpolarizations. Presumably, a similar effect occurs spontaneously in correlation with Type 1 behavior in freely moving animals.
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Phenomena of this kind are not restricted to the visual cortex. The responsiveness of units in both the somatosensory cortex (Chapin and Woodward, 1981) and the dorsal column nuclei (O’Keefe and Gaffan, 1971) to a sensory input have also been shown to vary with motor activity in a manner suggestive of the Type 1-Type 2 behavior classification derived from work on the hippocampus. Both of the latter studies noted that attention and arousal in immobile animals has no effect on unit responsiveness but that the occurrence of movement has a marked effect. The transcallosal evoked response, recordable in many areas of the neocortex following stimulation of the mirror image point in the opposite hemisphere, offers a number of advantages for the study of neocortical activity in relation to behavior. This system is relatively simple, involving fewer synapses than potentials evoked by stimulation of sensory pathways, and avoids the problems introduced by variations in sensory receptor sensitivity and orientation in behaving animals. Furthermore, extensive electrophysiological studies in anesthetized or immobilized animals have elucidated the cellular basis of the field potentials. An early component, deep-negative but positive or negative at the surface, is associated with depolarization and firing of pyramidal cells. At least part of this excitation is due to a monosynaptic input. A late longerduration component, positive in the depths of the cortex and negative at the surface, is associated with hyperpolarization and suppression of firing in pyramidal cells (Curtis, 1940a,b; Creutzfeldt et al., 1956; Latimer and Kennedy, 1961; Krnjevie et al., 1966a; Naito et al., 1970; Kitsikis and Steriade, 1975; Mamonets, 1981; Toyama et al., 1969). The latter potential can be regarded as a C-wave in the terminology of Calvet et al. (1964a). Racine et al. (1975) were the first to show that the transcallosal evoked potential varied strongly in relation to concurrent motor activity. More detailed analyses have followed their work, studying the relation between evoked potential development and the sleep-waking cycle as well as motor activity (Leung et al., 1982b; Vanderwolf et al.,1987). During slow wave sleep or 6- to 10-Hzspindle activity in the waking state, the early component of the transcallosal evoked potential is increased in comparison to its amplitude during waking immobility accompanied by LVFA (Fig. 7). This effect appears to be largely due to the evoked response being elicited during the course of C-waves during spindle activity or the irregular slow waves of slow wave sleep. The excitatory callosal afferents may provoke larger synaptic currents at these times, when pyramidal cells are hyperpolarized, than they do during periods of LVFA, when strong hyperpolarization is not likely to be present. In contrast to the early component, the late component displays only
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slight variations when comparisons are made across waking immobility accompanied by LVFA, waking immobility accompanied by rhythmical spindle activity, and quiet sleep accompanied by irregular slow waves. The duration of the late component increases by about 15% during the slow wave periods as compared to LVFA periods (Vanderwolf et al., 1987). Thus, the data show that during quiet or slow wave sleep, as compared to the immobile waking state, the early component of the transcallosal evoked potential is increased in amplitude and the late component is increased in duration. Since the presence of a background of 6- to 10-Hz spindle activity in the waking state has the same effects on the evoked potential as the presence of irregular slow waves during quiet sleep, it appears that the changes in the evoked potential are correlated with the state of the background electrocorticogram rather than the behavioral sleep-waking cycle as such. This conclusion is reinforced by the demonstration that during the occurrence of atropine-induced irregular slow waves in the waking state, the transcallosal evoked potential exhibits the same increases in early and late potentials as during natural slow wave sleep (see Fig. 7). In contrast to the rather minor changes (-15%) in the late component of the transcallosal evoked potential that correlate with the presence or absence of large slow waves in the electrocorticogram, major variations occur in correlation with various waking behaviors. In general, active Type 2 behaviors such as face washing, licking water, or biting a food pellet are associated with a large, long-duration, late component (C-wave)similar to what is seen during waking immobility. During Type 1 behaviors, such as walking, lateral or vertical head movements, or changes FIG.7. The transcallosal evoked response under various conditions of background electrocortical activity. Positivity up. Calibration: 100 msec; 0.5 mV. Top: Averages were derived from monopolar records of the right sensorimotor cortex; bipolar stimulation (S) at twice threshold intensity (80 PA) was applied to the left sensorimotor area. ILVFA, immobile and awake, with a background of low-voltage fast activity in the neocortex; SPINDLE, awake, immobile with a background of spontaneous 6- to 10-Hzspindle activity; SWS, lying down in quiet sleep with a background of large amplitude, irregular slow activity: ATROPINE I, awake, immobile, with a background of large amplitude, irregular slow activity resulting from treatment with atropine (50 mg/kg). The averages (32 sweeps/condition) were taken in sequence, as shown, over a period of several hours. The ILVFA condition was repeated to demonstrate the stability of the response. Bottom: Superimposition of 16 single sweeps of transcortical bipolar records on an oscilloscope during ILVFA and SPINDLE. During SPINDLE, SWS, and ATROPINE I, as compared to ILVFA, the amplitude of the early component of the transcallosal response, the duration of the late component, and the variability of both components are all increased. (From Vanderwolf et al., 1987, by permission of Elsevier Science Publishers B.V.)
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in posture, the late component is strongly suppressed (Figs. 8 and 9). In terms of deep multiunit activity (recorded among the large pyramidal cells in layer V), this means that during Type 2 behavior a callosal volley is followed by a long period of suppression of spike discharge, presumably correlated with widespread cell hyperpolarization, but that during Type 1 behavior this suppression is greatly reduced. Suppression of the late component of the transcallosal evoked response is greater during extensive movements such as walking than during more restricted movements such as movements of the head. This recalls an earlier observation that the hippocampal RSA associated with walking has a larger amplitude and a higher frequency than RSA associated with head movements (Vanderwolf, 1969). It appears that gross Type 1 movements are associated with a more intense activation of both neocortex and hippocampus than smaller Type 1 movements. The effect of passive movement on the transcallosal evoked response was studied in rats treated with trifluoperazine, a neuroleptic drug. This treatment makes it possible to move a rat’s limbs or head passively without provoking an active movement, something that invariably occurs in undrugged rats. However, trifluoperazine does not alter the patterns of cerebral activity that accompany active Type 1 movements whenever they do occur. The results of these tests indicate that purely passive movements have little or no effect on the transcallosal evoked potential (Vanderwolf et al., 1987). The studies reviewed in this section permit a number of tentative conclusions. 1. First, it is clear that the postsynaptic effects of afferent volleys to the neocortex vary greatly depending on the type of motor activity in progress at the time the afferent volley is initiated. Although the data are fragmentary, it appears that this is the case for visual, auditory, somesthetic, and transcallosal evoked potentials. Consequently, the effect cannot be interpreted simply as a “gating” of somatosensory input. Movement-related changes may occur as a result of a direct effect in the neocortex itself, as must be the case for the transcallosal evoked potential. In the case of cerebral potentials elicited by stimulation of sensory pathways, the opportunity exists for movement-related effects to be exerted at subcortical synapses as well. 2. During some behaviors (Type 2) an afferent volley appears to trigger a long-lasting synchronized hyperpolarization and suppression of spike discharges in deep pyramidal cells whereas during other behaviors (Type 1) this inhibitory response is strongly attenuated. It is unlikely that this suppression of a poststimulus inhibitory response is due to sensory
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FIG.8 . Effect of motor activity on the transcallosal response. EP, bipolar transcortical record of slow wave evoked response in left sensorimotor cortex, deep positivity up; MU, multiunit activity derived from the deep member of the same bipolar electrode pair: S, monopolar stimulation of right sensorimotor cortex at twice threshold (228 FA). Top: Waking immobility. Bottom: Walking elicited by pushing. Note large, late deep-positive component of the evoked potential and associated multiunit suppression during immobility and small, late deep-positive component and associated multinit activity during elicited walking. The early deep-negative component is only slightly altered. Four superimposed single sweeps in each behavioral condition. Calibration: EP, 0.5 mV MU, 0.05 mV 20 msec. (From Vandenvolf et al., 1987, by permission of Elsevier Science Publications B.V.)
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h
>
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FIG.9. Peak amplitude of the late component of the transcallosal evoked response in relation to various spontaneous behaviors in rats. For the transcortical bipolar recordings the stimuli were monopolar cathodal pulses (0.5-msec duration, 0.5 Hz, 2.5 X threshold). Means and standard deviations were derived from 40 single sweeps/ condition/ rat, five or six ratdcondition. IMM, alert immobility; CH, chewing food pellet; DW, drinking water; LP,licking forepaws; CFW, circus or rotary paw movements during face washing; BF, biting food; LH, lateral head movement; W, spontaneous walking. (From Vanderwolf et al., 1987, by permission of Elsevier Science Publishers B.V.)
feedback from an active movement since purely passive movements have little effect, at least in the case of the transcallosal evoked potential. Further, in the case of movement-related changes in evoked potentials recorded at the scalp in man (Coquery et al., 1972; Cohen and Starr, 1987) or at the medial lemniscus in cats (Ghez and Lenzi, 1971; Ghez and Pisa, 1972; Coulter, 1974), some aspects of the movement-related changes in potential have been shown to precede the onset of EMG activity. Thus, these changes cannot be due to sensory feedback from movement. The fact that some active movements, such as face-washing, have little effect on cortical evoked potentials even though they are (presumably) effective in eliciting sensory feedback also argues against the importance of direct sensory input in modifying the evoked potentials. The role of nonsensory factors must be considered. 3. The transition from sleep to waking appears to have only a small effect on transcallosal evoked potentials if care is taken to exclude movement in the waking condition. If this is also true of cerebral potentials evoked by other means, it may be suggested that in previous work in which large variations in evoked potentials were attributed to variations in “arousal level” and the sleep-waking cycle (Jouvet, 1967; Steriade and Hobson, 1976), it may be that the waking condition frequently involved movement.
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IV. Cholinergic and Serotonergic Control of Cortical Activation
A. HIPPOCAMPUS Following the pioneering studies of Lewis and Shute (1967) and Lewis et al. (1967) it has been convincingly shown that cholinergic pathways ascend from the' brainstem to the hippocampal formation. The dorsolateral tegmental and pedunculopontine nuclei contain neurons that react positively to monoclonal antibodies against choline-O-acetyltransferase (CUT) and stain for acetylcholinesterase (AChE). Anterograde and retrograde tracing techniques indicate that these cells project to a number of forebrain structures including the medial and lateral septal nuclei and the diagonal band (Satoh and Fibiger, 1986; Woolf and Butcher, 1986). Cells containing C U T and AChE and located in the medial septal nucleus and the vertical limb of the diagonal band, in turn, send fibers to the hippocampal formation, especially to stratum oriens and stratum radiatum of the hippocampus and the supra- and infragranular regions of the dentate gyrus (Lewis and Shute, 1967; Fibiger, 1982; Saper, 1984; Amaral and Kurz, 1985). High-affinity uptake of choline is present in the hippocampus and is reduced by septal lesions, suggesting that it occurs in septohippocampal neurons (Kuhar et al., 1973). Although it has not been rigorously shown how (or if) the brainstem cholinergic cells make contact with the medial septal and diagonal band cholinergic cells, it has been shown that many neurons in this region that project to the hippocampus (as shown by antidromic firing) are excited by cholinergic agonists. This excitation is blocked by atropine (Lamour et al., 1984; Segal, 1986). Thus, under normal physiological conditions, these cells may be driven by ascending brainstem cholinergic projections. In agreement with this, it has been found that stimulation of either the midbrain reticular formation or the medial septal nucleus produces a release of acetylcholine in the hippocampus (Dudar, 1975, 1977). Since both muscarinic and nicotinic receptors appear to be present on neurons in the hippocampal formation (Yamamura et al., 1974; Kuhar and Yamamura, 1976; Dudai and Segal, 1979; Kobayashi et al., 1978; Hunt and Schmidt, 1979; Rotter et al., 1979) and hippocampal cells respond to local iontophoresis of acetylcholine (Biscoe and Straughan, 1966; Bland et al., 1974; Segal, 1978b) all the components necessary for a functional reticulo-septo-hippocampalcholinergic pathway appear to be present. As a demonstration of one aspect of the function of this ascending pathway, early studies showed that hippocampal RSA could be produced
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in both waking and anesthetized animals by electrical stimulation of the reticular formation (Green and Arduini, 1954) or by systemic administration of cholinergic agonists such as eserine, pilocarpine, or diisopropylfluorophosphate(Stumpf, 1965). Acetylcholine also produces RSA when injected into the carotid artery of anesthetized animals (Stumpf, 1965). Antimuscarinic drugs such as atropine, scopolamine, or quinuclidinyl benzilate, given systemically, abolish all RSA in anesthetized rats or rabbits, resulting in a continuous pattern of large-amplitude irregular activity (Kramiset al., 1975; Vanderwolf et al., 1978a; Bland et al., 1981; Stewart et al., 1984; h u n g , 1985). Similarly, in waking animals, any RSA that is elicitable during Type 2 behavior is sensitive to antimuscarinic drugs. Thus, the RSA that is produced in immobile rabbits by novel sensory stimuli under certain conditions (Kramis et al., 1975) and the RSA produced in rats during immobility by electrical stimulation of the reticular formation (Vanderwolf, 1975) is readily abolished by antimuscarinic drugs. This is also true of any RSA occurring during Type 2 behavior following treatment with a variety of drugs including eserine, reserpine, and antipsychotic drugs such as chlorpromazine (Vanderwolf et al., 1975, 1978a). The RSA that can be elicited in rats or guinea pigs during immobility by the presence of such stimulus objects as cats, snakes, ferrets, or the sounds made by owls is also sensitive to centrally acting antimuscarinic drugs (Sainsbury and Montoya, 1984; Sainsbury et al., 1987). In all these situations, antimuscarinic drugs that fail to penetrate the blood-brain barrier do not affect RSA. Although centrally acting antimuscarinic drugs totally abolish the RSA occurring during anesthesia and waking Type 2 behavior, they have only minimal effects on the RSA accompanying Type 1 behavior (Kramis, et al., 1975; Vanderwolf, 1975; Vanderwolf et al., 1978a). A similar selective effect is observed when hemicholinium-3, a high-affinity choline uptake blocker, is injected into the lateral ventricles (Robinson and Green, 1980). Following treatment with hemicholinium, the RSA elicited by reticular stimulation in urethane-anesthetized rats is completely abolished while the RSA accompanying waking locomotion appears to be unaffected. A systemic injection of choline chloride restores RSA in the anesthetized rats, a finding that is consistent with the hypothesis that the effectiveness of hemicholinium in abolishing RSA in this condition is due to interference with choline uptake (Robinson and Green, 1980). Similar selective effects can be produced by destruction of cells in the dorsal medial septa1 region by a local injection of ibotenic acid (Stewart and Vanderwolf, 1987a). The experimental rats display RSA during waking Type 1 behavior but not during urethane anesthesia.
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These data indicate the existence of two distinct forms of hippocampal RSA, presumably produced by two different inputs to the hippocampus (Kramis et al., 1975; Vanderwolf, 1975; Vanderwolf et al., 1975). The atropine-sensitive input is presumed to be dependent on transmission at muscarinic cholinergic synapses, apparently located both in the hippocampus and in the septal nuclei. The atropine-resistant input is presumably not cholinergic since it is largely unaffected by antimuscarinic drugs, by antinicotinic drugs such as mecamylamine (Whishaw et al., 1978a), or by hemicholinium-3. Significant progress has been made in the identification of the neurochemical basis of atropine-resistant RSA. This type of RSA is abolished by anesthetics, since all RSA in anesthetized animals is sensitive to atropine, and also by subanesthetic doses of such psychotomimetic drugs as phencyclidine and cyclazocine (Vanderwolf and Leung, 1983; Vanderwolf, 1987a). Large doses of reserpine reduce but do not entirely abolish atropine-resistantRSA (Vanderwolf et al., 1978a; Leung, 1984a). However, treatment with large doses of paruchlorophenylalanine (an inhibitor of the synthesis of serotonin) can produce a total disappearance of atropine-resistant RSA (Vanderwolf and Baker, 1986). In rats treated with a combination of p-chlorophenylalanine plus atropine or scopolamine, hippocampal activity consists of an unchanging, irregular, largeamplitude wave form (resembling normal LIA) punctuated by the irregular occurrence of sharp waves. These observations suggest that atropine-resistant RSA may be dependent on the serotonergic pathways which ascend from the brainstem to the hippocampal formation (Dahlstrom and Fuxe, 1964; Andtn et al., 1966; Azmitia, 1978; Steinbusch and Nieuwenhuys, 1983). In keeping with this, it has been found that high-frequency stimulation of the median raphe nucleus in freely moving rats is very effective in eliciting atropine-resistant RSA and Type 1 behaviors such as locomotion (Robinson and Vanderwolf, 1978). The main serotonergic innervation of the dorsal hippocampus arises in the median raphe nucleus, courses through the lateral hypothalamic and septal regions, and loops caudally through the cingulum and supracallosal striae to enter the hippocampal formation via the entorhinal cortex (Azmitia and Segal, 1978; Gage et al., 1983). If this pathway is interrupted by lesions in the hypothalamus (Kolb and Whishaw, 1977; Whishaw and Kolb, 1979), in the cingulate cortex, or by surgical isolation or destruction of the entorhinal cortex (Vanderwolf and Leung, 1983; Vanderwolf et al., 1985), atropine-resistant RSA may be totally abolished. These findings are consistent with the view that atropine-resistant RSA is dependent on a serotonergic input to the hippocampus. However, they do not in themselves exclude other possibilities. For example, the
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ascending noradrenergic fibers from the locus coeruleus follow a path parallel to the ascending serotonergic fibers (And& et al., 1966; Lindvall and Bjorklund, 1974). However, pharmacological evidence indicates that noradrenalin does not play an essential role in the generation of atropineresistant RSA. Dopamine does not appear to be essential either, although it may play an indirect role (Vanderwolf and Stewart, 1986). Thus, blockade of the synthesis of catecholamines, blockade of catecholaminergic receptors, or destruction of catecholaminergic neurons by treatment with 6-hydroxydopamine or electrolytic lesions do not abolish atropine-resistant RSA (Kolb and Whishaw, 1977; Robinson et al., 1977; Whishaw et al., 1978b). Atropine-sensitive RSA is relatively unaffected by removal of the entorhinal cortex or by section of afferent pathways that run to it through the cingulum, supracallosal striae, and subcortical white matter (Vanderwolf and Leung, 1983; Vanderwolf et al., 1985) presumably because the main septohippocampal cholinergic input courses through the fimbria and dorsal fornix (Gage et al., 1983). However, some cholinergic fibers reach the hippocampal formation via a supracallosal trajectory (Gage et al., 1983; Saper, 1984). Interruption of these fibers may be responsible for the reduction in atropine-sensitive RSA (together with a complete loss of atropine-resistant RSA) following entorhinal lesions in the guinea pig (Montoya and Sainsbury, 1985). When atropine-resistant RSA has been abolished by isolation or removal of the entorhinal cortex, RSA maintains a relatively normal correlation with Type 1 behavior in waking rats (Vanderwolf and Leung, 1983; Vandenvolf et al., 1985) and guinea pigs (Montoya and Sainsbury, 1985). This may suggest that, in normal circumstances, a cholinergic septohippocampal input is active during Type 1 behavior. Several other pieces of information also point to this conclusion: (1) Single units in the medial septum fire rhythmically during locomotion (Ranck, 1975). (2) High levels of acetylcholine release are present in the hippocampus during locomotion (Dudar et al., 1979). (3) The population excitatory postsynaptic potential and the population spike evoked in CA1 by stirnulation of the Schaffer collaterals are smaller during locomotion than during immobility, and these differences are reduced by atropine (Leung, 1980; Leung and Vanderwolf, 1980). The data reviewed in this section suggest the followinggeneral hypotheses. During the occurrence of Type 1 behavior, RSA is produced by the joint action of cholinergic and serotonergic inputs to the hippocampus. If either of these inputs is experimentally inactivated, the other input, acting alone, will produce RSA during Type 1 behavior. Both the cholinergic and serotonergic inputs may be inactive during Type 2
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behavior (allowing large amplitude irregular activity to occur), but under some circumstances (depending on the environmental situation and the animal species) the cholinergic input may be active alone, producing RSA during Type 2 behavior. In anesthetized animals the serotonindependent pathway is inactivated and all RSA is produced by the cholinergic input. Although this review proposes that cholinergic and serotonergic inputs are responsible for all hippocampal RSA, the mechanisms by which these inputs are brought into action are rather obscure. The firing of pontine gigantocellar neurons and neurons in the nucleus raphe magnus have been reported to correlate with the occurrence of hippocampal RSA (Sheu et al., 1974; Vertes, 1977, 1979), but the relation between this activity and ascending cholinergic and serotonergic pathways is not well understood.
B. CELLULAR MECHANISMS IN THE HIPPOCAMPUS The cellular mechanisms by which acetylcholine induces rhythmical activity in the hippocampus are not entirely clear. Since destruction of the septal nuclei abolishes hippocampal RSA, and since some medial septal and diagonal band cells fire in rhythmical bursts in phase with RSA, Brucke, Petsche, Stumpf, and their colleagues proposed that septal neurons act as pacemakers for the hippocampal rhythm (Petsche et al., 1962; Stumpf, 1965). An important piece of evidence that supports this concept is the demonstration of slow rhythmical bursts of firing in neurons in an in Vitro septal slice preparation (Vinogradova et al., 1980). However, other evidence indicates that the hippocampus can generate RSA-like waves in the absence of a rhythmical cholinergic input. A nonrhythmical input may suffice. Microinjection of cholinergic agonists (acetylcholine, carbachol, eserine, methacholine, or oxotremorine) directly into the hippocampus produces RSA in both freely moving and in anesthetized animals (MacLean, 1957; Ott et al., 1977; Rowntree and Bland, 1986). Waveforms resembling RSA also occur in in vitro hippocampal slices when cholinergic muscarinic agonists are added to the superfusion fluid (Konopacki et al., 1987). Thus, it seems that mechanisms favoring rhythmical activity are present in both the septal nuclei and the hippocampus. Under normal conditions both may contribute to the development of rhythmical activity in the hippocampal formation. It has been known for many years that systemic injections of nicotine produce a brief episode of RSA (Stumpf, 1965), although injections directly into the hippocampus are ineffective (Ott et al., 1983). In
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contrast with muscarinic agonists, systemic injections of nicotine suppress activity in hippocampal 6' cells but are nonetheless effective in producing slow wave RSA, even in the presence of atropine (Bland and Colom, unpublished). The relation of this finding to normal function remains enigmatic. Fujita and Sato (1964) and Artemenko (1973) showed that hippocampal pyramidal cells display an intracellular RSA rhythm which is phasereversed with respect to the local extracellular 6' rhythm. L u n g and Yim (1986) have recently shown, in urethane-anesthetized rats, that if an IPSP provoked by stimulation of the alveus in impaled hippocampal pyramidal cells is reversed by current injection or by diffusion of acetate ions, then the phase of the intracellular RSA rhythm is inverted. Thus, with respect to the affected cell, the intracellular and local extracellular RSA rhythms are in phase. The reversal potentials for the intracellular RSA rhythm and the evoked IPSP do not differ significantly. Thus, the data suggest that the extracellular atropine-sensitive RSA in urethane-anesthetized rats results from a series of rhythmical IPSPs in pyramidal cells. These IPSPs, in turn, may result from rhythmical activity in inhibitory interneurons, as suggested by Spencer and Kandel(l962) and Artemenko (1973). Basket cells, possessing the property of rapid synthesis of the GABA-inactivatingenzyme GABAT (4-aminobutyrate aminotransferase) have been demonstrated in the hippocampus. It is probable that these cells contain GABA (y-aminobutyric acid) and may, therefore, act as inhibitory interneurons (Nagai et al., 1983). Precisely how acetylcholine modulates the activity of these interneurons to produce a rhythmical effect is not well understood. It has been shown that local iontophoresis of acetylcholine or medial septal stimulation reduces IPSPs evoked in pyramidal cells by stimulation of the fimbria or entorhinal cortex, perhaps by reducing the release of GABA (Ben-Ari et al., 1981; Krnjevid et al., 1981; Kmjevid and Ropert, 1981, 1982). These findings are consistent with other evidence indicating that many septal efferents end on interneurons in the hippocampus (Mosko et al., 1973; BuzsPki and Eidelberg, 1982; Buzsa'ki, 1984). Cholinergic septohippocampal fibers may modulate the effectiveness of inhibitory hippocampal interneurons in a rhythmical manner. However, Ben-Ari et al. (1981) and Cole and Nicoll (1984) found that acetylcholine also reduces K conductance in hippocampal pyramidal cells, as it does in many neocortical cells, thereby producing a slow depolarization. Thus, both interneurons and pyramidal cells may be affected directly by a cholinergic input. Both the depolarization and the reduction of inhibition presumably contribute to the increased firing observed in many hippocampal pyramidal cells during M A periods in urethane-anesthetizedanimals (Bland et al., 1980; Nuiiez +
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et al., 1987). A further complication is introduced by evidence that the application of acetylcholine to the apical dendrites of CA1 cells reduces the size of excitatory postsynaptic potentials (EPSPs) by a presynaptic action (Valentino and Dingledine, 1981). Thus, the total effect of physiological release of acetylcholine in the hippocampus is likely to be very complex. In the dentate gyrus, granule cell firing is increased during the presence of RSA in urethane-anesthetized animals (Bland et al., 1980). Consistent with this, medial septa1stimulation also excites granule cells, and the effect can be blocked by local application of atropine at the recording site (Wheal and Miller, 1980). There are indications that, as in CA1, acetylcholine release reduces the effectiveness of inhibition in the dentate gyrus (Bilkey and Goddard, 1985). Thus, rhythmical firing in cholinergic septohippocampal neurons might produce a rhythmical modulation of the effectiveness of inhibitory interneurons in both CA1 and the dentate gyrus. In freely moving animals the situation appears to be more complex than in urethane anesthetized preparations. Complex spike cells in CAI (presumably pyramidal cells) tend, as a class, to display reduced rates of discharge when RSA is present (Ranck, 1973; Suzuki and Smith, 1985). However, as discussed above (Section I1,C) some complex spike cells fire at relatively high rates during RSA, indicating great selectivity in the excitatory mechanicm. This phenomenon contrasts sharply with the nonspecific increase in cell firing that accompanies RSA in anesthetized animals. With respect to CA18 cells (presumed interneurons) and dentate 8 cells (presumed to be either local interneurons or granule cells) it is not clear that the elicitation of atropine-sensitiveRSA in waking rabbits produces any change in mean firing rate at all. When the RSA occurs, cell firing becomes rhythmic, but the overall rate is the same as it is during the presence of irregular slow wave activity (Sinclair et al., 1982; Bland et al., 1983). The RSA occurring during immobility, together with the associated rhythmic firing of 8 cells, is abolished by atropine (Bland et al., 1984). In this case, the data suggest that the cholinergic septohippocampal input serves only to synchronize and regularize firing without changing overall rate. During the occurrence of Type 1 behavior in the rabbits, the firing of 8 cells in both CAI and the dentate gyrus increases sharply in association with the appearance of atropine-resistant RSA (Sinclair et al., 1982; Bland et al., 1983). Rhythmical 8 cell firing persists after atropinization, although the rate declines significantly (Buzsaiki et al., 1983; Bland et al., 1984). As already mentioned, dentate cells in rats also fire at much higher rates during Type 1 behavior than during Type 2 behavior (Buzsaiki et al., 1983; Rose, 1983).
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Serotonin is widely regarded as having a predominantly inhibitory effect on hippocampal pyramidal cells (Biscoe and Straughan, 1966; Segal, 1975; Straughan, 1975) and on cells in the medial septa1nucleus (Segal, 1986). In CA1, this effect may be due to activation of K + channels leading to hyperpolarization (Segal, 1980). However, it is uncertain whether this represents its role under normal physiological conditions, since the experiments discussed by Segal and Straughan were carried out in anesthetized or in in vitro slice preparations. In the neocortex, serotonin has a predominantly inhibitory effect in anesthetized preparations but often has an excitatory effect in unanesthetized preparations (Roberts and Straughan, 1967; Johnson et al., 1969). Excitatory effects have been described in some cells in hippocampal slice preparations (Jahnsen, 1980). As already mentioned, atropine-resistant RSA, which appears to be dependent on serotonin, is abolished by anesthetics. It would be highly desirable to carry out studies on the effects of serotonin on hippocampal cells in unanesthetized in mvo preparations. An effect of anesthetics on central serotonergic mechanisms may account for a discrepancy between the findings of Assaf and Miller (1978) and Robinson and Vanderwolf (1978) on the effects of median raphe stimulation on hippocampal activity. Robinson and Vanderwolf, using freely moving rats, reported that 100-Hz raphe stimulation produced clear RSA, whereas Assaf and Miller, using urethane-anesthetized rats, reported that 100-Hz raphe stimulation produced low-amplitude fast waves. It is possible that the effects of serotonin are mediated in part via an interaction with glutamate. There is good evidence that perforant path fibers release glutamate in the hippocampus and dentate gyrus (Nadler et al., 1976; Storm-Mathisen, 1977). Excitation of the perforant path excites both hippocampal pyramidal cells and dentate granule cells (Andersen, 1975), and this excitatory effect may be due to the release of glutamate (Wheal and Miller, 1980). Cells in the entorhinal cortex, in which the perforant path originates, fire in a bursting pattern in phase with hippocampal RSA in waking rats (Mitchell and Ranck, 1980). If serotonin facilitates the action of glutamate in the hippocampus, as it does in the facial nucleus (McCall and Aghajanian, 1979), then a mechanism is suggested whereby serotonin release in the hippocampus would facilitate a rhythmical excitatory perforant path input to the hippocampus and dentate gyrus. In support of this, it may be pointed out that the serotonergic projections to CA1 appear to terminate heavily in stratum lacunosum-moleculare in CA1 (Lidov et al., 1980), which is also the zone of termination of the perforant path fibers (Blackstad, 1958; Steward, 1976). However, in the dentate gyrus the serotonergic input
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does not overlap with the perforant path input. Whatever the mechanisms ultimately turn out to be, there is, at present, a good deal of evidence that hippocampal RSA in waking animals is produced jointly by a series of IPSPs produced by a septohippocampal cholinergic input and a concurrent phase-locked series of EPSPs produced by an excitatory perforant path input (Buzsgki et al., 1983; Leung, 1984a,b).
C. NEOCORTEX: CHOLINERGIC CONTROL Following the early studies of Shute and Lewis (1967), it has recently been demonstrated that cholinergic pathways ascend from the brainstem to the neocortex. Cholinergic marker enzymes (ChAT and AChE) are present in neurons in the dorsolateral tegmental and pedunculopontine nuclei that project to basal forebrain structures such as the magnocellular preoptic and ventral pallidal areas (Satoh and Fibiger, 1986; Woolf and Butcher, 1986). These enzymes are also present in a second ascending cholinergic system that consists of neurons in the basal forebrain (including the substantia innominata, the magnocellular preoptic area, the ventral globus pallidus, and the nucleus basalis of Meynert as well as the diagonal band and medial septa1 nucleus) that project in a topographic manner to frontal, parietal, occipital, and temporal regions of the neocortex as well as to the cingulate and hippocampal formation cortices (Divac, 1975; Johnson et al., 1979, 1981; Lehman et al., 1980; Kimura et al., 1981; Big1 et al., 1982; Fibiger, 1982; Armstrong et al., 1983; Houser et al., 1983; McKinney et al., 1983; Mesulam et al., 1983; Saper, 1984; Butcher and Woolf, 1986; Luiten et al., 1987; Sofroniew et al., 1987). High-affinity choline uptake is present in the neocortex and, since it is reduced by basal forebrain lesions, it is assumed to be associated with the basal forebrain projections to the neocortex (Kuhar et al., 1973; Pedata et al., 1982). Muscarinic receptors are also present in the neocortex (Yamamura et al., 1974; Kuhar and Yamamura, 1975, 1976; Kobayashi et al., 1978; Rotter et al., 1979) and, since they persist with only slight reductions following lesions of the basal forebrain, it is presumed that they are located largely at postsynaptic sites (McKinney and Coyle, 1982; de Belleroche et al., 1985). Iontophoretic studies suggest that most neocortical neuronal responses to acetylcholine are dependent on muscarinic receptors (Kmjevie and Phillis, 1963a,b). Electrical stimulation of the reticular formation (Kanai and Szerb, 1965; Celesia and Jasper, 1966) or the basal forebrain (Casamenti et al., 1986) results in increased release of acetylcholine from the surface of the neocortex.
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Taken together, these data indicate that a functional cholinergic pathway runs from the reticular formation to the basal forebrain and thence, following one or more synapses, to the neocortex. Consistent with this, most basal forebrain units that can be driven antidromically from the neocortex are excited by acetylcholine or cholinergic agonists, and the effect can be blocked by atropine (Lamour et al., 1986). Since virtually all basal forebrain cells that can be labeled by retrograde transport from the neocortex also contain choline acetyltransferase (Saper, 1984), this indicates that the basal forebrain cholinergic cells are probably capable of being excited by the ascending brainstem cholinergic fibers. There is a great deal of evidence that cholinergic mechanisms play a role in the production of LVFA in the neocortex. Acetylcholine and cholinergic agonists such as eserine or pilocarpine produce LVFA when applied via several different routes, and the effects can be antagonized by atrophic drugs (Bonnet and Bremer, 1937; Bremer and Chatonnet, 1949; Monnier and Romanowski, 1961; Cuculic et al., 1968). Further, the release of acetylcholine from the neocortical surface is greater during periods of LVFA than during periods occupied by large slow waves in both anesthetized and freely moving animals (Kanai and Szerb, 1965; Sie et al., 1965; Celesia and Jasper, 1966; Szerb, 1967; Phillis, 1968; Jasper and Tessier, 1971). Natural slow wave sleep is associated with low levels of release of acetylcholine in the neocortex. Thus, the release of acetylcholine from nerve terminals in the neocortex may be a cause of the occurrence of LVFA. Behavioral studies in freely moving rats show that LVFA is invariably present during the performance of Type 1 behavior and is often present during Type 2 behavior as well (Fig. 10). Spontaneous spindle bursts of 6-10 Hz occur in rats only during waking immobility, but their occurrence is very sporadic in most individuals. However, following the administration of a centrally acting antimuscarinic drug such as atropine, a strong correlation can be demonstrated between spontaneous motor activity and the electrocorticogram, especially if bipolar surface-to-depth (surface to layer V) records of neocortical activity are taken (Vanderwolf, 1975; Schallert et al., 1980; Vanderwolf and Pappas, 1980). During the occurrence of Type 1 movements, such as postural changes, locomotion, or head movement, LVFA (atropine-resistant LVFA) and an associated continuous multiunit activity (MUA) discharge are present, just as they always are in undrugged animals (see Figs. 6 and 10). However, during immobility, there is large-amplitude, irregular slow wave activity associated with a burst-suppression pattern of MUA. Large irregular slow waves also occur during active Type 2 behaviors such as chattering or gnashing of the teeth, tremor, and face washing (if it occurs for several
CONTROL OF CEREBRAL ACTIVITY AND BEHAVIOR
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FIG.10. Effects of atropine sulfate on neocortical electrical activity in relation to behavior. CTX, neocortical slow wave activity from a surface-to-depth electrode pair; MVMNT, output of magnet-and-coil type of movement sensor. Upper traces: Undrugged rat displays continuous LVFA with no relation to motor activity. Lower traces: After atropine treatment (50 mg/kg, ip) large amplitude, irregular slow waves occur during immobility but LVFA persists during spontaneous motor activity (head movements and stepping). (From Vanderwolf, 1984, by permission of Humana Press Inc.)
seconds in the absence of postural shifts). The correlation between the electrocorticogram and concurrent motor activity in atropinized rats can be demonstrated not only in frontal (motor) cortex but also in parietal (somesthetic), occipital (visual), and temporal (auditory) regions of the neocortex (Vanderwolf, 1975). As in the case of atropine-resistant hippocampal RSA, the occurrence of atropine-resistant neocortical LVFA is not due to sensory feedback from moving body parts. Passive movement does not elicit atropine-resistant LVFA in rats (Vanderwolf, 1975), but the waveform occurs spontaneously in rats paralyzed by a large dose of gallamine (Whishaw et al., 1976). Therefore, atropine-resistant LVFA may be related to the causes of active Type 1 movement and not to its consequences. Rats treated with centrally acting antimuscarinic drugs are more active than normal, spending much of their time moving the head, sniffing, stepping with the forelimbs, and walking (Van Der Poel and
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Remmelts, 1971;Stewart et al., 1984). Occasionally, they stand immobile or lie down, full length, with the head on the substrate. If an actively moving atropinized rat is startled by a loud noise it may either continue moving or freeze for several seconds. Large slow waves do not occur in the first case but do occur during behavioral immobility in the second case (Vanderwolf et al., 1973). This shows that ‘‘attention’’and “arousal” are irrelevant to the state of the electrocorticogram in atropinized rats since these concepts, presumably, apply in both cases (atropinized rats always display vigorous startle responses to loud sounds, regardless of their subsequent behavior). In fact, it could be said that the reaction of the rat that becomes immobile is especially indicative of “attention,” even though slow waves appear in the electrocorticogram. The classical descriptions of the effect of atrophic drugs on the electrocorticogram in behaving animals noted that the large slow waves occurred in the absence of sleep (Wikler, 1952; Bradley and Elkes, 1957) but did not mention the close correlation of the electrocorticogram with concurrent motor activity. This failure to observe a rather striking phenomenon may have been due to two factors: (1) The records were taken from the surface of the cortex, a derivation that does not reveal the effect as clearly as a bipolar surface-to-depthrecord does (Vanderwolf, 1975; Vanderwolf and Pappas, 1980). (2) In the absence of any tradition of observing the details of motor activity closely, the effect may simply have been overlooked. The pattern of blockade of LVFA during Type 2 behavior and preservation of LVFA during Type 1 behavior that occurs following the administration of atropine also occurs following adequate doses of a variety of other drugs with central antimuscarinic effects including Ditran, promethazine, quinuclidinyl benzilate, and scopolamine (Vanderwolf, 1975;Vanderwolf and Pappas, 1980;Stewart et al., 1984;Vanderwolf and Stewart, 1986). Peripherally acting antimuscarinic drugs such as atropine methyl nitrate, as well as a variety of other centrally acting drugs, do not have this effect, even in extremely large doses. Figure 11 shows the extent of suppression of LVFA during immobility as a function of dose for atropine, quinuclidinyl benzilate, and scopolamine. It is evident that the three drugs have very similar effects on neocortical activity in rats but that atropine is less potent by a factor of 10 or more than quinuclidinyl benzilate or scopolamine. In all cases the doses required to affect cortical activity are much larger than those required to block peripheral autonomic muscarinic receptors or alter brain function in humans. One factor that may contribute to this is poor penetration of the drugs into the brain relative to other tissues (Albanus et al., 1968a,b;Herz et al., 1965;Kalser et al., 1957;Ternnesen, 1948).
271
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80
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FIG. 11. Dose-response curves for the effects on neocortical electrical activity of atropine sulfate during immobility (A)and walking (A), quinuclidinyl benzilate during immobility ( 0 )and walking (0),and scopolamine hydrobromide during immobility ( W ) and walking (0).Points shown are means (five or six rats per point) plus standard error of the mean. C, control injection. All three drugs produced increases in largeamplitude, irregular slow wave activity as a function of dose during immobility, although atropine is only about one-tenth as potent as the other drugs on a milligram per kilogram basis. Large doses of atropine produce a slight increase in slow wave activity during walking but QNB and scopolamine did not do this at the highest doses tested (4 and 16 mg/kg, respectively). Data from Stewart et al. (1984) and Vandenvolf and Stewart (1986).
Also, a number of plant-eating animals including rabbits, rats, and guinea pigs have evolved a specific hydrolytic enzyme, atropine esterase, which inactivates atropine and scopolamine. Consequently, these animals are much less sensitive to such drugs than, for example, cats and humans, which generally do not possess atropine esterase (Sz6ra’dy et al., 1970). The large doses of atropine and scopolamine that are required to affect cortical activity in rats appear to correspond closely to doses required to produce significant changes in behavior (Overton, 1966). For example, recent work by P. Prior (unpublished) shows that the dose-effect curve for disruption of maze-running by scopolamine in rats
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corresponds closely to the dose-effect curve for blockade of LVFA during immobility that is shown in Fig. 11. Consequently, it appears that rather large doses of antimuscarinic drugs are necessary to disrupt cerebral function in rats. It is interesting that the blocking effect of centrally acting antimuscarinic drugs on LVFA during immobility does not increase the time spent in immobility during spontaneous behavior to more than about 70% (see Fig. 11). This is largely due to the fact that the large irregular slow waves take time to develop when a rat stops moving and becomes totally immobile. Thus, there may be a delay of up to 3 sec (usually considerably less than this) after the end of a movement before conspicuous slow waves appear. There is no comparable delay in the appearance of LVFA when a motionless atropinized rat begins to move about. LVFA always occurs at about the same time as the onset of motor activity. However, no detailed studies of the exact temporal relation have been carried out as yet. The foregoing studies of the effects of antimuscarinic drugs on neocortical activity in freely moving rats indicate that the systems that activate the neocortex are organized in the same way as those that activate the hippocampus. An atropine-sensitivesystem is responsible for any activation that may be present during Type 2 behavior, but in addition, an atropine-resistant system comes into play during Type 1 behavior. A further parallel is demonstrated by the observation that atropine-resistant neocortical LVFA (like atropine-resistant hippocampal RSA) is abolished when rats are anesthetized with a volatile anesthetic such as diethyl ether (Vanderwolf et al., 1975). Thus, unlike the situation in freely moving animals, the LVFA that is normally present during such anesthesia (Bremer, 1936; Beecher and McDonough, 1939; Rossi and Zirondoli, 1955) is entirely abolished by atropinic drugs (Vanderwolf et al., 1975). This suggests that any cerebral activation that occurs during surgical anesthesia is due to a cholinergic input to the neocortex. After experimental lesions of the substantia innominata, the levels of the cholinergic marker enzymes and high-affinity choline uptake are sharply reduced in the neocortex and the release of acetylcholine is reduced as well (Wenk et al., 1980; LoConte et al., 1982a,b; Pedata et al., 1982). Such lesions may produce changes in the electrocorticogram that resemble those produced by systemic injection of atropinic drugs. Figure 12 illustrates this in a chronic rat preparation in which acetylcholinesterase-positiveneurons in the substantia innominata had been unilaterally destroyed by a local injection of kainic acid as described by Stewart et al. (1984) and Borst et al. (1987). Large-amplitude irregular activity is usually present ipsilateral to the lesion during behavioral
-273
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immobility and other Type 2 behavior but LVFA is usually present during locomotion, head movements, and other Type 1 behaviors. Systemic injections of pilocarpine (20-100 mg/kg) abolished these slow waves, resulting in bilateral LVFA (Stewart et al., 1984; Vanderwolf and Stewart, unpublished observations). This effect may be due to a direct action on muscarinic receptors that persist in the neocortex following lesions of the basal forebrain (McKinney and Coyle, 1982; de Belleroche et al., 1985). Extracellular recording in freely moving cats has revealed a population of basal forebrain cells that is most active during periods of neocortical LVFA and a second population that is most active during episodes of large slow waves occurring during quiet sleep (Detari et al., 1984; Szymusiak and McGinty, 1986). Experiments with urethane-anesthetized rats (Detari and Vanderwolf, 1987)(Fig. 13) have revealed that basal forebrain cells of the first type can be driven antidromically by stimulation of the neocortex, suggesting that they are cholinergic. (As already mentioned, virtually all basal forebrain cells that project to the neocortex
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C. H. VANDERWOLF
FIG. 13. Antidromically identified cortically projecting cell. (A) Collision test. Stimuli delayed by 10 msec after spontaneous spikes evoke antidromic responses, but there is no response if the delay is decreased to 9 msec. Both photographs depict eight successive stimuli. Note the constant latency driving on the left photograph. Calibration, 10 msec. (B) Electrode track and localization of the cell on a camera lucida drawing of the histological section. (C)Four continuous traces from the original record. In each trace the upper row shows pulses corresponding to cell firing, the middle row shows time marks, and lower row shows the electrocorticogram. Note the close correlation between temporary changes in the electrocorticogram and the cell firing. The record is contaminated by 0 activity during low-voltagefast activity, as the deep electrode was close to the dorsal hippocampus. Calibration, 0.5 mV and 5 sec. (From Detari and Vandenvolf, 1988.)
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275
also contain ChAT and AChE.) The firing rate in such cells is about 5-fold higher during LVFA periods (occurring spontaneouslyor as a result of a tail pinch or stimulation of the midbrain reticular formation) than during periods occupied by large slow waves. Neurons that fire at high rates during the occurrence of large slow waves in the neocortex cannot be driven antidromically from the neocortex. Perhaps these cells are interneurons that inhibit the cortically projecting cholinergic cells. Probable GABA-ergic neurons that have been demonstrated in the basal forebrain (Nagai et al., 1983) may be such inhibitory interneurons. In agreement with this, the firing rate of cortically projecting basal forebrain cells is strongly decreased by the application of GABA (Lamour et al., 1986), and the release of acetylcholine from the neocortex is diminished by injection of muscimol (a GABA agonist) into the basal forebrain (Casamenti et al., 1986). These findings are consistent with the view that the activity of the cortically projecting cholinergic basal forebrain neurons is regulated by GABA-ergic inhibitory interneurons. The foregoing data provide strong evidence for the concept that the atropine-sensitive component of neocortical low-voltage fast activity is due to the activity of the cortically projecting cholinergic neurons of the basal forebrain. The activity of these cells appears to be controlled by a variety of inputs, probably including local inhibitory interneurons, ascending cholinergic neurons from the brainstem, and, indirectly, dopaminergic fibers from the midbrain (Casamenti et al., 1986).
D. NEOCORTEX: SEROTONERGIC CONTROL The atropine-resistant component of neocortical LVFA which is discussed in the preceding section appears to be dependent on a monoamine. This was first suggested by experiments (Vanderwolf et al., 1978a) showing that atropine-resistant LVFA could be abolished by reserpine, a nonspecific depletor of a variety of amines (Shore and Giachetti, 1978; Boulton, 1979). Thus, following treatment with a combination of reserpine plus atropine or scopolamine all LVFA disappears (Fig. 14). The electrocorticogram consists of alternating B- and C-waves (see Section II1,A) which cannot be blocked by any form of peripheral stimulation or by strong electrical stimulation of the reticular formation (Vanderwolf and Pappas, 1980). Reserpine alone in large doses (5-10 mg/kg) increases the occurrence of large irregular slow waves and spindles, but episodes of LVFA lasting many seconds continue to occur during either immobility or movement.
276
C. H. VANDERWOLF
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FIG.14. Neocortical slow wave activity in a rat following reserpine (10 mg/kg) or pimozide (5 mg/kg) plus atropine (50 mg/kg) treatment. Upper traces: One hr after pimozide and 30 min after atropine treatment the rat is severely cataleptic but makes occasional spontaneous head movements (h) that are associated with low-voltage fast activity (LVFA). Walking and struggling are also associated with LVFA but large slow waves occur during immobility. Lower traces: Same rat 24 days later; 48 hr after reserpine and 30 min after atropine treatment, the rat displays moderate spontaneous activity, moving the head, stepping, and walking. LVFA is absent. Time marks indicate 1- and 5-sec intervals. (From Vanderwolf and Pappas, 1980, by permission of Elsevier Science Publishers B.V.)
One approach used to identify the transmitters involved in the production of atropine-resistant LVFA has consisted of attempts to restore such LVFA in reserpinized rats (Vanderwolf et al., 1980;Vanderwolf, 1984). L-Dopa was of particular interest since it will restore spontaneous motor activity in rats rendered immobile and akinetic by a large dose of reserpine (Carlsson et al., 1957). Figure 15 shows that L-dopa, given together with benserazide (a peripherally acting inhibitor of L-aromatic
277
CONTROL OF CEREBRAL ACTIVITY AND BEHAVIOR
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FIG.15. Effects of reserpine, L-dopa, and atropine sulfate on neocortical electrical activity and behavior. CTX, neocortex; MVMNT, movement sensor output. Top traces: After treatment with reserpine (10 mg/kg, ip) rat is cataleptic and akinetic. Note (1) low-voltage fast activity (LVFA) during immobility; (2) LVFA during spontaneous movement; (3) irregular slow waves during immobility; (4) rhythmical spindle activity during immobility. As in normal rats, LVFA is always present during Type 1 movement but either LVFA or slow waves can occur during immobility. Middle traces: Sixty minutes after injection of Ro-4-4602 (benserazide, 50 mg/kg, ip, a peripheral blocker of L-aromatic amino acid decarboxylase) and 30 min after L-dopa (300 mg/kg), LVFA is nearly continuous. The rat moves its head, rears, and walks. Bottom traces: Ten minutes after the administration of atropine (50 mg/kg, ip) all LVFA is abolished although the rat is very active, walking and gnawing at the apparatus. (From Vanderwolf et al., 1980, by permission of Elsevier Science Publishers B.V.)
278
C. H. VANDERWOLF
amino acid decarboxylase) (Pletscher and Gey, 1963) produces an impressive increase in the occurrence of both LVFA and spontaneous motor activity in reserpinized rats. This agrees with previous work (Takagi et al., 1968). Other dopaminergic agonists such as D-amphetamine or apomorphine produce similar effects. The increase in LVFA is probably due to the release of acetylcholine in the neocortex since dopaminergic agonists are known to increase such release (Pepeu and Bartolini, 1968; Pepeu, 1973). However, when atropine is injected, either before or after treatment with dopaminergic agonists, all LVFA disappears even though the animals are extremely active (Fig. 15). Consequently, atropineresistant LVFA is not restored by L-dopa and other dopaminergic compounds. The amino acids D,L-tyrosine, m-tyrosine; L-phenylalanine, and D-phenylalanine are also completely ineffective, as are clonidine (a noradrenergic agonist) and octopamine (Vanderwolf, 1984). In contrast, 5 -hydroxytryptophan (5-HTP) produces a significant restoration of atropine-resistant LVFA (Vanderwolf, 1984) (Fig. 16), suggesting that serotonin is responsible for such activity. Reserpine-treated rats display a clear increase in the occurrence of LVFA following treatment with a combination of benserazide (50 mg/kg) and 5-HTP (300 mg/kg). This is accompanied by the well-known serotonin syndrome (Jacobs, 1976), which includes resting tremor, side-to-side head movements, splaying of the hind limbs, and alternating treading movements of the forelimbs. The addition of atropine sulfate (50 mg/kg) did not block the behavioral syndrome and reduced but did not abolish LVFA, which continued to occur on 77.8 f 8.9% of a series of trials in which the rats were pushed in an attempt to induce locomotion or stepping. A normal rat treated with atropine alone will display LVFA on 100% of such trials. If rats are initially treated with a combination of reserpine plus atropine, all LVFA disappears, as already noted. If benserazide and 5-HTP are added, LVFA is restored (see Fig. 16). 5-Methoxy-N,N-dimethyltryptamine and fl-phenylethylamine, compounds that have a serotonergic agonistic effect (Jacobs, 1976; Sloviter et al., 1978, 1980), will also restore atropine-resistantLVFA in reserpinized rats (Figs. 16 and 17). Pargyline, nialamide, and tranylcypromine have a similar effect (Fig. 18), producing quite clear LVFA in 15-30 min in rats pretreated with reserpine and atropine. The probable explanation of this effect is that in reserpinized rats, in which dopamine, norepinephrine, and serotonin are all severely depleted, treatment with a monoamine oxidase inhibitor leads to a rapid increase in brain serotonin levels but little or no change in the levels of the catecholamines (Fuxe, 1965). The ability of serotonergic agonists to produce LVFA in rats pretreated with reserpine and atropine appears to be a genuine restoration and
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FIG.16. Restoration of atropine-resistant LVFA in reserpinized rats by treatment with serotonin agonists. (A) Sixteen hours after treatment with reserpine (10 mg/kg, ip) and 15 min after treatment with scopolamine hydrobromide (5 mg/kg, sc), the parietal neocortex displays continuous irregular slow waves (SW) associated with a burst-suppression pattern of deep multiunit activity (MUA). (B)A few minutes after the administration of 5-methyoxy-N,N-dimethyltryptamine (4 mg/kg, ip), LVFA reappears and is associated with a continuous MUA discharge. Bipolar transcortical record. Calibration in A and B for SW, 0.5 mV; for MUA, 0.050 mV 200 msec. (C) Rat prepared with reserpine (as in A) plus atropine (50 mg/kg, ip) plus benserazide (50 mg/kg, ip) displays continuous large irregular slow waves. MVMT, output of movement sensor. At arrows, rat is pushed forward to elicit walking or struggling, but no LVFA occurs. (D) After addition of 5-HTP (300 mg/kg, ip). Beginning after a delay of about 35 min, and increasingly thereafter, brief episodes of LVFA occur spontaneously or in response to pushing (at arrow). The amplitude of the irregular slow waves is also decreased. Calibration for C and D, 1 mV, 5 sec.
not merely a reduction in wave amplitude, since a continuous multiunit discharge accompanies the restored activity. It is unlikely that the effect is due to some sort of peripheral action of the drugs, since peripheral stimulation does not produce LVFA in rats treated with a combination of reserpine and atropine and since significant peripheral effects of serotonin would not occur when 5-HTP is combined with benserazide (Pletscher and Gey,1963; Butcher et al., 1972). Further, the effect cannot be due to interaction of the serotonergic drugs with the catecholamine systems, such as a release of dopamine by 5-HTP (Butcher et al., 1972; Ng et al., 1972; Fuller and Perry, 1981),since catecholaminergic agonists themselves do not restore atropine-resistantLVFA. Still another possibility is that the serotonergic agonists restore LVFA in rats treated with a
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FIG.17. Effects of atropine and fl-phenylethylamine (PE) on spontaneous neocortical electrical activity in a reserpinized rat. SW, slow wave activity; band pass set at . 3 Hz to 3 kHz; MUA, multiunit activity, band pass set at .3-10 kHz. Both records from same bipolar surface-to-depth electrode pair. Upward deflection indicates positivity in deep cortical layers. MUA derived from deep electrode. (A, B) Sixteen hours after reserpine (10 mg/kg) treatment the neocortex may display low-voltagefast activity (LVFA) associated with continuous MUA (A) or large-amplitude SW associated with bursting pattern of MUA (B). Units discharge during SW deep negativity and are silent during SW deep positivity. (C) Thirty minutes after atropine SO4 (50 mg/kg) treatment, the large SW and bursting MUA are continuous; LVFA has disappeared. (D) Ten minutes after PE (80 mg/kg) treatment, LVFA and continuous MUA have reappeared. (From Vanderwolf, 1984, by permission of Humana Press Inc.)
combination of reserpine and atropine by an antiatropine effect rather than by an antireserpine effect. However, none of the serotonergic agonists used are effective as anticholinesterases and have no known action on muscarinic synapses. Consequently, it is probable that the restoration of LVFA in rats treated with reserpine and atropine is due to a restoration of function at central serotonergic synapses. It should be noted however, that the restorative effect is never complete. So far, in reserpinized rats, it has not been possible to restore the good correlation that exists between neocortical activity and motor activity in the atropinized but nonreserpinized rat.
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FIG. 18. Pargyline-induced restoration of atropine-resistant LVFA in the right neocortex (R CTX)of a reserpinized rat. Upper traces: Control rat (701). After reserpine (10 mg/kg), electrocorticogram includes LVFA (l),rhythmical spindles (2), and irregular SW (3) all occurring during behavioral immobility. After atropine treatment (50 mg/kg), all LVFA is abolished, even during trials in which the rat is pushed to elicit walking (p). No further change after saline administration. Lower traces: Experimental rat (670) resembles control rat after reserpine and reserpine plus atropine but displays prominent LVFA during spontaneous behavior after administration of pargyline (50 mg/kg). h, Head movement; step, stepping; hs, head shake. (From Vanderwolf, 1984, by permission of Humana Press Inc.)
These findings provide strong evidence that atropine-resistant neocortical LVFA is dependent on serotonin rather than on catecholamines. Experiments involving selective depletion of specific amines point to the same conclusion. Systemic injection of a-methyl-p-tyrosineor treatment with 6-hydroxydopamine (given systemically in neonatal rats or in the cerebral ventricles in adult rats) does not abolish atropine-resistantLVFA in the neocortex even though catecholamine levels are reduced by as much as 99% (Robinson et al., 1977; Whishaw et al., 1978b). However, systemic injection of p-chlorophenylalanine, a relatively specific inhibitor of tryptophan hydroxylase and, by this means, of the synthesis of serotonin (Koe and Weissman, 1966), can produce a total disappearance of atropine-resistant LVFA (Vanderwolf and Baker, 1986; Vandenvolf et al., 1987). As in the case of reserpine, the result is the occurrence of a continuous sequence of large-amplitude B- and C-waves in the neocortex when p-chlorophenylalanine is combined with a muscarinic blocking drug such as scopolamine (Fig. 19). Injection of 5,7-dihydroxytryptamine, a specific serotonergic neurotoxin, into the cerebral ventricles (together with systemic injection of desipramine to reduce
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FIG. 19. Effects of atropine and PCPA on neocortical electrical activity. CTX, record from surface-to-depth electrode in sensorimotor neocortex; M, output of movement sensor; h, spontaneous head movement; t, turn; w, walk. Upper traces: Neocortical activity in relation to behavior following atropine (50 mg/kg, ip) treatment. Lower traces: Neocortical activity in relation to behavior following PCPA (500 mg/kg/day for 3 days) plus atropine (50 mg/kg) treatment. The lower records were taken 6 days after the upper records. Note that atropine-resistant LVFA has disappeared. Calibration: 0.5 mV, 5 sec. (From Vanderwolf and Baker, 1986, by permission of Elsevier Science Publishers B.V.)
destruction of noradrenergic neurons, as recommended by Bjorklund et al., 1975) can produce the same result (Vanderwolf et al., unpublished data). In rats treated with a combination of reserpine or p-chlorophenylalanine plus atropine or scopolamine the spontaneous electrocorticogram consists of uninterrupted slow waves regardless of the concurrent behavior. The modulation of the transcallosal evoked potentials that normally correlates with motor activity (see Section II1,B) also disappears. The late component of the evoked potential and the related multiunit suppression are both large and well developed regardless of concurrent motor activity in rats treated with this drug combination (Vanderwolf et al., 1987). It is interesting that systemic treatment with p-chlorophenylalanine or intracerebral injections of 5,7-dihydroxytryptamine have no obvious
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gross effect on the electrocorticogram in the absence of antimuscarinic drugs. As in normal rats, LVFA is usually present during immobility in the waking state and is always present during locomotion or extensive head movement. This suggests that cholinergic inputs to the neocortex are normally active during walking and other Type 1 behavior as well as during immobility. This is similar to the situation in the hippocampus (see Section IV,A) in which cholinergic inputs are active during Type 1 behavior. As we have seen, several converging lines of evidence point to the conclusion that the atropine-resistant LVFA that occurs in correlation with Type 1 behavior in rats is dependent on central serotonergic pathways. Other indoleamines, such as tryptamine, appear to be ruled out by the following observations: (1) p-chlorophenylalanine blocks the atropine-resistant effects and (2) 5-hydroxytryptophan restores the atropine-resistant effects in reserpinized rats. It is likely that the atropine-resistant effects on the neocortex are exerted via the ascending serotonergic projections from the brainstem (Dahlstrom and Fuxe, 1964; And& et al., 1966; Azmitia, 1978; Steinbusch and Nieuwenhuys, 1983). A number of findings suggest this. Highfrequency (100-Hz) stimulation of the median raphe nucleus can produce LVFA, usually in association with Type 1 movement, in waking atropinized rats (Robinson and Vanderwolf, 1978). Also, Jacobs and his colleagues have provided independent evidence that is consistent with this view. They have identified serotonergic units in the brainstem of freely moving cats by the following criteria: (1) regular firing at about 3-5 Hz, (2) depression of firing following systemic administration of 5-methoxy-N,N-dimethyltryptamine, and (3) anatomical location. The activity of units in the dorsal and median raphe nuclei that meet these criteria correlates, in a general way, with motor activity. Higher firing rates occur during active waking than during quiet waking and the rate in most cells is increased (approximately doubled) by visual and auditory stimuli that elicit orienting movements. (An orienting movement of the head is a Type 1 movement in rats.) The occurrence of sleep spindles is frequently preceded and accompanied by a transitory decrease in firing rate (Trulson and Jacobs, 1979, 1981; Heym et al., 1982; Rasmussen et al., ,1984). A possible correlation of unit activity with specific behaviors such as walking or face washing does not appear to have been looked for in these studies, but one of their figures (Fig. 6 in Trulson and Jacobs, 1981) shows a dorsal raphe unit in a cat that had a distinctly higher rate during the digging of a hole than during a subsequent period of immobility during which the cat defecated. The rate increased again as the cat moved about subsequent to defecation. This type of correlation with behavior is strongly reminiscent of the Type 1-Type 2 behavior
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classification suggested by work on hippocampal and neocortical slow wave activity in rats. Additional detailed observations in this field would be very desirable. One potential difficulty with the theory that atropine-resistant activation of the neocortex and hippocampus is dependent on central serotonergic pathways is that a number of supposed serotonergic antagonists are completely ineffective in blocking the atropine-resistant waveforms. Methysergide, cyproheptadine, metergoline, and spiroperidol fail to block either atropine-resistant RSA or atropine-resistant LVFA (Vanderwolf, 1984), even at dose levels well in excess of those inhibiting 5-hydroxytryptophan-induced head twitches (Peroutka et al., 1981). There appear to be several different types of serotonin receptors in the brain (Peroutka and Snyder, 1979; Aghajanian, 1981; Barret et al., 1982; Nelson et al., 1983; Robaut et al., 1985), possibly differing from those located peripherally. Since the classical peripherally active serotonin antagonists do not appear to be very effective in blocking central serotonergic effects (Haigler and Aghajanian, 1974), it is perhaps not surprising that they also fail to block atropine-resistant RSA and atropine-resistant LVFA. A factor that may be important in this connection is that atropineresistant hippocampal RSA and atropine-resistant neocortical LVFA are both abolished by a variety of anesthetics (Vanderwolf et al., 1975, 1978a), suggesting that central serotonergic function is grossly altered by anesthetics. Studies on the elicited activity of dorsal raphe units (Heym et al., 1984) and iontophoretic studies of the effect of serotonin on neocortical neurons (Roberts and Straughan, 1967;Johnson et al., 1969) also point to this conclusion. Consequently, the results of studies of serotonergic systems carried out under conditions of anesthesia may not be fully applicable to normal physiology. Conceivably, a compound that antagonizes serotonin in anesthetized animals might not be effective in waking animals. Another potential difficulty with the views presented here is that lesions of the median raphe nucleus do not abolish atropine-resistant RSA, although its frequency is reduced (Maru et al., 1979). The probable explanation of this is that large numbers of serotonergic neurons that project to the cerebral cortex are located outside the mesencephalic raphe nuclei (Fuxe and Jonsson, 1974; Steinbusch and Nieuwenhuys, 1983). As a result, raphe lesions produce only moderate depletions of forebrain serotonin. E. NEOCORTEX: MECHANISMS OF ACTIVATION The mechanisms through which acetylcholine and serotonin cause the occurrence of LVFA in the neocortex are not well understood.
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According to several authoritative reviews (Steriade and Hobson, 1976; Steriade, 1978, 1981) neocortical projection neurons usually fire at a higher mean rate and appear generally more excitable during quiet waking than during quiet (slow wave) sleep. In contrast, neocortical interneurons fire at lower mean rates during waking than during quiet sleep. In agreement with this, neocortical activation is associated with an increased release of glutamate, probably derived from cortical efferent neurons (McGeer et al., 1977) and a decreased release of GABA, probably derived from interneurons (Jasper and Koyama, 1969). However, it is not clear what significance should be attached to neuronal firing rates averaged over periods of several seconds. Projection neurons fire at high rates during bursts during slow wave episodes and may be totally silent between bursts, as discussed above (see Section 111,A). Under these circumstances, the mean firing rate may have little meaning in a physiological sense. It is perhaps more meaningful to analyze the electrocorticogram wave by wave, as recommended by Calvet et al. (1973), than it is to treat long segments of record as if they were homogeneous. Furthermore, it is unlikely that the mean firing rate of projection neurons is of much significance for the appearance of either large slow waves or LVFA. It is well known that anesthetics depress markedly the activity of neocortical neurons (Mountcastle et al., 1957; Murata and Kameda, 1963), but this does not prevent the appearance of LVFA in anesthetized animals (Bremer, 1936; Beecher and McDonough, 1939; Rossi and Zirondoli, 1955). Perhaps the most striking feature of unit activity during LVFA episodes is the smoothing out of the burst-suppression pattern of firing seen during slow wave episodes, thus yielding a continuous pattern of unit firing (see Section 111,A). The reduction of phasic synchronized inhibition, which is probably largely responsible for this, appears to be much greater during Tn>e 1 behavior, when both cholinergic and serotonergic systems are active, than it is during waking Type 2 behavior, when activation is largely or entirely controlled by cholinergic afferents to the neocortex. Thus, the period of multiunit suppression following an excitatory callosal volley is longer during immobility or face washing than during walking (see Section 111,B). When the cholinergic and serotonergic inputs to the neocortex are suppressed by pharmacological means this phasic synchronized inhibition is allowed free rein. Continuous slow waves and a lack of behavioral modulation of the transcallosal evoked potential are the result. It is well known that acetylcholine has an excitatory effect on some neocortical neurons when applied by iontophoresis (Krnjevie and Phillis, 1963a,b; KrnjeviC et al., 1971) but inhibits other neocortical neurons (Randic et al., 1964; Phillis and York, 1967). In vitro studies indicate
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that acetylcholine produces (1) a slow excitation of neocortical pyramidal cells, (2) a blockade of Ca2+-activated K + conductance, (3) inhibition of pyramidal cells, possibly via a rapid muscarinic activation of GABAcontaining interneurons, and (4) a possible presynaptic effect on the release of other transmitters (McCormick and Prince, 1986). The relation of these effects to the regulation of the gross electrocorticogram is not clear. Intracellular studies and a cellular model of cortical activation have been presented by Inubushi et al. (1978a,b). Serotonin has not been studied as intensively as acetylcholine, but it too inhibits some neocortical neurons and excites others (Roberts and Straughan, 1967; Johnson et al., 1969; Jones, 1982). Interpretation of such findings would be greatly facilitated if tests were carried out on neurons that could be identified as projection cells or interneurons, a point that has been stressed by Steriade (1978).
F. CINGULATE CORTEX Very little work has been done as yet on the electrical activity of the cingulate cortex. A recent study by L u n g and Borst (1987) of rats shows that cingulate slow wave activity resembles hippocampal activity in several respects. Irregular slow waves, accompanied by irregularly occurring sharp waves of about 20-msec duration, occur during waking immobility, grooming, drinking, and eating (Type 2 behavior). The sharp waves are suppressed and a rhythmical waveform of 6-10 Hz that resembles hippocampal RSA occurs during head movements, walking, rearing, and changes in posture (Type 1 behavior). Type 1 behavior is also associated with increased fast waves ( > 30 Hz)as compared to Type 2 behavior. In addition, the cingulate transcallosal evoked potential displays different waveforms during Type 1 and Type 2 behavior. h u n g and Borst suggest that these waveforms are generated in the cingulate cortex, since (1) the sharp waves (EEG spikes) display opposite polarity at the surface and in the depths of the cingulate cortex, (2) the sharp waves correlate with bursts of multiunit activity, (3) the RSA-like waves are phase locked to bursts of multiunit activity, and (4) small medial septa1 lesions which abolish hippocampal RSA leave intact, or actually increase, the amplitude of the RSA-likewaveform in the cingulate cortex (Borst et al., 1987; Leung and Borst, 1987). A series of pharmacological and lesion experiments showed that the correlations observed between behavior and the electrical activity of the cingulate cortex are largely due to the activity of an atropine-sensitive cholinergic input from the region of the diagonal band but that an
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atropine-resistant input is present as well (Stewart et al., 1985; Borst et al., 1987). Whether or not this atropine-resistant input is dependent on serotonin is not known. The discovery of close correlations between spontaneous motor activity and the slow wave and multiunit activity of the cingulate cortex suggests the necessity of a reinterpretation of the results of experiments attempting to relate cingulate cortex activity to psychological concepts (Gabriel et al., 1980, 1986). Training an animal in a conventional conditioning task, as Gabriel and his colleagues have done, produces complex changes in its motor activity. Unless experiments of the type developed by work on the hippocampus (see Section I1,D) are carried out, it is impossible to distinguish neural activity related to overt motor activity from neural activity that may reflect the operation of psychological processes. G. ROLEOF THE THALAMUS IN THE CONTROL OF NEOCORTICAL ACTIVITY A review by Andersen and Andersson (1968) summarizes early woFk showing that the spindle bursts occurring in the neocortex of barbiturateanesthetized cats are dependent on a rhythmical input from the thalamus. This concept was supported by the following facts: (1) Thalamic neurons, but not striatal, midbrain, pontine, or hypothalamic neurons, fire in a bursting pattern, phase-locked to the waves of neocortical spindles. (2) This rhythmical bursting persists in the thalamus after removal of the neocortex. (3) Unilateral thalamic lesions abolish spindles ipsilaterally; bilateral thalamic lesions abolish them bilaterally. (4) A singlepulse electrical stimulus applied to the thalamus will often trigger a complex waveform that closely resembles spontaneous barbiturate spindles. ( 5 ) Cooling of the whole brain, including the thalamus, reduces the frequency of neocortical spindles but local cooling of the neocortex does not alter spindle frequency. These findings indicate that the thalamus acts as a pacemaker for the neocortex in the generation of barbiturate spindle activity. The rhythmical spindle activity that occurs during natural slow wave sleep also appears to be dependent on the thalamus, since it disappears ipsilaterally following a large unilateral thalamic lesion (Angeleri et al., 1969) and disappears bilaterally after a bilateral thalamic lesion (Villablanca and Salinas-Zeballos, 1972). Thalamic projection cells tend to fire in a bursting pattern during slow wave sleep and during spindles (Verzeano and Calma, 1954; Hubel, 1960; Sakakura, 1968; Benoit and Chataignier, 1973) and, under favorable
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conditions, rhythmical bursting in phase with concurrent neocortical spindle waves can be demonstrated (Steriade and Hobson, 1976). Andersen and his colleagues (Andersen and Sears, 1964; Andersen and Andersson, 1968) proposed that synchronized rhythmical bursting in thalamic neurons was brought about by both inhibitory interneurons with a widespread recurrent effect; and post-anodal exaltation leading to rebound firing after inhibition in thalamocortical neurons. Thus, thalamocortical neurons would fire in synchronized periodic bursts as a result of the combined action of these two mechanisms. More recent in vilro studies have developed this hypothesis by showing that individual thalamic cells possess intrinsic properties that permit either tonic firing or burst firing in either of two frequency ranges, 5-6 Hz and 9-11 Hz (Deschhes et al., 1982; Jahnsen and Llinis, 1984a,b). However, under in vivo conditions, the development of 7- to 14-Hz rhythmical activity in the thalamus is strongly dependent on intercellular connectivity since such activity is abolished when the reticular nucleus is destroyed or disconnected from the rest of the thalamus (Steriade et al., 1985). Steriade et al. suggest that neurons in the thalamic reticular nucleus regulate the activity of local inhibitory interneurons in other thalamic nuclei. Rhythmical spindling in the intact thalamus thus appears to be dependent on both the properties of intrinsic rhythmical processes in the membranes of thalamic cells and on the properties of the network of intrathalamic connections. In contrast to the rhythmic spindling type of wave form, the large amplitude irregular waveform of the neocortex is probably not primarily dependent on input from the thalamus. That there may be important differences between spindles and irregular slow waves was noted by Dempsey and Morison (1942), who observed that the latter (referred to as “projection activity”) are more sensitive to anesthetics than the former. This has been contirmed repeatedly (Calvet et al., 1973). The opposite type of dissociation was observed by Angeleri et al. (1969), who noted that thalamic lesions abolish rhythmical spindles but increase the occurrence of irregular 2- to 5-Hz waves in the neocortex. Similarly, large doses of atropine abolish 6- to 10-Hz spindles in waking rats but increase the occurrence of large, irregular, 2- to 6-Hz slow waves (Vandemolf, 1975). The study of the electrical activity of the isolated cerebral cortex is of particular interest with respect to the genesis of the large amplitude irregular type of activity. Small isolated slabs of neocortex may not display spontaneous activity, especially if the circulation has been interfered with (Andersen and Andersson, 1968). In contrast, large slabs of neocortex (Ingvar, 1955) or an entire isolated cerebral hemisphere (Kellaway et al., 1966; Villablanca, 1966/1967) may display continuous spontaneous
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activity. In the experiments of Kellaway et al. (1966) the neocortex of one hemisphere was isolated by section of the callosum plus removal of much of the striatum, hippocampal formation, and other tissue to reach the base of the anterior fossa. Completeness of the section was confirmed by the failure of seizures to propagate from the intact to the isolated cortex and by subsequent histological study. In isolated neocortex preparations with good circulation (assessed by intravenous or intra-aortic injection of India ink at the end of the experiment), the electrocorticogram consisted of continuous irregular slow waves of 0.5-4.0 Hz with some superimposed fast activity. Extracellular unit discharges in layer VI tended to occur on the surface-positive component of the slow waves (Frost and Gol, 1966), suggesting the presence of alternating B and C generators according to the terminology of Calvet (see Section 111,A). However, in a later paper, using the same isolated hemisphere preparation, Frost (1967) reported intracellular recordings from a group of 16 neurons that discharged predominantly during the surface-negative component of the slow waves. Rhythmical spindles and low-voltagefast activity were not observed in the isolated hemisphere preparation, although there were silent periods (i.e., episodes of no spontaneous activity), especially in cases with defective circulation (Kellaway et al., 1966). These findings suggest the hypothesis that large-amplitude irregular slow activity is endogenous to the cerebral cortex. There is good evidence that inhibitory effects operate in the neocortex (Krnjevit et al., 1966a,b), probably as a result of the activity of GABA-containing interneurons (Ribak, 1978; Freund et al.,1983). One can hypothesize that alternating bursts of activity and episodes of widely synchronized recurrent and feedforward inhibition might provide the basis of a burst-suppression pattern of activity in pyramidal cells, thus giving rise to a large-amplitude irregular electrocorticogram. It is a striking fact that in the same series of experiments, the isolated cerebral cortex produced irregular patterns of slow waves while the thalamus, isolated by sections rostra1 and caudal to it, gave rise to rhythmical 6- to 8-Hz activity (Kellaway et al., 1966). This may suggest that the properties of neocortical networks differ from those of thalamic networks in some important respects. In a normal animal the activity of the thalamus and neocortex are closely coupled as a result of reciprocal thalamocortical and corticothalamic connections (Jones, 1983). One is led to the conjecture (undoubtedly simplistic) that during spindle bursts, a thalamic pattern of activity is imposed on the neocortex, whereas during large amplitude irregular waves a cortical pattern of activity may be imposed on the thalamus. It is widely held that the main ascending pathway leading to the generation of LVFA in the neocortex consists of inputs from the
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midbrain reticular formation to the medial thalamus, especially the intralaminar nuclei, which then relay the activating effect to the neocortex (Moruzzi and Magoun, 1949; Jasper, 1949; Steriade, 1981; Steriade and Deschtnes, 1984). High-frequency electrical stimulation (100-300 Hz) of the reticular formation or medial thalamus is capable of activating this pathway artificially. However, there are many findings that contradict this hypothesis. Unilateral surgical lesions of the thalamus including the intralaminar nuclei do not prevent the eventual occurrence of good LVFA ipsilateral to the lesion although there may be little or no LVFA in the immediate post-operative period (Angeleri et al., 1969). Villablanca and Salinas-Zeballos (1972) removed virtually the entire thalamus (sparing part of the geniculate bodies) by suction through an opening in the corpus callosum. The electrocorticogram at first consisted exclusively of large-amplitude irregular activity but after a recovery period of 2-3 months, clear LVFA reappeared. Spindle bursts remained absent during survivals of up to 189 days. This suggests that the thalamus is essential for the occurrence of spindle bursts but not for the occurrence of large amplitude irregular slow waves; is not essential for LVFA; but may contribute in some way to the occurrence of LVFA, since LVFA recovered slowly after the thalamic lesion. Schlag and Chaillet (1963) found many sites in the intralaminar thalamic region at which low-frequency electrical stimulation produced good recruiting responses in the neocortex but high-frequency stimulation failed to produce good LVFA. This suggests that the thalamic neurons that mediate the recruiting response are not responsible for the activation of the cortex. In confirmation of this, Schlag and Chaillet (1963) found that transvem cuts placed just caudal to the thalamus could abolish neocortical activation produced by high-frequency stimulation of the intralaminar region but had no effect on cortical recruiting responses produced by low-frequency thalamic stimulation. This suggests that the recruiting response is mediated by ascending thalamocortical pathways but that any activation that is elicitable from the medial thalamus is dependent on a descending pathway. In earlier sections (IV,C and IV,D), evidence has been presented to show that the occurrence of LVFA in the neocortex is dependent on ascending cholinergic pathways from the basal forebrain and ascending serotonergic pathways from the brainstem. This hypothesis does not afford the thalamus a major role in the control of neocortical activation, since neither cholinergic nor serotonergic cell bodies are located there. Since this view disagrees with traditional teaching, a reinvestigation of the role of the thalamus in neocortical activation was carried out (Stewart et al., 1984; Vanderwolf and Stewart, 1988).
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In one component of this investigation, intrathalamic injections of kainic acid were made to produce a widespread loss of thalamic neurons while sparing fibers of passage (Coyle et al., 1978). Such fibers would have been severed in previous work that relied on the technique of surgical destruction of the thalamus. Extensive unilateral lesions, including most of the rostra1 intralaminar nuclei, were found to produce only a shortlived increase in irregular slow wave activity ipsilateral to the lesion (Stewart et al., 1984). These large slow waves disappear within 2 weeks after surgery. Bilateral lesions are more difficult to study since they are often fatal and produce a chronic epileptic condition in many of the survivors. Both behavioral and electrographic seizures have been observed. However, following recovery from a large bilateral lesion destroying almost the entire thalamus except for caudally placed nuclei such as the geniculate bodies, part of the habenula, the caudal extremities of the ventral nuclei, and part of the parafascicular nucleus (Fig. 20) quite good LVFA could be demonstrated (Fig. 21). A second strategy for investigating the role of the thalamus in neocortical activation consisted of the study of thalamocortical transmission in rats following pharmacological blockade of activation (Figs. 22 and 23). Following a large dose (10 mg/kg) of reserpine, good LVFA can be produced by tactile stimulation or by high-frequency (100 Hz) stimulation of the intralaminar region. Low-frequencystimulation of this region produced recruiting responses in the frontal neocortex. Figure 23 illustrates the gradual development of the typical long-latency, surfacenegative recruiting potentials during the course of a brief train of lowfrequency thalamic stimuli. Sensory evoked potentials could be demonstrated in somatosensory, visual, or auditory cortex following stimulation of the appropriate sensory modalities (Fig. 22). These observations are evidence of intact thalamocortical transmission. After the addition of scopolamine, all activation disappeared. No trace of LVFA could be produced by tactile or painful stimulation or by 100-Hzstimulation of the intralaminar region. Despite this, clear-cut sensory evoked potentials and recruiting responses could be readily elicited (Figs. 22 and 23). This means that transmission through the classical thalamocortical pathways (as indicated by sensory evoked potentials) or through the so-called thalamic nonspecific system (as indicated by the recruiting response) is insufficient to produce LVFA. Sensory stimulation produced a clear increase in multiunit activity in the neocortex but was incapable of smoothing out the burst-suppression pattern of firing that occurs continuously in the neocortex when ascending cholinergic and serotonergic transmission is blocked (Fig. 24). The results of these investigations are clear. The occurrence of LVFA
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FIG.20. Reconstructions in the sagittal and coronal planes of cell loss in a rat after bilateral intrathalamic injection of 1 jig of kainic acid dissolved in 1 ~1 of Locke’s solution. Black areas, virtually total cell loss; cross-hatchedareas, partial cell loss. ac, Anterior commissure; CC, corpus callosum; h, hippocampus; hit, habenulointerpendunculartract; sm, stria medullaris thalami. The reconstruction is based on the study of gallocyaninstained coronal brain sections. (From Vanderwolf and Stewart, 1988.)
after near-total destruction of thalamic neurons indicates that the thalamus is not essential for neocortical activation. The fact that thalamocortical transmission survives when neocortical activation has been totally suppressed by a combination of reserpine plus scopolamine indicates that thalamic inputs to the neocortex are not sufficient to produce activation. Taken together, these facts indicate that thalamocortical transmission does not play a major role in the production of LVFA in the neocortex. Nonetheless, it is well established that thalamic projection neurons, like neocortical projection neurons, fire in a burstsuppression pattern when large slow waves are present in the electrocorticogram and fire in a continuous irregular pattern when LVFA is present
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FIG. 21. Neocortical electrical activity in a rat 54 days after infliction of the thalamic lesion illustrated in Fig. 20. L. CTX, left sensorimotor area; time in sec; MVMNT, output of magnet-and-coil movement detector; voltage calibration, 0.5 mV. The lower set of polygraph records are a continuation of the upper set. Note (1) that LVFA occurs spontaneously and when the rat is picked up (p) and (2) that 3- to 4-Hz spindle bursts occur spontaneously. (A) Oscilloscope recording of slow waves (SW) and associated multiunit activity (MUA, derived from layer V of neocortex); note continuous MUA discharge during LVFA. (B) Similar record (same calibration) during spontaneous spindle burst. Note epileptiform “wave and spike” morphology of the slow waves and correlated burst-silence pattern of MUA. All surface-to-depth records; deep negativity is up in the polygraph records but deep positivity is up in the oscilloscope records (A and B). (From Vanderwolf and Stewart, 1988.)
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(Hubel, 1960; Sakakura, 1968; Benoit and Chataignier, 1973; Steriade and Hobson, 1976). Although this correlation between thalamic and neocortical activity has usually been regarded as evidence of thalamic control of the neocortex, it is equally plausible to suppose that activity in both the thalamus and neocortex are regulated in parallel by factors originating outside these structures. It is well established that ascending brainstem cholinergic fibers innervate the thalamus as well as the basal forebrain (Satoh and Fibiger, 1986; Woolf and Butcher, 1986), from which cholinergic control of the neocortex appears to be exerted. Similarly, serotonergic fibers innervate both the thalamus and the cerebral cortex (AndCn et al., 1966; Steinbusch and Nieuwenhuys, 1983). Perhaps the neocortex and thalamus, two structures with abundant reciprocal connections uones, 1983), are regulated in parallel by ascending cholinergic and serotonergic inputs. The occurrence of a continuous irregular discharge in projection neurons (the pattern associated with LVFA) in one of these structures might reinforce the occurrence of a similar pattern in the other. This might help account for the long delay in the reappearance of LVFA in the neocortex following the creation of a large thalamic lesion without the necessity of assuming that thalamic activity is the primary cause of the LVFA pattern in the neocortex. The observation that increases in neocortical unit activity are insufficient to produce LVFA in rats treated with reserpine and atropine is of considerable interest. Taken in the context of the other data reviewed here, the phenomenon suggests that thalamocortical inputs regulate the
FIG. 22. Effects of reserpine and scopolamine on neocortical electrical activity. (A) Surface-to-depthbipolar polygraph recording from left sensorimotor neocortex in a rat about 16 hr after treatment with reserpine (10 mg/kg, ip). Rat is immobile and cataleptic but handling (p, horizontal line) elicits LVFA. (B) After treatment with scopolamine (5 mg/kg, sc) handling does not elicit LVFA. (C) Similar polygraph record from another reserpinized rat: stimulation of the nucleus centralis medialis (125 pA, 0.5-msec rectangular pulses, 100 Hz) at S (horizontal line) produces LVFA. (D) After treatment with scopolamine, stimulation is ineffective. (A-D) Calibration is 0.5 mV, time in seconds. (E) Upper traces: monopolar record from layer V of left sensorimotor cortex; lower trace: bipolar record of multiunit activity derived from layer V of right sensorimotor cortex in rat following treatment with reserpine plus scopolamine. At “0” a 0.5-msec shock was delivered to the sole of the right hind paw, four successive sweeps at irregular intervals. Positivity at active electrodes is up. Calibration: 20 msec, 0.5 mV for upper trace, 0.050 mV for lower trace. Slow wave and multiunit responses occurred in both hemispheres. (F) Average monopolar records from deep layen of lateral (auditory) neocortex (16 sweeps), positivity up; at “X”a 0.5-msec pulse was applied to a speaker directly in front of the rat. Upper trace taken after reserpine treatment; lower trace taken after reserpine plus scopolamine treatment. Calibration: 0.25 mV, 20 msec. (From Vanderwolf and Stewart, 1988.)
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A
B
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FIG. 23. Recruiting responses in a rat treated with reserpine and scopolamine. Monopolar records from deep and superficial (surf) layers of the right sensorimotor cortex. At “o”,single monopolar cathodal pulses are applied to the medial thalamus (nucleus centralis medialis and nucleus rhomboideus) at 8 Hz,170 pA, and 0.5-msec duration. (A and C) After reserpine (10 mg/kg, ip) treatment. (B and D) After reserpine plus scopolamine (5 mg/kg, sc) treatment. Time calibration: 100 msec in A and B; 10 msec in C and D. Voltage calibration: 1 mV for all. In C and D, only the last four to six sweeps of a series of 10 successive shocks are shown. Positivity up. Note that good recruiting responses occur under both drug conditions. (From Vandenvolf and Stewart, 1988.)
firing rate of specific neocortical cells but that cholinergic and serotonergic inputs regulate the overall pattern of firing throughout the neocortex.
H. BEHAVIORAL EFFECTSOF CENTRAL AND SEROTONERGIC BLOCKADE
CHOLINERGIC
Until quite recently, the only known means of producing a total loss of cortical activation was deep anesthesia or acute transection of the
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FIG. 24. Effects of reserpine and atropine sulfate on slow wave and multiunit activity in the somatosensory neocortex. MUA, multiunit activity; SW, slow wave activity. Each deflection of the MUA record represents four unit spikes. (A) Polygraph record 22 hr after reserpine (10 mg/kg) treatment. The ECG consists of mixed LVFA and large amplitude slow waves. Handling the rat (pick up between arrows) increases MUA. (B) Thirty minutes after the administration of atropine (50 mg/kg), LVFA is abolished but handling the rat still increases MUA. (C) Oscilloscope record of normal LVFA pattern with an associated continuous MUA discharge taken the day before reserpine treatment. (D) Oscilloscope record of large amplitude, irregular slow wave pattern with an associated burs-suppression pattern of MUA taken after treatment with reserpine plus atropine. Calibration for A and B: 1 sec and 0.5 mV for C and D: MUA, 0.050 mV; SW, 0.50 mV, 200 msec. (From Vanderwolf and Stewart, 1988.)
brainstem (Bremer, 1935, 1936). Even following very large lesions of the midbrain tegmentum or the medial and ventral diencephalon, some neocortical LVFA persists during the application of auditory or somatic stimulation (Lindsley et al., 1949, 1950). Anesthesia and brainstem lesions both produce a state of immobilization and coma. In contrast, a combination of central cholinergic and serotonergic blockade is capable
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of eliminating all neocortical LVFA (as well as all hippocampal RSA), without eliminating spontaneous motor activity. This makes possible behavioral studies of the effects of a selective loss of all cortical activation in the absence of a generalized depression of central nervous function. Following systemic injection of a large dose of reserpine, rats become akinetic and cataleptic (Bein, 1956). Restoration of catecholaminergic function by injection of L-dopa (Carlsson et al., 1957) or dopaminergic agonists (Rech, 1964; Anden et al., 1973) restores spontaneous motor activity and the ability to perform well in simple behavioral tasks. For example, the ability to perform hypothalamic self-stimulationin a leverpressing task is temporarily restored to normal levels in reserpinized rats following an injection of D-amphetamine (Stein, 1964; Vanderwolf et al., 1984). However, if reserpine is combined with atropine or scopolamine so that all neocortical activation is abolished, amphetamine does not restore self-stimulation. The treated rats walk about continuously in an aimless fashion, or gnaw at the apparatus, but do not consistently remain near the experimental lever (Vanderwolf et al., 1984). Amphetamine and atropine, alone or in combination, do not prevent high levels of performance in the self-stimulation task. A combination of p-chlorophenylalanine and scopolamine, which provides a convenient means of abolishing cerebral cortical activation, produces deficits in selfstimulation behavior that are similar to those produced by a combination of reserpine with atropine or scopolamine plus amphetamine (Vanderwolf, 1987b). Rats treated with a combination of p-chlorophenylalanine and scopolamine are interesting preparations from a behavioral point of view (Vanderwolf and Baker, 1986; Vanderwolf, 1987b). Occasionally, they lapse into a state of immobility, resting on the floor with the eyes closed, but more frequently, they walk about actively. When placed on a table, they will walk over the edge without hesitation if they are not physically restrained. When placed in a tank filled with water, they swim as rapidly as normal rats but are completely unable to find an elevated platform that is plainly visible in the center of the tank (Fig. 25). The ability to acquire or retain the behavior of jumping out of a box to avoid an FIG.25. Top view of an aquarium used in behavioral tests. Top: Rat treated with parachlorophenylalanine(500 mg/kg/day X 3, ip) plus scopolamine (5 mg/kg, sc) swims in circles around the aquarium, failing to find the wire mesh platform in the center. Bottom: Parachlorophenylalanine plus scopolamine-treatedrat has just swum the length of the tank and climbed up the wire mesh screen. (From Vanderwolf, 198713, by permission of Elsevier Science Publishers B.V.)
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electric shock is also lost, although the rats are physically able to jump. Vehicle-injected normal rats, placed in an unfamiliar observation box after receiving a soaking in a water tank, engage in long sequences of grooming behavior, for example: sit up, paw shake, face-wash,postural change, lick back, postural change, paw shake, face-wash, lick paws, postural change, lick abdomen, etc. Such sequenceswere found to contain a mean of 13.4 f 1.9 items of grooming behavior and are ended by the occurrence of episodes of head movement, sniffing, and walking. Rats treated with a combination of @-chlorophenylalanineand scopolamine, tested in the same way, display attenuated sequences of grooming containing only 2.8 f 1.1 grooming behavior items (Vandenvolf, 1987b). Very similar attenuated sequences occur in rats that have been surgically decorticated (Vanderwolf et al., 1978b). It may be that in both cases the grooming sequences are shortened as a result of loss of cerebral control of the postural changes (Type 1 behaviors) that link the various Type 2 behavior components of grooming together. In sum, it can be said that treatment with a combination of 9-chlorophenylalanine and scopolamine or atropine, resulting in a loss of both types of activation of the hippocampus and neocortex, produces a severe generalized impairment in the organization of behavior. A similar behavioral impairment and loss of cerebral activation appears to be present in rats treated with a combination of reserpine and atropine, plus amphetamine to reverse the reserpine-induced catalepsy and akinesia. Motor performance at a low level is largely intact in both types of preparation. Sensory input, at least at a simple level, is also intact when activation is completely abolished, since visual, auditory, and somesthetic evoked potentials can be demonstrated readily in the appropriate cortical areas in rats treated with a combination of reserpine and scopolamine (see Section IV,G). What appears to be lost when cortical activation is abolished is the ability to perform specific motor acts in adaptive relation to the environment and to other behavior. The overall effect is strongly reminiscent of the generalized behavioral deterioration or dementia produced by extensive cortical lesions (Lashley, 1929; Vandenvolf et al., 1978b; Whishaw et al., 1983). Can the deficits produced by a blockade of cerebral cortical activation be attributed to a loss of the capacity for learning and memory? This question is ambiguous since the terms “learning” and “memory” may refer to either a change in behavior as a result of individual experience, or a hypothetical change in the nervous system, that is, formation of an engram (Vanderwolf, 1987b). “Learned’ behavior is often contrasted with “instinctive”behavior, which is performed appropriately without prior experience (Alcock, 1984, p. 87).
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With respect to acquired behavioral performance, there have been numerous demonstrations that centrally acting antimuscarinic drugs impair the ability of rats to perform well in various mazes, discrimination tests, and shock avoidance tests (Macht, 1924;Bures'ovai et al., 1964; Longo, 1966;Brimblecombe, 1974;Sutherland et al., 1982;Spencer and Lal, 1985; Whishaw et al., 1985). Deficits are not found in all tests, however. Longo (1966)pointed out that the drug-induced impairments are more evident in difficult tasks than in simple ones and also if the animals receive no training prior to the drug treatment. Destruction of the basal forebrain, damaging the corticopetal cholinergic projections, has effects which resemble, in some respects, the effects of systemic administration of antimuscarinic drugs (Lo Conte et al., 1982b;Mitchell et al., 1982;Hepler et al., 1985a,b;Lerer et al., 1985; Whishaw et al., 1985; Dunnett et al., 1987). Accentuation of cholinergic function by treatment with cholinergic agonists or high dietary choline or lecithin, however, can improve behavioral performance in aged rats (Bartus et al., 1980, 1982)or in rats with lesions of the basal forebrain (Murray and Fibiger, 1985, 1986). Interference in central serotonergic function may have effects that are partially similar to the effects of interfering with central cholinergic function. For example, there is evidence that manipulation of central serotonergic pathways affects shock avoidance behavior (Open, 1982). Destruction of serotonergic neurons by treatment with p-chloroamphetamine impairs subsequent avoidance responding in a shuttle box (Kijhler et al., 1978). Further, performance in a maze test is severely impaired if brain serotonin is depleted by 70% or more as a result of intracerebroventricular injections of 5,7-dihydroxytryptamine. Depletion of central noradrenalin had very little effect in the same maze test (Pang et al., 1985). The behavioral impairments produced by specific blockade of either central cholinergic or central serotonergic systems are not restricted to tests involving training. Centrally acting antimuscarinic drugs block untrained or instinctive behavior such as grooming, aggressive attacks, and mating behavior (Soulairac and Soulairac, 1957;Singer, 1968;Van Der Poel and Remmelts, 1971; Bell and Brown, 1985). Similarly, the serotonin-depleting drug p-chlorophenylalanine has been reported to disturb various untrained or instinctive behaviors, although the nature of the disturbances seems quite different from those produced by antimuscarinic drugs. Different investigators have described increased male-male sexual mounting (Sheard, 1969; Tagliamonte et al., 1969; Ferguson et al., 1970); increased predatory attack (Sheard, 1969;Ferguson et al., 1970);increased social grooming and body contact (Shillito, 1969);
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increased spontaneous locomotor activity (Fibiger and Campbell, 1971; BorbCly et al., 1973; Marsden and Curzon, 1976); occurrence of behavior (in cats) suggesting hallucinations (Ferguson et al., 1970; Dement et al., 1972); and increased reactivity to painful stimuli (Tenen, 1967). Confusion and hallucinations have also been reported following treatment with parachlorophenylalanine in human carcinoid syndrome patients (Engelman et al., 1967). In addition, Miliaressis et al. (1975) and Miliaressis (1977) have reported that median raphe stimulation will support self-stimulation behavior and that p-chlorophenylalanine will block this without affecting self-stimulation at ventromedial tegmental sites in the same animals. Consequently, it is probable that blockade of central cholinergic and serotonergic transmission produces a generalized deterioration of both learned and instinctive behavior rather than a specific loss of learning and memory. Conversely, although no evidence on the topic appears to exist, it may be expected that manipulations that improve subnormal function of central cholinergic and serotonergic transmission would produce improvements in instinctive behavior as well as in learned behavior. The defects in instinctive and learned behavior following central cholinergic or serotonergic blockade may have a common basis. It is widely recognized that instinctive predispositions play a major role in performance in tests of learning and memory (Seligman and H a p , 1972; Hinde and Stevenson-Hinde, 1973; Barker et al., 1977). Consequently, if a pharmacological procedure produces impaired performance in a test of “learning” or “memory” it may be that the deficit is due to interference at nonplastic (innately organized) synapses rather than at plastic (learning) synapses in a complex mechanism in which both types of synapses are necessary for successful performance. It is very difficult to devise a procedure that will permit the unambiguous identification of a specific class of synapses as the site of plastic change during behavioral training in mammals (Vanderwolf, 1987b). Therefore, it is unclear whether or not acetylcholine and serotonin are directly involved in the formation of engrams. Blockade of cerebral cortical activation may be the principal (though undoubtedly not the only) mechanism by which centrally acting antimuscarinic and antiserotonergic drugs affect both learned and unlearned behavior. Impairment of behavior is especially severe when all activation is abolished by a combination of cholinergic and serotonergic blockade. It is probably impossible for the neocortex to carry out its normal function in the presence of continuous large-amplitude slow wave activity, a condition during which the activity of vast numbers of its output neurons is cut short for 100 msec, or more, two or more times
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per second. Similarly, in the hippocampus deprived of cholinergic and serotonergic inputs, the continual irregular occurrence of sharp waves, associated with undifferentiated bursts of large numbers of cells of different types, may be incompatible with normal function. As a result, the analysis of sensory input, the organization of motor output, the longterm storage of information, or whatever else the cerebral cortex does, cannot proceed normally in the absence of cholinergic and serotonergic inputs. Experiments carried out on normally sleeping humans indicate that cerebral activation may be a necessary condition for the occurrence of learning and memory, at least according to the behavioral definition mentioned above. Verbal statements presented during slow wave sleep (monitored with a polygraph) cannot be correctly repeated or selected from among a list of alternatives upon subsequent awakening unless the presentation of the statements during sleep has produced activation of the electrocorticogram. Further, the probability that a test statement will be correctly repeated or identified upon awakening from sleep is strongly and positively related to the duration of the activation period that occurred when the statement was presented during sleep (Emmons and Simon, 1956; Simon and Emmons, 1956a,b; Koukkou and Lehmann, 1968). If cholinergic and serotonergic control of cerebral activity is similar in humans and in rats, the foregoing data on cerebral activation and learning in humans are consistent with at least two hypotheses. (1) Cholinergic and serotonergic cerebral activating mechanisms play a direct role in the formation of engrams. (2) Cholinergic and/or serotonergic activation of the cerebrum provides conditions under which noncholinergic and nonserotonergicmechanisms can form engrams. That is, activation may be permissive rather than causative with respect to engram formation. As yet, there is no evidence that would compel rejection of either hypothesis. However, since cerebral activation appears to be required for effective performance during instinctive behavior as well as during learned behavior it is unlikely that the cholinergic and serotonergic activating systems are involved exclusively in learning and memory. Study of the role of cholinergic and serotonergic systems in the control of cerebral activity and behavior in animals may contribute to current studies of dementia in man. Since the pioneering investigations of Berger, it has been known that the electroencephalogram (EEG) of patients with Alzheimer’s disease shows a progressive slowing and a development of irregular rhythms (Mahendra, 1984). This suggests an increasing occurrence of the large-amplitude irregular waveform that occurs continuously
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in the neocortex in rats following central cholinergic and serotonergic blockade. A basis for the generalized EEG changes in dementia is suggested by recent evidence that Alzheimer’sdisease and related disorders are characterized by a reduction of central cholinergic and serotonergic function (Davies and Maloney, 1976; Spillane et al., 1977; Whitehouse et al., 1981, 1982; Benton et al., 1982; Bowen et al., 1983; Katzman, 1983; Hutton and Kenney, 1985; Scheibel et al., 1986; Palmer et al., 1987a,b). Central noradrenergic function is reduced as well, but the studies in animals suggest that this is relatively unimportant for either behavior or the electrocorticogram. Further, the work in animals shows that blockade of central cholinergic and serotonergic function alone, without the degeneration of cortical or other neurons that occurs in Alzheimer’s disease, is sufficient to produce a severe disorganization of behavior. Consequently, it is probable that a reduction in central cholinergic and serotonergic function is at least partly responsible for the disturbance of behavioral performance that occurs in Alzheimer’s disease. Other forms of dementia or psychosis may also be related to disturbances of cholinergic or serotonergic activation. Recent evidence (Vanderwolf, 1987a) indicates that the behavioral disturbances produced by phencyclidine (angel dust) and the psychotomimetic opioids may be partially attributable to a blockade of serotonin-dependent hippocampal and neocortical activation. This may be relevant to dementia or psychosis in man since the behavioral syndrome produced by phencyclidine is said to be indistinguishablefrom schizophrenia (Luisada, 1978; Snyder, 1980).
V. Sleep and Waking
A. Is THERE A “SLEEP CENTER” IN
THE
BASAL FOREBRAIN?
In the preceding sections it is argued that the cholinergic projections from the basal forebrain to the cerebral cortex are responsible for an atropine-sensitivecomponent of cortical activation. A different view of the function of this part of the brain was proposed many years ago by Nauta (1946), who showed that surgical transection of the rostra1 half of the hypothalamus in rats resulted in a total loss of behavioral sleep. Death occurred in 3 days, on the average. The syndrome was attributed to inactivation of a sleep center in the preoptic area. However, subsequent work revealed that lesions of the preoptic region result in pulmonary edema (often rapidly fatal after bilateral lesions), gastric ulceration, and behavioral hyperactivity (Gamble and Patton, 1953;
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Maire and Patton, 1956). Consequently, sleeplessness may be a secondary consequence of other disturbances after preoptic lesions rather than a primary effect. Nonetheless, a number of researchers have supported the concept of a basal forebrain mechanism that promotes neocortical slow wave activity and behavioral sleep. One line of evidence that may appear to support this concept is the finding that bilateral lesions of the preoptic area or hypothalamus produce a long-lasting increase in the occurrence of LVFA which develops slowly during a period of 2-3 weeks after surgery (McGinty and Sterman, 1968; McGinty, 1969). In contrast, it has been reported that acute lesions in the preoptic area tend to produce large slow waves or spindles in the neocortex (Bach y Rita et al., 1969). The latter finding cannot readily be interpreted as being due to a transient irritative effect of the lesions, since in the same study acute lesions of the subthalamus and the reticular formation also produced increases in slow waves and spindles. In a study of the effect of acute preoptic lesions by Bremer (1973), one of a group of five cats displayed “EEG and ocular signs of a rise in the brain waking level” but we are not told what happened in the other four cases. However, Bremer concluded that acute preoptic lesions did not increase waking. How can all this be interpreted? Large, unilateral, kainic acid-induced lesions of the basal forebrain produce chronic slow wave activity in the neocortex ipsilateral to the lesion (Stewart et al., 1984). In our experience, only large lesions achieve this effect in the rat, presumably owing to the widespread distribution of corticopetal cholinergic cells in the preoptic area, globus pallidus, substantia innominata, and diagonal band. Large bilateral lesions of the basal forebrain are invariably fatal, as has recently been confirmed by Lo Conte et al. (1982b). Consequently, the rather small basal forebrain lesions that are compatible with survival can inflict only partial damage on the cortically projecting cholinergic system. Ascending fibers from the brainstem may also be partially destroyed by such lesions. It is conceivable that a pathological process, such as the development of denervation supersensitivity (Stavraky, 1961), the growth of abnormal connections, which has been demonstrated in the medial septal nucleus following section of septal afferents by Raisman (1969) and Raisman and Field (1973), or a loss of inhibitory interneurons might lead to the gradual development of increased activity in the surviving cholinergic neurons several weeks after the original lesion. This might account for the slow increase in LVFA observed by McGinty and Sterman (1968) and McGinty (1969). A slow development of recovery processes in the basal forebrain might also be responsible for the reappearance of spontaneous LVFA in the
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chronic cerveau isole preparation, in which all ascending influences from the reticular formation are cut off by transection of the upper brainstem. A freshly prepared cerveau isolC displays continuous slow waves and spindle activity (Bremer, 1935), but, despite this, high-frequency electrical stimulation of the preoptic area or hypothalamus still produces LVFA in the neocortex (Belardetti et al., 1977). If the animals (cats or dogs) are kept alive for periods of 10 days or more, episodes of spontaneous LVFA, often lasting many minutes, begin to appear (Batsel, 1960, 1964; Villablanca, 1962, 1965). Thus, the facts can be accounted for reasonably well on the assumption that basal forebrain cholinergic neurons normally produce LVFA in the neocortex but become inactive when ascending excitatory inputs to them are destroyed. In time, they recover and may even become abnormally active after a period of several weeks, thus producing long periods of neocortisal LVFA. A paper by Lucas and Sterman (1975) which appears to contradict this generalization reported that lesions made in the preoptic region of waking cats produced an activated electrocorticogram for the next 2-12 hr. This contradicts the report by Bach y Rita et al. (1969) that preoptic lesions produce increased spindling and slow wave activity. An important difference between these studies is that Bach y Rita et al. made their lesions using a 50-KHz alternating current while Lucas and Sterman relied on anodal DC currents. It is well known that anodal DC lesions can have an irritative effect, mimicking the effect of electrical stimulation, due to the local deposit of metallic ions (Whishaw and Robinson, 1974). The thermal lesion produced by the high-frequency current used by Bach y Rita et al. is unlikely to have such an irritative effect. Thus, the Lucas and Sterman (1975) experiment can be regarded as a demonstration that chemical irritation of the preoptic region produces longlasting LVFA. Another factor that needs to be considered in this field is that a component of the decrease in sleep following preoptic lesions may be due to a disturbance of temperature regulation. Following preoptic damage, sleep duration becomes abnormally sensitive to environmental temperature (Szymusiak and Satinoff, 1984). Other reports that have encouraged belief in the concept of a basal forebrain sleep mechanism are those describing the elicitation of sleep following electrical or chemical stimulation of the basal forebrain. Sterman and Clemente (1962a,b) reported a rapid onset of largeamplitude slow waves in the neocortex accompanied by behavioral sleep following low-frequency electrical stimulation of the preoptic area or diagonal band. Hernandez- Peon et al. (1963) reported similar results following the injection of cholinergic agonists into the same general
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region, but this was not confirmed by MacPhail (1968) and MacPhail and Miller (1968). A major problem in all such studies is that cats left undisturbed in familiar surroundings tend to fall asleep spontaneously in a short time. Consequently, it is necessary to apply control procedures (sham stimulation trials) rigorously in such work and evaluate the results statistically. This was not done by Sterman and Clemente or by Hernandez-Peon et al. In a review of this field, Jouvet (1967, p. 135) concluded: “Our personal experience, during which we have stimulated hundreds of chronic cats over a period of several years, has not convinced us that a cat asleep after any stimulation (other than the painful ones) would not have gone to sleep spontaneously.” Support for this view is provided by LoPiccolo (1977), who carried out systematic experiments on the possibly hypnogenic effects of lateral preoptic or medial thalamic electrical stimulation (6 or 150 Hz and several different waveforms) in cats. Stimulation trials and control trials (equal in duration to stimulation trials but without stimulation) were run in counterbalanced blocks during an 8-hr daily test period. Stimulation of either the preoptic area or the medial thalamus led to spindles and recruiting waves during the period of stimulation but did not increase the occurrence of either behavioral sleep or long trains of slow waves continuing after stimulation. In conclusion, it appears that there is no firm basis for the belief that electrical stimulation of the preoptic area can cause either behavioral sleep or long-continuing trains of large slow waves in the electrocorticogram after stimulation is discontinued. In fact, high-frequency stimulation of this part of the brain results in a release of acetylcholine in the neocortex (Casamenti et a l . , 1986) and in the production of LVFA (Belardetti et al., 1977). On critical examination, interpretation of the effects of preoptic lesions also does not require the assumption that basal forebrain neurons promote either behavioral sleep or the occurrence of slow waves. Rather, the evidence is consistent with the view that the cholinergic projections from the basal forebrain to the neocortex are essential for the occurrence of an atropine-sensitive component of LVFA which is important in the control of adaptive behavior.
B. DOESSEROTONIN PROMOTE SLOWWAVESLEEP? A series of experiments by Jouvet and his collaborators, published about 20 years ago, aroused great interest in the theory that the release of serotonin in the brain initiates slow wave sleep (Jouvet, 1969, 1972, 1977, 1984). Several converging lines of evidence appeared to support this theory. (1) Systemic injection of 5-hydroxytryptophan (5-HTR the
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immediate precursor of serotonin) or serotonin itself or direct application of serotonin to various sites in the brain was said to produce behavioral sleep and slow waves in the neocortex. (2) Treatment with @-chlorophenylalaninewas said to produce insomnia but subsequent treatment with 5-HTP was said to restore slow wave sleep as a result of replenishment of central serotonin. The latter effect was described by Jouvet (1972, p. 204) as one of the crucial findings pointing to a role of serotonin in sleep. (3) Surgical destruction of the raphe nuclei of the brainstem was said to produce a total insomnia lasting 3-4 days, with some recovery later on. The conclusions drawn from these data of 10-20 years ago appear quite doubtful today. In the following section, both old and new data on the proposed role of serotonin in sleep are examined critically. First, although it is undoubtedly true that cats treated with large doses of @zruchlorophenylalaninedisplay very little sleep for several days, the effects observed on sleep in rats following treatment with parachlorophenylalanine (Rechschaffen et al., 1973) or intraventricular 5,7-dihydroxytryptamine(Ross et al., 1976) tend to be minimal or nonexistent. Further, it is not improbable that the insomniac effect in cats is secondary to a variety of other disturbances in behavior (see Section IV,F) as suggested by Ferguson et al. (1970) and Dement et al. (1972). The same could be said of the insomnia produced by raphe lesions, since surgical destruction of the median raphe, in particular, produces a variety of changes in behavior, including a sharp increase in locomotor activity, much as parachlorophenylalanine (PCPA) does (Kostowski et al., 1968; Lorens et al., 1971;Jacobs et al., 1974a,b). There is evidence that while part of the lesion effect may be due to a reduction in serotonergic activity (Sainati and Lorens, 1982), nonserotonergic elements are also damaged by the lesions and contribute to the hyperactivity (Asin and Fibiger, 1983). Further, the data on the effects of treatment of normal or of (PCPA)pretreated animals with serotonin or 5-HTP do not, in fact, offer much support for the view that central serotonergic systems promote behavioral sleep and large-amplitude slow wave activity in the neocortex. Previous reviews of this field have not dealt adequately with the complex nature of the evidence. In one of the early studies in this field, Monnier and Tissot (1958) reported that although small doses of 5-HTP (5-20 mg/kg, iv) tended to produce slow waves in the electrocorticogram (ECG) of the unanesthetized rabbit, a larger dose (30 mg/kg, iv) produced, after a delay of 10-20 min, a long-lasting period of LVFA. 5-Hydroxytqpophan retained its activating effect in a cerveau isole preparation, suggesting a direct action in the forebrain. Rather similar results were obtained
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by Schweigerdt and Himwich (1964), who obtained LVFA beginning about 50 min after intracarotid (44 mg) or intravenous (165-187 mg) injections of 5-HTP in curarized rabbits. This effect persisted if the brainstem was sectioned caudal to the midbrain but disappeared following a section rostra1 to the midbrain. Cuculic et al. (1968) also reported LVFA following intracarotid injections of 5-HTP but Glasser and Mantegazzini (1960) reported only slow waves following intracarotid (via the lingual artery) 5-HTP administration to cats prepared with a midpontine pretrigeminal section. This effect may have been due to their use of rather small doses (2-20 mg), since Costa et al. (1960) observed an early increase of neocortical slow wave activity if 22 mg of 5-HTP was injected into the carotid artery of curarized rabbits, but LVFA often occurred after a delay if 44 mg was injected. It should be noted that the largest of these doses is less than 20 mg/kg. Macchitelli et al. (1966), using rhesus monkeys, observed an initial period of champing, head shaking, retching, and occasional vomiting following intramuscular injection of 50-100 mg/kg of 5-HTP. After a delay of about 30 min the monkeys became quiet, and large slow waves appeared in the electrocorticogram. Intravenous or intra-arterial injections of serotonin itself in cats or rabbits have been reported to produce LVFA (Monnier and Tissot, 1958; Glasser and Mantegazzini, 1960) or LVFA (usually) followed by a period of slow wave activity (Koella and Czicman, 1966). This brief review shows that it is by no means the case that systemic injections of serotonin or 5-HTP generally result in behavioral or electrographic signs of sleep. Either slow waves or LVFA may occur, depending on the dose, route of administration, and perhaps on the species studied. However, the early work does not permit any firm conclusions concerning the effect of enhanced serotonergic transmission in the brain. Serotonin itself has a very limited ability to penetrate the brain (Oldendorf, 1971) and produces widespread peripheral effects such as complex changes in the tone of various vascular beds, contraction of the ureters and bronchioles, and stimulation of the gut (Page, 1958; Kosterlitz and Lees, 1964). These actions may secondarily affect the electrocorticogram. Bronchiolar constriction is of especial importance since it may lead to cerebral hypoxia. Spooner and Winters (1965, 1967) observed that subcutaneous injections of serotonin produced behavioral sedation as well as slow waves in the brain of the chick and argued that this was due to a central action of the amine, since the blood-brain barrier is not developed in chicks and recorded changes in blood pressure did not correlate with the changes in brain activity. These facts are not sufficient to exclude the possibility that the effects observed were due to some
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peripheral action of serotonin. Serotonin has also been described as having a sedative action following systemic injection in adult mice, in which the blood- brain barrier is, presumably, well developed (Kobinger, 1958). It seems likely that acute visceral distress can result in a state that has a superficial resemblance to natural sleep. Systemic injection of 5-HTP presents problems similar to those attending injection of serotonin since most of an injected dose is decarboxylated outside the blood-brain barrier, forming serotonin in peripheral tissues and brain capillary walls. Very little actually penetrates into the brain. This is revealed clearly in histofluorescent studies that show a strong accumulation of yellow fluorescence in the pericytes and endothelial cells of brain capillaries following systemic injections of 5-HTP (Corrodi et al., 1967; Fuxe et al., 1971; Butcher et al., 1972). Doses of more than 500 mg/kg are necessary to produce detectable increases in fluorescence in the raphe neurons. If peripheral decarboxylation is prevented by benserazide or other peripherally acting aromatic amino acid decarboxylase inhibitors (Bartholini and Pletscher, 1975), the amount of 5-HTP that is decarboxylated in the brain parenchyma is greatly increased (Fuxe et al., 1971; Butcher et al., 1972). This treatment makes it possible to distinguish between central and peripheral effects of serotonin, although the possibility that 5-HTP itself may have significant pharmacological effects must also be kept in mind. A further complication is introduced by fluorescence histochemical studies indicating that following treatment with benserazide plus large doses of 5-HTP (500-1000 mg/kg), the 5-HTP enters catecholamine-containing neurons and is decarboxylated there, resulting in displacement and release of the endogenous amine (Lichtensteiger et al., 1967; Fuxe et al., 1971; Butcher et al., 1972). These histochemical studies indicate that doses of 5 -HTP below 500 mg/kg do not result in accumulation of serotonin in catecholaminergic neurons. However, biochemical studies suggest that doses of 5-HTP as low as 30 mg/kg (ip) may result in significant displacement and metabolism of dopamine (Fuller and Perry, 1981). Behavioral experiments have shown that 5 -HTP, given alone, tends to depress spontaneous motor activity over a wide range of doses (Modigh, 1972). Systemic injections of serotonin also have this effect (Kobinger, 1958; Brown, 1960), as already noted. However, if 5-HTP treatment is preceded by an injection of a peripherally acting decarboxylase inhibitor (benserazide or carbidopa) motor inhibition is prevented (Modigh, 1972; Carter and Appel, 1976; Carter et al., 1978; Barret et al., 1982). In fact, as already noted in a preceding section (IV,C) a combination of a peripheral decarboxylase inhibitor plus an adequate dose of 5-HTP produces a peculiar syndrome of hyperactivity in rats involving alter-
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nating treading movements of the forelimbs, splaying of the hind limbs, side-to-side head movements, tremor, and muscular spasms. Similar behaviors are produced by a combination of tryptophan and a monoamine oxidase inhibitor such as tranylcypromine or pargyline or by putative serotonergic agonists such as 5-methoxy-N, N-dimethyltryptamine or P-phenylethylamine(Grahame-Smith, 1971;Jacobs, 1976; Sloviter et al., 1980). It is probable that these behaviors are due to activation of descending bulbospinal serotonergic fibers, since the syndrome can still be elicited by a combination of pargyline and L-tryptophan in rats in which the brainstem has been sectioned rostra1 to the pons (Jacobs and Klemfuss, 1975). Consistent with this, serotonin has been shown to have a facilitatory effect on spinal and cranial a-motor neurons (Anderson, 1972; Anderson and Proudfit, 1981; Aghajanian, 1981).There is evidence that some components of the behavioral serotonin syndrome are not expressed if dopaminergic transmission is blocked (Green and Grahame-Smith, 1974; Marsden and Curzon, 1976; Tricklebank et al., 1985), but it appears that the characteristic aspects of the syndrome are due to serotonin receptor activation (Sloviter et al., 1978). These facts show that, far from producing sleep, central serotonergic stimulation produces motor excitation. However, since this excitation is at least partly dependent on descending bulbospinal serotonergic fibers, its occurrence does not tell us anything about the effects of excitation of serotonergic projections to the forebrain. A good source of information on the behavioral correlates of serotonergic activity in the forebrain is provided by studies of the firing rate of serotonergic neurons in the dorsal and median raphe nuclei. Studies of this type have been unanimous in showing that presumed serotonergic neurons in these locations fire at higher rates during the waking state (especially during active waking or orienting behavior; see Section IV,C) than during slow wave sleep (Sheu et al., 1974; McGinty and Harper, 1976; Trulson and Jacobs, 1979). This provides strong evidence that serotonin release is not a cause of sleep. Some defenders of the view that serotonin release in the forebrain promotes sleep might wish to argue that serotonin release from axon terminals is independent of the occurrence of action potentials in the parent cell body. Some support for such a view is provided by evidence indicating that activation of catecholamine receptors on serotonergic terminals can produce or regulate the release of serotonin (Raiteri et al., 1983; Balfour and Iyaniwura, 1985). Fortunately, there is independent biochemical evidence of serotonin release during the sleep-waking cycle. HCry et al. (1972) conducted a series of experiments in rats on the rate of synthesis of serotonin and on brain levels of serotonin and
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5-hydroxyindoleacetic acid (5-HIAA) at various times during the day and night. They concluded that serotonin synthesis is relatively greater during the day but that serotonin release is greater during the night and suggested that the enhanced release may be correlated with the wellknown nocturnal motor activity of rats. Another mechanism by which serotonergic transmission may be enhanced in the rat brain at night is an increased sensitivity of neurons to applied serotonin (Mason, 1986). A direct demonstration that serotonin release from the neocortex is lower during sleep than during waking has been made by Puizillout et al. (1979), who made use of cortical superfusion and a radioenzymatic assay in work on encephale isole cats. Voltametric measures of serotonin release yield similar results (Cespuglio et al., 1984). Support for a correlation between motor activity and serotonin release is provided by studies showing increased levels of 5-HIAA in the brains of rats walking in a treadmill (El0 and Tirri, 1972) or in the lumbar cerebrospinal fluid of patients who exercise for several hours (Post et al., 1973). Szostak et al. (1986) obtained evidence of increased forebrain serotonin turnover (as indicated by serotonin to 5-HIAA ratios) in rats performing in a conditioned circling task. These studies are valuable but not fully conclusive. Under some circumstances the levels of brain 5-HIAA appear to be a reliable index of serotonin release (Shannon et al., 1986), but under other circumstances they may reflect the level of monoamine oxidase activity (Wolf et al., 1985). In the case of measurements in human lumbar cerebrospinal fluid, 5-HIAA is probably derived partly from the brain and partly from the spinal cord (Garelis et al., 1974). Direct measurement of serotonin release by microdialysis or superfusion of brain structures in behaving animals would provide an important extension of knowledge in this field. Localized electrical stimulation of the dorsal and median raphe nuclei has provided a means of experimentally activating the ascending serotonergic projections. In anesthetized rats and cats such stimulation, at 2-100 Hz,produces an elevation in forebrain 5-HIAA levels, frequently accompanied by a decline in serotonin levels. Stimulation of the lateral midbrain in intact preparations or stimulation of the raphe in preparations in which the medial forebrain bundle has been sectioned do not affect forebrain serotonin or 5-HIAA levels. These data indicate that stimulation localized in the raphe nuclei results in the release of serotonin from ascending serotonergic fibers (Aghajanian et al., 1967; Sheard and Aghajanian, 1968a; Sheard and Zolovich, 1971). Increases in 5-HIAA and decreases in forebrain serotonin also occur following raphe stimulation in unanesthetized rats (Kostowski et al., 1969). In contrast to the effects on serotonin metabolism, the effects on behavior appear to vary widely depending on stimulation frequency.
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Stimulation at 1-3 Hz is reported to produce sedation and sleep (Kostowski et al., 1969; Gomulka et al., 1971), whereas stimulation at 10 Hz or more is reported to produce “behavioral excitation”or the occurrence of Type 1 behavior, together with activation of the electrocorticogram (Kostowski et al., 1969; Gomulka et al., 1971;Jacobs et al., 1973; Robinson and Vanderwolf, 1978). Sheard and Aghajanian (196813) reported minimal behavioral effects in response to stimulation, but the current level used appears to have been quite low. Graeff and Silveira Filho (1978), using 60-Hz sine wave stimulation of the median raphe in rats, observed either continuous circling behavior or a syndrome of immobility, piloerection, and defecation. The latter effect was abolished in some animals following treatment with PCPA. Finally, Polc and Monnier (1970), stimulating the raphe magnus nucleus in rabbits, observed a behavioral orienting response accompanied by neocortical LVFA and hippocampal RSA at stimulation frequencies of 6-150 Hz. It is difficult to decide which of these various effects is the best indicator of the physiological role of the ascending raphe projections. Some of the behavioral or electrographic effects may be due to concurrent activation of nonserotonergic neurons. Since raphe neurons tend to fire spontaneously at rates of 2-4 Hz in freely moving animals (Trulson and Jacobs, 1979), it is rather surprising that raphe stimulation at such frequencies has any effect at all on serotonin metabolism. The main effect of stimulation should be to synchronize firing in the affected population of cells. In fact, Shannon et al. (1986) found that 5-Hz stimulation of the dorsal raphe had only slight effects on serotonin metabolism but that stimulation at 10 Hz produced substantial increases in brain 5-HIAA levels together with small decreases in serotonin levels. Kostowski et al. (1969) found that stimulation at 10 Hz,which produced the strongest effects on serotonin metabolism, also produced behavioral excitation and neocortical LVFA. Therefore, at the very least, the data are not incompatible with the view that excitation of ascending serotonergic fibers produces a form of neocortical LVFA. In fact, they offer considerable support for such a view. The finding that ingestion of L-tryptophan tends to promote sleep in some animals and some people (Smith and Prockop, 1962; Radulovacki, 1982) is a final piece of evidence that might be cited in support of the hypothesis that central serotonergic systems act to promote sleep. While this effect is interesting and may have practical value (Hartman, 1983), it is unlikely to be related to brain serotonin. The increased serotonin that is formed as a result of tryptophan loading is deaminated intraneuronally and does not lead to increased serotonin release (Grahame-Smith, 1971). Further, following a series of experiments with various enzyme-blocking drugs, Modigh (1973) concluded that the
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depression of motor activity by tryptophan in mice is due to an action of the amino acid itself and not to an action of any of its metabolites, such as serotonin. In conclusion, it appears that there is no compelling reason to believe that central serotonergic pathways play any special role in the occurrence of sleep. On the contrary, a critical review of the evidence that has accumulated in this field tends to support the conclusion, arrived at in Sections IV,A and D, that serotonin mediates an atropine-resistant form of neocortical and hippocampal activation that is closely related to some types of waking motor activity. A final point that should be mentioned here concerns the possible functions of serotonin during active sleep (paradoxical or rapid eye movement sleep). The theory that cholinergic and serotonergic inputs to the cerebrum are responsible for activation in the waking state naturally suggests the question of whether the same inputs are responsible for cerebral activation during active sleep. The existing evidence (Robinson et al., 1977; Nakahara et al., 1977; Usui and Iwahara, 1977; Vandenvolf and Robinson, 1981; Leung, 1984c; Stewart and Vanderwolf, 1987b)suggests that cerebral activation during active sleep is dependent on atropinesensitive and atropine-resistant inputs, just as in the waking state. The atropine-resistant inputs produce hippocampal RSA and neocortical LVFA in correlation with the occurrence of phasic bursts of muscular twitches, whereas the atropine-sensitive inputs produce RSA and LVFA in the intertwitch intervals. This is consistent with the hypothesis that atropine-resistant activation is related to motor activity. In agreement with the hypothesis that RSA is generated by the same systems during waking and sleep, a recent study by Welsh et al. (1985) suggests that RSA during both active sleep and active waking is controlled by a single circadian oscillator. There is no evidence, as yet, as to whether or not serotonin is responsible for the atropine-resistant activating input to the cerebral cortex during active sleep. The fact that the firing rate of putative dorsal raphe serotonergic neurons falls to a very low level during active sleep (McGinty and Harper, 1976; Trulson and Jacobs, 1979; 1981) does not support the involvement of serotonin. However, according to Trulson and Jacobs (1981, p. 552) “some units occasionally discharged in short bursts in conjunction with eye movement or head and limb twitches” during active sleep. Therefore, serotonin may play a role in the production of atropineresistant activation of the cerebrum during active sleep after all. Further work is necessary here.
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VI. Interpretation and Discussion
The data and ideas discussed in the preceding sections can be summarized very simply. The spontaneous slow wave activity of the neocortex appears to comprise three principal waveforms that are generated by different circuits. (1) Large amplitude irregular slow activity (LISA, 0.5to 6-Hz waves, including the classical 6 wave band) appears to be generated by circuitry endogenous to the neocortex. (2) Rhythmical spindle bursts occur in response to rhythmical inputs from thalamocortical neurons. (3) Low voltage fast activity (LVFA) occurs in response to a cholinergic input from the basal forebrain or a serotonergic input from the brainstem (Fig. 26). Hippocampus and cingulate cortex generate two principal waveforms: (1) an irregular pattern associated with sharp waves (or EEG “spikes”) which is probably analogous to the LISA pattern of the neocortex, and (2) a pattern of rhythmical slow activity (RSA) which, like the LVFA of the neocortex, is probably generated in response to a cholinergic input from the basal forebrain or a serotonergic input from the brainstem (Fig. 26). Each of these waveforms is associated with distinctive local patterns of neuronal discharge in the three types of cortex. Slow waveforms and the correlated unit activities in the neocortex, hippocampus, and cingulate cortex are closely correlated with concurrent spontaneous behavior. In normal animals, Type 1 (voluntary) movement is always accompanied by the occurrence of LVFA in the neocortex
FIG.26. Diagram illustrating part of the proposed pathways that determine the occurrence of low-voltagefast activity in the neocortex and rhythmical slow activity in the hippocampus. ( O ) ,cholinergic pathway: active during Type 1 behavior and active sleep, may be active during Type 2 behavior. (O), serotonergic pathway: active during Type 1 behavior and phasic events of active sleep.
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and RSA in the hippocampus and cingulate cortex. The occurrence of these waveforms appears to be a result of the joint action of cholinergic and serotonergic inputs to the cerebrum. Type 2 behavior (immobility and reflexive, automatic, or consummatory acts) is largely independent of any specific cerebral waveform. Any LVFA or RSA that occurs during Type 2 behavior in the absence of Type 1 behavior appears to be due mainly to cholinergic inputs to the cerebral cortex. Combined pharmacological blockade of central cholinergic and serotonergic systems results in severe disturbances of adaptive behavior but does not produce paralysis or coma. It is probable that disturbances of central cholinergic and serotonergic transmission play a significant causative role in dementia and psychosis in man. How can these data be integrated harmoniously with other information on the function of the brain? As one example of the difficulties to be encountered, some authors (Gray, 1982, p. 194) believe that hippocampal RSA cannot possibly play a role in the control of movement, since animals move about actively after the hippocampus is destroyed. Since totally decorticate or even high-decerebrate rats and cats are able to walk about (Bard and Rioch, 1937; Woods, 1964; Vanderwolf et al., 1978b), the same argument could be applied to the whole forebrain, leading to the conclusion that it does not play any role in the control of movement. Few would agree with this. A solution to the problem lies in the concept of a multilevel hierarchical control of movement, as envisaged by HughlingsJackson over a century ago (Taylor, 1958). According to this conception, the coordination of muscular activity at a local or segmental level is accomplished by relatively simple spinal and bulbar reflexes. A middle level of integration, partly represented in the brainstem and cerebellum, coordinates the activity of lower-levelreflexes to produce such complex motor patterns as locomotion. The highest levels of sensorimotor integration, located partly in the cerebral cortex, control those below, thereby producing movements that are adapted to external circumstances and internal requirements. A central concept in Hughlings Jackson’s ideas was that destruction of a part of the brain produces two kinds of symptoms. Negative symptoms comprise the loss of specific abilities resulting from loss of the damaged structures while positive symptoms comprise such phenomena as enhanced motor activity resulting from the unregulated activity of the lower levels of the nervous system. The existence of impaired performance in maze tests (Kaada et al., 1961) and in sexual, aggressive, and maternal behavior (Kimble et al., 1967; Sainsbury and Jason, 1976; Terlecki and Sainsbury, 1978) together with high levels of spontaneous locomotor activity (Teitelbaum and Milner, 1963) in rodents following
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hippocampal damage is, therefore, not incompatible with Hughlings Jackson’s concepts of what is to be expected following damage to mechanisms involved in high-level control of movement. Experience with the precentral gyrus in primates has resulted in general acceptance of the idea that a “motor cortex” should produce movements when it is stimulated. This idea is only partly correct since the principal effect of electrical stimulation of any part of the cortex is a paralysis of function. During stimulation of the motor cortex, a human patient is unable to move the affected part voluntarily. Such interference can occur in the absence of elicited movement (Penfield, 1958). Electrical stimulation of the hippocampus is complicated by the low seizure threshold of this type of cortex. However, Votaw (1959) reported movements of the face and upper limb following hippocampal stimulation in monkeys and Motles and Gonzalez (1984) reported contralateral head and eye movement following hippocampal stimulation in cats. However, stimulation was only occasionally effective in the latter study, and the movements were always accompanied by epileptiform afterdischarges in the hippocampus. Local chemical stimulation of the hippocampus has been more effective than electrical stimulation. Intrahippocampal injection of carbachol reliably induces movements of the head and eyes in cats. The movements can be blocked by atropine and are usually not accompanied by afterdischarges (Motles and Gonzalez, 1984). Injection of carbachol into the dentate gyrus of anesthetized rats produces RSA (Rowntree and Bland, 1986), as noted above (Section IV,B). Bilateral injection of carbachol in the dentate gyrus of freely moving rats produces a behavioral syndrome that includes high levels of locomotion and rearing (Flicker and Geyer, 1982). Thus, appropriate pharmacological stimulation of the hippocampus activates both a naturally occurring electrophysiological activity (RSA) and some of the Type 1 behavior that normally accompanies RSA when it occurs spontaneously. On the other hand, local interference with RSA may result in a selective suppression of Type 1 behavior. This was demonstrated by Bland and Vanderwolf (1972b) who found that stimulation of the dentate gyrus in approximately the middle of the septo-temporal extent of the hippocampal formation produced a profound behavioral arrest. Effective stimulation sites tended to be located in the hilus of the dentate gyrus, as shown by subsequent histology and by the fact that recordings from such sites revealed a pattern of fast waves, up to 50 Hz, with an amplitude of as much as 1 mV. This type of waveform is localized in the hilus of the rat dentate gyrus (Bland and Whishaw, 1976). Stimulation of the
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hilar region produced short-latency (2.5-7.0 msec) evoked potentials bilaterally in the CA1 area over a septo-temporal extent of at least 5 mm (Fig. 27). Stimulation producing these evoked potentials had no behavioral effect at frequencies below 5 Hz, but at rates of 5-15 Hz behavioral arrest became more prominent as the frequency increased (Fig. 28). Stimulation at higher frequencies (up to 100 Hz) also produced behavioral arrest. The behavioral arrest produced by dentate hilar stimulation was quite general, producing a total cessation of struggling against manual restraint; jumping out of a box (a previously established shock avoidance response); lever pressing (self-stimulation of the hypothalamus); swimming in a water-filled T-maze (even when the maze was filled with ice water); and climbing up over the edge of a table (after the rat had been suspended by the forepaws). When stimulation was applied, the rats ceased moving instantly, remaining as though frozen in whatever posture they occupied at the moment of stimulation onset. This effect continued throughout the duration of stimulation (usually no more than a few seconds). There was no postural collapse at any time. The eyes remained open and the heart rate and respiratory rate were not affected if stimulation was applied during a period of behavioral immobility. However, not all movements were affected. Application of stimulation during the drinking of water had little or no effect on the lapping movements of the tongue (Fig. 29). Similarly, unpublished experiments by B. H. Bland and I. Q. Whishaw showed that the pattern of EMG bursts associated with shivering in a chilled rat was not affected, although this fact was not mentioned in the original Bland and Vanderwolf (1972) paper. Some clarification of these findings has been provided by subsequent work. Autoradiographic investigations by Swanson et al. (1978) have shown that cells in the hilus of the dentate gyrus, especially those in the middle of the septo-temporal extent of the hippocampal formation, project to a very wide region of the ipsilateral and contralateral dentate gyrus, Ammon’s horn, and subiculum. Hilar cells also give rise to fibers that join the Schaffer collateral projections to CAI. There is good evidence that the commissural component of these projections, at least, has a powerful inhibitory effect on dentate granule cells, probably mediated by the release of GABA (BuzsPki, 1984). Thus, the evoked potentials observed by Bland and Vanderwolf (197213) probably have a complex origin, requiring much additional analysis, but may have involved inhibition of dentate granule cells and hippocampal pyramidal cells. When these evoked potentials occupy a large part of the hippocampal formation and occur with a frequency sufficient to ensure that each successive
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NO 78
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FIG.27. Evoked responses in the hippocampus during stimulation of the dentate hilar CA4 region. Arrows indicate the time at which the stimulus was administered. (A and B) Responses recorded in CA1 contralateral to the stimulating electrode. Stimulus intensity: 2.2 V and 3.2 V in A and B, respectively. (C) Upper beam illustrates an ipsilateral response, lower beam illustrates a contralateral response. Stimulus intensity: 5.0 V. Each record consists of about 25 superimposed sweeps. (From Bland and Vanderwolf, 1972b, by permission of Elsevier Science Publishers B.V.)
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10
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FIG. 28. Effects of the evoked responses produced by dentate hilar CA4 area stimulation of rat 78 on rhythmical slow activity (RSA) in the dorsal hippocampus. (A) Irregular activity present while the animal was motionless (no stimulus). (B) Rhythmic slow activity during walking (no stimulus). ( C ) Animal struggling while receiving dentate CA4 stimulation at 1 Hz. (D) Animal struggling while receiving stimulation at 5 Hz.(E) Effect of stimulation at a frequency of 10 Hz.The struggling was arrested at 10 Hz and also at 20 Hz as illustrated in F. Stimulus intensity was 2 V throughout. (From Bland and Vanderwolf, 1972b. by permission of Elsevier Science Publishers B.V.)
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L HlPP
1
I
S T l M (4V.100ppr)
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FIG.29. Response of the CA1 hippocampal fields to unilateral stimulation of the dentate hilar CA4 area as recorded with an ink-writingoscillograph. L HIPF', left hippocampus; R HIPP, right hippocampus. Note that 100-Hz stimulation has no detectable effect on licking movements of the tongue. Lower frequency stimulation (10-20 Hz) was also completely ineffective. (Taken from Bland and Vanderwolf, 1972b, by permission of Elsevier Science Publishers B.V.)
RSA wave cycle is interrupted by an evoked potential, RSA is suppressed. Correlated with this, all Type 1 behavior stops but at least two Type 2 behaviors are unaffected. In conclusion, the effects of eliciting RSA by local pharmacological stimulation of the hippocampus and the effects of disrupting RSA by electrical stimulation both point to the conclusion that the hippocampal formation exerts some form of higher level control over the occurrence of Type 1behavior. The effects of hippocampal damage in animals are not incompatible with this conclusion. The observation that stimulation of the hilus of the dentate gyrus suppresses walking, spontaneous head movements, lever pressing, etc., although it has no effect on licking and shivering will not suprise anyone familiar with Jackson's concepts of sensorimotor control. Jackson was well aware that a cerebral lesion that produces a paralysis of voluntary movement of the tongue in man may have no effect at all on the more automatic movement of licking water from the lips (Taylor, 1958, p. 153). Similarly, shivering is normal on both sides of the body in cases of hemiplegia (Uprus et al., 1935). A modern study that is consistent with Jackson's concepts is the demonstration by Hoffman and Luschei (1980) that many precentral cortical cells in a monkey fire at high rates during the performance of a task requiring fine control of the force
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exerted by the jaws but usually do not increase their firing rate during ordinary chewing. Thus, it may be that both the precentral gyrus and the hippocampus are relatively uninvolved in the performance of automatic or Type 2 behavior. Anatomical considerations provide some clues concerning the circuitry in which the hippocampus is involved. Widespread areas of the neocortex are in potential communication with the hippocampus via a multisynaptic pathway through the entorhinal cortex (Leichnetz and Astruc, 1975; Van Hoesen and Pandya, 1975a,b; Van Hoesen et al., 1975; Deacon et al., 1983). These findings, raising the possibility that the hippocampus represents one of several efferent pathways from the neocortex (others would include the striatum, thalamus, brainstem, spinal cord, and the amygdala) have led to suggestions that the hippocampus is to be regarded as a “supramodal association cortex” (Swanson, 1983) or “nonspecific cortex” (Vanderwolf et al., 1978b). In a historically important review of the hippocampus, Brodal(l947, p. 204) stated: “from a morphological point of view this part of the brain must be considered a clear-cut example of a purely effectory structure” (italics in original). The use of different terminologies gives an illusory impression of widely divergent points of view. In fact, a “supramodal association cortex” is not very different from a “motor” cortex. Motor control structures are necessarily nonspecific in their response to sensory input, since a great variety of sensory inputs are capable of eliciting a given type of motor output. If the hippocampus is to be regarded as a structure with an important role in the control of movement, one would like to h o w the pathways by which such control is effected. This topic is poorly understood. A recent review by Mogenson (1987) emphasizes the importance of projections to the nucleus accumbens, but outputs to the hypothalamus (with or without a relay in the septa1 nuclei) and the neocortex (Swanson and Kohler, 1986) should also be considered. A part of the data discussed in this review indicates that activity in auditory, somesthetic, and visual cortex is regulated in close correlation with motor activity. How is this to be interpreted? Anatomical findings (Diamond, 1979) show that layer V pyramidal cells in “sensory” areas of the neocortex project to centers in the tectum, pons, and medulla which have direct or indirect access to spinal and cranial motor neurons. These connections may provide the anatomical basis for the finding that in ununesthetized animals, stimulation of virtually any point in the neocortex will produce a movement of some sort (Lilly, 1958). In anesthetized animals the effect of cortical stimulation is very much reduced but movements can still be elicited from the classical frontal
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motor area, presumably owing to oligosynaptic connections to bulbospinal motor neurones. The idea that “sensory” cortex also has “motor” functions should not be surprising. If the purpose of vision, for example, is the guidance of motor activity, then the efferents from visual cortex might be expected to be capable of exerting a powerful control over motor neurons. It is not possible to draw a sharp distinction between “sensory” and “motor” processes anywhere in the brain (Sperry, 1952). In very general terms, the data and concepts discussed in this review offer a new approach to the generalized regulation of cerebral activity that is represented by the electrocorticogram and the neuronal activity that is correlated with it. Our understanding of these gross aspects of brain activity has been in a state of confusion for many decades. It is widely believed that the varying patterns of the electrocorticogram have some sort of special relation to sleep, despite repeated warnings to the contrary. According to N. Kleitman, a pioneer in sleep research, “It is clear that the EEG by itself not only fails to gauge the depth of sleep but the very presence of behavioral sleep” (Kleitman, 1963, p. 30). Another eminent sleep researcher agrees: “Yet in no case does the state of the corticogram allow us to presume whether an animal is asleep or awake” (Jouvet, 1967, p. 119). Persistent failure to establish a consistent and comprehensive relation between the electrocorticogram and behavior in the past has led to a widespread assumption that brain waves are epiphenomena with only a marginal relation to brain function. However, examination of the evidence shows that the electrocorticogram, gross measure though it may be, is closely related to unitary and neurochemical activity. The difficulties experienced in past attempts to relate the electrocorticogram to behavior appear to have been due to an inadequate approach to the study of behavior rather than to the limitations of the electrocorticogram as a measure of brain activity. I believe that a major impediment to progress in the brain-behavior field is an uncritical implicit or explicit adherence to traditional folk psychological concepts that provide misleading guides to the investigation of cerebral function (Vanderwolf, 1983). The advances in the understanding of how brain activity is related to behavior that are discussed in this review were made possible by an experimental approach, originating in the field of animal behavior, that emphasizes descriptions of what animals actually do. Folk psychological traditions tend to discourage this. If one is intent on solving the grand problems of attention or memory, there seems to be little point in detailed investigations of head movements, walking, and face-washingin rats. It is to be hoped
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that the data and concepts discussed here will encourage a more descriptive and naturalistic approach to animal behavior in future studies of brain function. Acknow ledgments
Research reported in this paper was supported by a grant from the Natural Sciences and Engineering Research Council. I thank R. Cooley for technical assistance and L. Mitchell for typing the manuscript. L.-W. S. Leung made many helpful suggestions, both while the article was being written and after the first draft had been completed.
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INDEX
A
N-Acetylated, a-linked, acidic dipeptidepreferring peptidase N-acetylaspartylglutamate degradation, 75-81 amino acid and peptide inhibitors, 77-79 regional distribution, table, 80 structure -activity relationships, 78 Acetylcholine application to CAl-cell apical dendrites, 265 excitatory effects on neocortical neurons, 285-286 inhibitory effects on neocortical neurons, 285-286 low-voltage fast activity, production in neocortex, 268 neocortical neuronal responses, 267 release from neocortex, muscimol effects, 275 rhythmical slow activity, hippocampal, 260 cellular mechanism, 263-267 production by microinjection, 263 tritiated, probe for nicotinic acetylcholine receptors, 7 Acetylcholinesterase in basal forebrain, 267 in cerebrospinal fluid, 115-116 in C-6 glial cells, 188 in dorsolateral tegmental and pedunculopontine nuclei, 267 in lateral tegmental and pedunculopontine nuclei, 259 in medial septa1 nucleus and vertical limb of diagonal band, 259 Adherence, neurons on poly-L-lysine, collagen, and laminin, 154-155 ADP-ribosyltransferase toxins, 128-130 Aging, glial cell changes during, 198-207 Alzheimer’s disease, electroencephalogram changes, 303-304 a-Amidating monooxygenase in cerebrospinal fluid, 110-111 properties in human cerebrospinal fluid, table, 110
Abrin, RNA N-glycosidase toxin, 130-131 Acetazolamide, anticonvulsant actions, astrocyte role, 182 N-Acetylaspartate developmental patterns, 50-51 phylogenetic and regional distribution in neural tissue, 46-49 N-Acetylaspartylglutamate colocalization in different neuronal systems, table, 64 degradation by N-acetylated, a-linked, acidic dipeptide-preferring peptidase, 75-81 developmental patterns, 50-51 electrophysiological effects, 81-82 in Vitro, 84-87 in Vivo, 82-83 endogenous, subcellular compartmentation, 75 enzymatic biosynthesis, 72-75 function, in vitro biochemical approaches, 87-88 as neuropeptide, 88 neurotransmission at glutamatergic pathways/synapses, 45-46, 89 pathways, lesion studies, 69-71 phylogenetic and regional distribution in neural tissue, 46-49 as precursor for transmitter glutamate, 90 receptors, identification, 91-92 ribosomal biosynthesis, 72-75 N-Acetylaspartylglutamate-like immunoreactivity in brainstem and cerebellum (rodent), 58-65 colocalization in different neuronal systems, table, 64 in forebrain nuclei and retina, 65-69 in spinal cord (rodent), 54-58 visualization, 51-52 visualization techniques, 52 -54 341
342
INDEX
Amino acids, acidic, neurotransmission. 41-45 4-aminobutyrate aminotransferase, synthesis by hippocampal basket cells, 264 y-Aminobutyric acid, uptake and release by astroctyes, 188-191 Aminopeptidases, in cerebrospinal fluid, 114-115 2-Amino-4-phosphonobutyryicacid, aspartate and glutamate antagonist, 44 2-Amino-7-phosphonoheptanoate, antagonist of excitatory amino acid neurot ransmission, 45 2-Amino-5-phosphonovaleric acid, aspartate and glutamate antagonist, 44 Anesthetics. effect on atropine-resistant hippocampal rhythmical slow activity, 284 atropine-resistant neocortical low-voltage fast activity, 272, 284 central serotonergic functions, 284 central serotonergic mechanisms, 266 Angiotensin-converting enzyme brain distribution (mammalian), 107 function, 107 in cerebrospinal fluid, 105-108 levels, in Parkinson’s disease, 107 marker role of, 107 substrate specificity, 107-108 Antibodies chimeric, 142-143 mAb35, to agonist receptor in chick brain, 23 nicotinic acetylcholine receptors agonist receptor, 19-20 probes for, 14-15 toxin receptor, 17-19 Antibody-toxin chimeras, 135 Antimuscarinic drugs abolishment of rhythmical slow activity, 260 centrally acting behavioral effects, 301 blocking effect during immobility, 272 effect on activity, 269-270 low-voltage fast activity, 270 rhythmical slow activity accompanying Type 1 behavior, 260 effects, blockade of cerebral cortical activation as mechanism of, 302-303
peripherally acting, effect on low-voltage fast activity, 270 Antineoplastic drugs, chimeric toxins, 137-138 Antiserotonergic drug effects, cerebral cortical activation blockade as mechanism of, 302-303 Aspartate neurotransmitter role, 41-45 uptake by astroctyes and cerebellar granule cells, 192 P-D-Aspartyl-P-alanine, antagonist of amino acid-induced excitation, 45 - D -Asparty1- aminome thylphosphonate, antagonist of amino acid-induced excitation, 45 Astrocytes activated, 197 carbonic anhydrase in primary cultures of, 183 chlorine ion transport, 183 glutamine synthetase biochemical marker, 168-169 growth and differentiation, influence of neurons, 167-170 identification with glial fibrillary acidic protein, 171 membrane permeability to Na+, 181-182 neural cell adhesion molecule (N-CAM), 158 neurotransmitter receptors, 193 role in anticonvulsant actions of acetazolamide, 182 development, regenerative failure, and induced regeneration upon transplantation, 195-196 myelin basic protein expression in cultured oligodendrocytes, 174-175 neurotransmission processes. 183-194 synaptic density regulation, 180 uptake of dopamine, 186 norepinephrine, 184-185 serotonin, 186-187 Astroglia differentiation-promotingeffect of neurons, 170 functions affected by receptor stimulation, 193-194
INDEX
effect of neurotransmitters, 173 neurotransmitter receptors, 192-194 Astroglia growth factor, 167 Atropine abolishment of hippocampal rhythmical slow activity, 260 block of brainstem neurons excited by cholinergic agonists, 259 dose required to affect cortical activity, 271-272 effects on spontaneous neocortical activity in reserpinized rats, 280 inactivation by atropine esterase, 271 Atropine esterase, inactivation of atropine and scopolamine, 271 Atropine methyl nitrate, effect on low-voltage fast activity, 270 Atropine-resistant rhythmical slow activity, see Rhythmical slow activity, atropine-resistant Atropine-sensitive rhythmical slow activity, see Rhythmical slow activity, atropine-sensitive Atropine sulfate, effects on neocortical electrical activity and behavior, 277 slow wave and multiunit activity in somatosensory neocortex, 297 Atrophic drugs, effect on electrocorticogram in behaving animals, 270 Augmenting responses, electrocorticogram, 242-243 A-wave generators, 243-244 Axonal membranes, development and organization, glial contact and, 176-177 Axonal regeneration, role of glial cells, 194-197
B Basket cells, hippocampal, synthesis of 4-aminobutyrate aminotransferase, 264 Behavior disturbances induced by phencyclidine and psychotomimetic opioids, 304 effects of atropine sulfate, 277 basal forebrain destruction, 301 blockade of cholinergic and serotonergic systems, 296-304
343 centrally acting antimuscarinic drugs, 301 L-dopa, 277 interference with central serotonergic and cholerinergic function, 296-304 peripheral decarboxylase inhibitors and 5-hydroxytryptophan, 310-311 reserpine, 277 reserpine with atropine or scopolamine, 298 scopolamine with p-chlorophenylalanine, 298-300 hippocampal activity and complex spike cells, 235-237 place cells, 235-237 slow waves, 228-235 theta cells, 235-237 unit activity, 235-237 immobility, rhythmical slow activity during, 233 large-amplitude irregular activity, 228 mental processes and, 238-241 metabolic requirements of motor activities, 230 rhythmical slow activity, 228-233 theta rhythm and, 228-233
Type 1 antimuscarinic drug effects on rhythmical slow activity, 260 firing characteristics of complex spike cells, 235 local interference effects on rhythmical slow activity, 317-318 low-voltage fast activity preservation during, 270 rhythmical slow activity during, 260, 262-263, 317-318 theta cell firing characteristics, 235 Type 2 blockade of low-voltage fast activity, 270 firing characteristics of complex spike cells, 235 rhythmical slow activity during, 262-263 theta cell firing characteristics, 235 waking, variation in transcallosal evoked response, 254 (3 Rhythm, blockade prior to and during voluntary movement, 250 Biosynthesis, N-acetylaspartylglutamate enzymatic, 72-75 ribosomal, 72-75
344
INDEX
Botulinum binary toxin, 129 Botulinum C, toxin, 129 Botulinum neurotoxin as ADP-ribosylating toxin, 129 blocker of transmitter release, 141-142 chimeric drug, 142 chimeric molecule with tetanus toxin, 142 mechanism of action, 131-134 pheochromocytoma treatment, 140-141 Brain, see also specific brain region glial cells from different regions, properties defining neuronal polarity, 157-158 nicotine-binding sites, 5-6 partial destruction, symptoms produced by, 316-317 penetration by 5-hydroxytryptophan, 310 by serotonin, 309-310 Brainstem N-acetylaspartylglutamate-like immunoreactivity (rodent), 58-65 neurons excited by cholinergic agonists, 259 Bulbectomy, olfactory, effect on N-acetylaspartylglutamate levels, 70-71 a-Bungarotoxin, probe for nicotinic acetylcholine receptors in neuromuscular junction, 10-12 a-Bungarotoxin 3.3, 13-14 x-Bunprotoxin, 3, 13 v-Bungarotoxin, 3, 13 Bungarotoxin (Bgt) 2.2, 13 Bungarotoxin (Bgt) 3.1, 13 a-Bungarotoxin receptors cerebellar (rat), 29-30 in higher vertebrates, 24-25 hypothalamic, 29 in lower vertebrates and invertebrates, 24-25 member of cholinergic receptor family, 26 nicotine target, 26-27 physiological significance, 28 purification and characterization, 10-11 synaptic and extrasynaptic protein, 27-28 Butyrylcholinerase, in C-6 glial cells, 188 B-wave generators, 244245
C Calcitonin gene-related peptide cleavage by substance P-converting enzyme, 109
immunoreactivity in vertebrate motoneurons, 56, 58 Carbachol, rhythmical slow activity production by microinjection into hippocampus, 263 Carbonic anhydrase, in cultured astrocytes, 183 Catecholamines, uptake by neuronal and glial cells, 184 Central nervous system, axonal regeneration, 194-195 Cerebellar cortex, neuronal migration, 159-160 Cerebellum N-acetylaspartylglutamate-like immunoreactivity (rodent), 58-65 a-bungarotoxin receptors, 29-30 distribution of N-acetylaspartylglutamatelike immunoreactive neurons (rat), table, 55 Cerebral activity, cholinergic and serotonergic control, 303 Cerebral cortex, endogenous large-amplitude irregular slow activity, 289 Cerebral evoked potentials, nature of, 250-251 Cerebrospinal fluid neuropeptide-transforming enzymes acetylcholinesterase, 115-116 a-amidating monooxygenase, 110-111 aminopeptidases, 114-115 angiotensin-converting enzyme, 105-108 characterization, 104-105 dynorphin-converting enzyme, 111-114 isolation, 105 membrane metallo-endopeptidase, 114 substance P-converting endopeptidase, 108-110 proteases-peptidases, 116-118 Cerveau isolC preparations, spontaneous lowvoltage fast activity, 305-306 Channel formation, diphtheria toxin, 127 Chimeric antibodies, 142-143 Chimeric drug, pheochromocytoma treatment, 140-141 Chimeric drugs blockers of transmitter release, 141-142 botulinum neurotoxin, 142 brain tumor treatment, 139-140 import, 143-144 neural tumor treatment, 139-140 ricin, 140 tetanus toxin, 142
INDEX
Chimeric toxins, 134 antibody-toxin chimeras, 135 antineoplastic drugs, 137-138 drugs, see Chimeric drugs Chlorine, transport by astrocytes, 183 P-Chlorophenylalanine with atropine or scopolamine, effects on hippocampal activity, 261 effect on atropine-resistant low-voltage fast activity, 281 atropine-resistant rhythmical slow activity, 261 electrocorticogram without antimuscarinic drugs, 282-283 sleep behavior, 308 with scopolamine, behavioral effects, 298-300 Cholera toxin, as ADP-ribosylating toxin, 128-129 Choline high-affinity uptake in hippocampus, 259 high-affinity uptake in neocortex, 267 metabolism in brain under depolarizing conditions, 190 under nerve-resting conditions, 189 uptake by neuronal and glial cells, 187-188 Choline acetyltransferase, colocalization with N-acetylaspartylglutamate-like immunoreactivity in rat ventral horn neurons, 56 Choline 0-acetyltransferase in basal forebrain, 267 in donolateral tegmental and pedunculoponine nuclei, 267 in medial septa1 nucleus and vertical limb of diagonal band, 259 neurons in lateral tegmetal and pedunculopontine nuclei, 259 Cholinergic agonists brainstem neurons excited by, 259 production of hippocampal rhythmical slow activity, 260 neocortical low-voltage fast activity, 268 rhythmical slow activity by microinjection into hippocampus, 263 Cholinergic cerebral activating mechanisms, role in engram formation, 303 Cholinergic control hippocampus, 259-261 neocortex, 267-275
345
Cholinergic function. behavioral effects of interference, 301 tests involving training, 301 untrained or instinctive behavion, 301-302 Cholinergic ligands, probes for nicotinic acetylcholine receptors, 5-9 Cholinergic pathways, from brainstem to hippocampal formation, 259 Cholinergic systems control of cerebral activity, 303-304 and serotonergic systems, behaviorial effects of blockade, 296-304 Choroid plexi, dynorphin-converting enzymes, 113 Cingulate cortex, electrical activity, 286-287 Clostridial chimeras, 142 Clostridium botulinum binary toxin, 129 Clostridium botulinum C, toxin, 129 Clostridium botulinum neurotoxin as ADP-ribosylating toxin, 129 blocker of transmitter release, 141-142 chimeric drug, 142 chimeric molecule with tetanus toxin, 142 mechanism of action, 131-134 pheochromocytoma treatment, 140-141 Clostridium perfringens iota toxin, 129 Clostridium spiroforme iota-like toxin, 129-130 Cognitive map, pyramidal cell elements, 237 Collagen, neuron adherence, 154-155 Complementary DNA, probe for nicotinic acetylcholine receptor genes and transcripts, 15-16 Complex spike cells firing characteristics during Type 1 and Type 2 behavior, 235 firing rate and pattern, 235 firing rate during rhythmical slow activity, 265 place fields, 237 Cortical activation blockade deficits, loss of learning and memory and, 300 principal mechanism of antimuscarinic and antiserotonergic drug effects, 302-303 hippocampal cholinergic control, 259-261 serotonergic control, 261-262 for occurrence of learning and memory, 303
346
INDEX
reticular activating system actions, 227 total loss of, 298-299 Cortisol glial cell response, 173 inhibition of astrocyte uptake of norepinephrine, 184 Curanmimetic protein toxins, 10-14 C-wave generators, 245-246 Cyclazoncine, effect on atropine-resistant rhythmical slow activity, 261 2 ' 3'Cyclic-nucleotide 3 '-phosphodiesterase, 174 biochemical marker for oligodendrocytes, 168-169, 199-200 Cyproheptadine, lack of effect on blockade of atropine-resistant waveforms, 284 Cytisine, probe for nicotinic acetylcholine receptors, 7
D Decarboxylase inhibitors, and 5-hydroxytryptophan, behavioral effects, 310-311 Dentate g y r u s granule cell firing during rhythmical slow activity, 265 hilar region projections, 318 stimulation, behavioral arrest produced by, 318 hilar region, CA4 stimulation effects of evoked responses on rhythmical slow activity, 320 hippocampal evoked responses produced during, 319 response to CA1 hippocampal fields, 321 Depolarization effects, on electro. corticograms, 243 Differentiation astrocytes, influence of neurons, 167-170 Schwann cells, influence of neurons, 166-167 Dihydro-0-erythroidine probe for nicotinic acetylcholine receptors, 8-9 target, 16 3,4-Dihydroxyphenylacetic acid, levels in Parkinson's disease, 107 5,7-Dihydroxytryptamine, effect on
atropine-resistant low-voltage fast activity, 281-283 electrocorticogram without antimuscarinic drugs, 282-283 sleep behavior, 308 Diisopropylfluorophosphate, production of hippocampal rhythmical slow activity, 260 Diphtheria toxin, 125-126 as ADFribosylating toxin, 128 antagonists, 127 elongation factor 2, 125-126 functional domains, 125 insertion into endosomal membrane, pH dependence, 127 Diphthimide, occurrence and function, 126 Ditran, effect on low-voltage fast activity, 270 L-Dopa effects on neocortical electrical activity and behavior, 277 restoration of catecholaminergic function, 298 Dopamine role in atropine-resistant rhythmical slow activity, 262 uptake by astrocytes, 186 Dopaminergic agonists, restoration of catecholaminergic function, 298 Dorsal column nuclei, unit response variation with motor activity, 253 Dynorphin A, preproenkephalin B. schematic, 103 Dynorphin B conversion to Leu-enkephalin-Arg6, effects of protease inhibitors and synthetic peptides, table, 113 preproenkephalin B, schematic, 103 Dynorphin-converting enzymes in cerebrospinal fluid. 111-114 properties in human cerebrospinal fluid and choroid plexus, table. 112
Electrocorticograms atrophic drug effects in behaving animals, 270 augmenting and recruiting responses, 242-243
347
INDEX
A-wave generators, 243-244 bipolar transcortical records, 247-248 B-wave generators, 244-245 correlation of slow wave spindle activity with rhythmical discharges, 249 C-wave generators, 245-246 depolarization effects, 243 gross potentials at surface and in deep layers, 246-247 hyperpolarization of apical dendrites, 246 large-amplitude irregular slow waves, 249 main source, 241 origin of low-voltage fast activity, 249 role of thalamorcortical pathways, 242 spindle bursts dependent on, 290 Type 1 and Type 2 waves, 241-242 Type 1 spindles dependent on repetitive thalamocortical discharges, 242 Electroencephalograms, changes in Alzheimer’s disease, 303-304 Elongation factor 2, 125-126 Energy coupling, between neurons and glia, 172 Engrams, role of cholinergic and serotonergic cerebral activating mechanisms, 303 Enkephalin, preproenkephalin A, schematic, 103 Enkephalinase, see Membrane metalloendopeptidase Entorhinal cortex, removal effects on rhythmical slow activity, 262 Enucleation, eye, effect on N-acetylaspartylglutamate levels, 71 Enzymatic biosynthesis, N-acetylaspartylglutamate, 72-75 Enzymes, neuropeptide-transforming in cerebrospinal fluid, 104-105 isolation from cerebrospinal fluid, 105 Escherichia coli enterotoxin, as ADP-ribosylating toxin, 128-129 Eserine hippocampal rhythmical slow activity production, 260, 263 neocortical low-voltage fast activity production, 268 Ethograms, 238 l-Ethyl-3-(3-dimethyl-amino-propyl)carbodiimide, 53 Evoked potentials, cerebral, nature of, 250-251
Excitatory postsynaptic potentials, reduction by acetylcholine application to apical dendrites of CA1 cells, 265
F x-Flavitoxin, 13 Forebrain N-acetylaspartylglutamatecolocalization, table, 64 N-acetylaspartylglutamate-like immunoreactive neurons, distribution (rat), table, 55 basal acetylcholinesterase, 267 cells active during large slow wave episodes during quiet sleep, 273-274 cells active during neocortical low-voltage fast activity, 273-274 choline 0-acetyltransferase, 267 cholinergic cells, stimulation by ascending brainstem cholinergic fibers, 268 destruction, behavioral effects, 301 electrical stimulation, effect on neocortical acetylcholine, 267 sleep center, 304-307 nuclei, N-acetylaspartylglutamate-like immunoreactivity, 65-69
G Galactocerebroside, expression by cultured oligodendrocytes, 171 Ganglionic blockers probes for nicotinic acetylcholine receptors, 7-8 target, 16 Genes, nicotinic acetylcholine receptor agonist receptor, 22-23 cDNAs as probes for, 15-16 Glial cells age effects, 198-207 on glial fibrillary acidic protein levels, 202-204 on glycerol phosphate dehydrogenaserhodamine levels, 202-204 C-6 cell line acetylcholinesterase and butyrylcholinerase in, 188
348
INDEX
choline uptake, 187 competition with afferent fibers for apposition to neuronal surfaces, 179-180 from different brain regions, properties defining neuronal polarity, 157-158 effect on neuritic growth, 153-159 neuronal migration and guidance, 159-165 neuronal phenotypic expression, 151-152 energy coupling with neurons, 172 factors secreted by, 158-159 ionic regulation, 181-182 membrane specialization at neuron-glia contacts, 178-179 metabolic responses, neuronal signals and, 172-174 neurite-glial cell surface interaction, 156 neurotransmitter receptors, 192-194 regulatory role extracellular pH, 181-182 extracellular potassium concentration, 181-182 extracellular space volume, 181-182 response to neurohonnones, 173 neurotransmitters, 173 RN-22 cell line, choline uptake, 187 role in axonal regeneration, 194-197 neural repair, invertebrate animal models, 196-197 synaptic remodeling and synaptic turnover, 207-208 surfaces, effect on aggregation and neurite fasciculation, 154-155 synaptic contacts with neuronal processes, 177-178 uptake of y-aminobutyric acid, 188-192 aspartate, 188-192 catecholamines, 184 choline, 187-188 glutamate, 188-192 monoamines, 183-187 Glial fibrillary acidic protein age effects, 202-204 astrocyte identification with, 171 Glial growth factor, 166 Glia maturation factor, 167
Gliosis, 194 activated, 197 Glutamate neurotransmitter role, 41-45 release-associated neocortical activation, 285 serotonin interaction, 266 uptake by astroctyes and neurons, 191-192 Glutamatergic synapses, N-acetylaspartylglutamate neurotransmission, 45-46 Glutamine synthetase, biochemical marker for astrocytes, 168-169, 201
0-o-Glutamyl-aminomethylphosphonate, antagonist of amino acid-induced excitation, 45 6-D-Glutamyl-glycine,antagonist of amino acid-induced excitation, 45 Glutathione, distribution and function, 45-46 Grafting, of neural tissue, 197 Granule cells, firing in dentate gyrus during rhythmical slow activity, 265 Growth astrocytes, influence of neurons, 167-170 neurites, glial cell influence, 153-159 Schwann cells, influence of neurons, 166-167 Guidance, neuronal. influence of glial cells, 159-165
H Hemicholinium-3, effects on rhythmical slow activity, 260 Hexamethonium, probe for nicotinic acetylcholine receptors, 7-8 High-performance liquid chromatography, N-acetylaspartylglutamate from forebrain extract, 42 Hippocampus N-acetylaspartylglutamate levels, excitoxin lesion effects, 69-70 atropine-resistant rhythmical slow activity, see Rhythmical slow activity, atropine-resistant basket cell synthesis of 4-aminobutyrate aminotransferase, 264 cellular mechanism of acetylcholineinduced rhythmical activity, 263-267 cognitive map of pyramidal cells, 237
349
INDEX
complex spike cells, 235-237 cortical activation cholinergic control, 259-261 serotonergic control, 261-262 dorsal rhythmical slow activity, effects of evoked responses produced by dentate hilar CA4 area stimulation, 320 serotonergic innervation, 261 as effectory structure, 322 electrical stimulation, 317 high-affinity uptake of choline, 259 inhibitory interneurons, 264-265 irregular pattern associated with sharp waves, 315 local chemical stimulation, 317 place cells, 237-238 pyramidal cells phase-reversed intracellular rhythmical slow activity rhythm, 264 rhythmical inhibitory postsynaptic potentials, 264 serotonin inhibitory effect, 266 rhythmical slow activity, 315 abolishment by antimuscarinic drugs, 260 anticipatory activity, 230-231 atropine-resistant, see Rhythmical slow activity, atropine-resistant atropine-sensitive, 260 during behavioral immobility, 233 complex spike cell firing rate during, 265 correlation with motor activity, 233-234 dorsal hippocampus, effects of evoked responses produced by dentate hilar CA4 area stimulation, 320 effects of anesthetics, 284 central cholinergic and serotonergic blockade, 297-298 generation of voluntary movements and, 231-233 granule cell firing in dentate gyrus during, 265 local interference, behavioral effects, 317-318 phase-reversed rhythm in hypocampal pyramidal cells, 264 postural change or locomotion accompanied by, 229-230 production by reticular formation stimulation, 260
relation to behavior, 228-233 relation to mechanisms controlling voluntary motor activity, 231 role in movement control, 316 slow wave sleep and, 237 still alertness and, 237 during Type 1 and Type 2 behaviors, 262-263 waves without rhythmical cholinergic input, 263 septa1 neurons as pacemakers, 263 slow waves, 228-235 small amplitude irregular activity, 237 as supramodal association cortex, 322 theta cells, 235-237 unit activity, 235-237 Histrionicotoxin, probe for nicotinic acetylcholine receptors, 8 Homovanillic acid, levels in Parkinson’s disease, 107 6-Hydroxydopamine, effect on atropineresistant low-voltage fast activity, 281 5-Hydroxytryptamine, immunoreactive neurons from raphe nuclei, morphological differentiation, 156-157 5-Hydroxytryptophan brain penetration ability, 310 effect on atropine-resistant low-voltage fast activity, 278 and peripheral decarboxylase inhibitor, prevention of motor inhibition by, 310-311 systemic injection, behavioral and/or electrographic sleep signs and, 308-309 Hyperpolarization, apical dendrites, 246 Hypothalamus, a-bungarotoxin receptors, 29
I Ibotenic acid, effects on rhythmical slow activity, 260 Import, drugs and toxins, 143-144 Interneurons, hippocampal, inhibitory, 264-265 Ionic regulation, roles of glial cells, 181-182
K Kainic acid, antagonist for amino acid receptors, 45
350
INDEX
L Laminin, neuron adherence, 154-155 Large-amplitude irregular activity, 249, 315 antimuscarinic drug-induced, 260 neocortical, 288-289 relation to behavior, 228 static posture accompanied by, 229 Lectins, plant, RNA N-glycosidase toxins,
130-131 Lesions effect on N-acetylaspartylglutamate-containing pathways, 69-71 neocortical low-voltage fast activity,
272-273 median raphe nucleus, atropine-resistant rhythmical slow activity and, 284 preoptic and/or hypothalamic, low-voltage fast activity after, 305 Leu-enkephalin-Arg, in cerebrospinal fluid, 111-113 from dynorphin B, effects of protease inhibitors and synthetic peptides, table, 113 Locustu migrutonk motor nerve terminals, morphological relationship with Schwann cell processes, 178 Lophotoxin, probe for nicotinic acetylcholine receptors, 8 Low-voltage fast activity, 227, 315 atropine-resistant, 269 p-chlorophenylalanine effects, 281 5,7-dihydroxytryptamine effects, 281-283 effect of anesthetics, 272 6-hydroxydopamine effects, 281 5-hydroxytryptophan effects, 278 identification of transmitters, 276-278 5-methoxy-N,N-dimethyltryptamine effects, 278 a-methyl-p-tyrosine effects, 281 neocortical, identification of transmitters, 276-278 nialamide effects, 278 pargyline effects, 278 0-phenylethylamine effects, 278 restoration by pargyline in right neocortex in resperinized rats, 281 sloviter effects, 278 tranylcypromine effects, 278 blockade during Type 2 behavior, 270
after destruction of thalamic neurons, 291 effects of anesthetics, 284 central cholinergic and serotonergic blockade, 297-298 reserpine plus atropine or scopolamine,
275 effects on centrally acting and peripherally acting antimuscarinic drugs, 270 after intravenous or intra-arterial serotonin injections, 309 lesion effects, 272-273, 305 origin, 249 after preoptic and/or hypothalamic lesions, 305 preservation during Type 1 behavior, 270 production by acetylcholine and cholinergic agonists, 268 cholinergic mechanisms, 268 restoration by serotonin agonisk in reserpinized rats, 278-280 spontaneous, in chronic cerveau isole preparations, 305-306 during Type 1 and Type 2 behaviors,
268-269 unit activity during, 285
M Magic bullets, toxins as, 134-138 Mecamylamine, target, 16 Medulla-pons N-acetylaspartylglutamatecolocalization, table, 64 N-acetylaspartylglutamate-like immunoreactive neurons, distribution (rat), table, 55 Membrane metallo-endopeptidase, in cerebrospinal fluid, 114 Membrane perturbation, diphtheria toxin,
127 Metergoline, lack of effect on blockade of atropine-resistant waveforms, 284 Methacholine, rhythmical slow activity production by microinjection into hippocampus, 263 5-Methoxy-N,N-dimethyltryptamine, effect on atropine-resistant low-voltage fast activity, 278
351
INDEX
N-Methyl-carbarnoylcholine,tritiated, probe for nicotinic acetylcholine receptors, 7 N-Methyl-D-aspartate receptors, N-acetylaspartylglutamate and, 68 a-Methyl-9-tyrosine, effect on atropine-resistant low-voltage fast activity, 281 Methysergide, lack of effect on blockade of atropine-resistant waveforms, 284 p rhythm, blockade prior to and during voluntary movement, 250 Midbrain N-acetylaspartylglutamatecolocalization, table, 64 N-acetylaspartylglutamate-likeimmunoreactive neurons, distribution (rat), table, 55 Migration, neuronal cerebellar cortex, glial cell influence, 159-160 developing murine spinal cord, glial cell influence, 160-161 glycogen as ultrastructural marker, 161 influence of glial cells, 159-165 in vitro studies, 162 optic system, glial cell influence, 161-162 weaver and normal mice, glial cell influence, 164 Monoamines, uptake by neuronal and glial cells, 183-187 Morphology, neuron-glia interactionassociated synaptic events, 177-181 Motor activity correlations with serotonin release, 312 folk psychology-derived descriptions, 239, 323-324 Motor excitation, produced by serotonergic stimulation, 311 Movement control, rhythmical slow activity role, 316 different types, effect on afferent pathway transmission, 252 multilevel hierarchical control, 316 relation to neocortical activity, 249-258 voluntary blockade of 0 rhythm prior to and during, 250 blockade of wicket rhythm prior to and during, 250 pyramidal tract neurons firing prior to and during, 250
readiness potential prior to, 250 slowly developing surface negativity prior to, 250 Multiunit activity, somatosensory neocortex, effects of reserpine and atropine sulfate, 297 Muscarinic receptors, in neocortex, 267 Muscimol, effect on acetylcholine release from neocortex, 275 Myasthenia gravis, immunotoxins, 144 Myelin, protein synthesis, role of neurons, 176 Myelin-associatedglycoprotein, 174 Myelination, neuron -glia interrelationship, 174-177 Myelin basic protein, 174 expression by cultured oligodendrocytes, 171 role of neurons and astrocytes, 174-175
N Neocortex, 227 acetylcholine release, muscirnol effects, 275 activation blockade, thalamocortical transmission after, 291 atropine-resistant effects via ascending serotonergic projections, 283 cholinergic control, 267-275 electrocorticograms, 241-249 glutamate release-associated activation, 285 high-affinity choline uptake, 267 large-amplitude irregular slow activity, 315 large-amplitude irregular waveform, 288-289 low-voltage fast activity, 227, 315 ascending pathway, inputs to, 289-290 atropine-resistant, 269 effect of anesthetics, 272 identification of transmitters, 276-278 blockade during Type 2 behavior, 270 effect of anesthetics, 284 effects of reserpine plus atropine or scopolamine, 275 lesion effects, 272-273 preservation during Type 1 behavior, 270
352
INDEX
production, cholinergic mechanisms, 268 during Type 1 and Type 2 behaviors, 268-269 unit activity during, 285 mechanisms of activation, 284-286 muscarinic receptors, 267 neuronal responses to acetylcholine, 267 origin of low-voltagefast activity, 249 parallel regulation with thalamus, 294 relation to movement, 249-258 rhythmical inhibitory postsynaptic potentials, 252 rhythmical potentials, nature of, 252 rhythmical spindle bursts, 315 serotonergic control, 275-284 serotonin release during sleep-waking cycle, 311-312 slow wave activity after reserpine or pimozide plus atropine treatment, 276 slow wave sleep associated with acetylcholine release, 268 thalamic role in electrical activity, 287-296 transcallosal evoked response, see Transcallosal evoked response a-Neoendorphins, preproenkephalin B, schematic, 103 Neosurugatoxin probe for nicotinic acetylcholine receptors, 8 target, 16 Nerve growth factor, glia-secreted, 158-159 Neural cell adhesion molecule (N-CAM), 158 Neurites growth, glial cell influence, 153-159 neurite- glial cell surface interaction, 156 Neurohomones, glial cell response, 173 Neuromuscular junction botulinum toxin and tetanus toxin mechanisms of action, 132-134 nicotinic acetylcholine receptor probes antibodies, 14-15 a-bungarotoxin, 10-12 cDNAs for genes and transcripts, 13 curarimimetic protein toxins, 10-14 Neuron-conditioned medium, effect on glialcell growth and differentiation, 169-170 Neurons adherence on poly-L-lpine, collagen, and laminin, 154-155 brainstem, excited by cholinergic agonists, 259 effect on glial cells
growth and differentiation astrocytes, 167-170 oligodendrocytes, 170-172 Schwann cells, 166-167 metabolic response to neuronal signals, 172-174 myelination, 174-177 energy coupling with glia, 172 5-hydroxytryptamine immunoreactive from raphe nuclei, morphological differentiation, 156-157 identification with neuron-specific enolase, 171 neocortical, acetylcholine excitatory and inhibitory effects, 285-286 neuron- oligodendrocyte relationship, 175 phenotypic expression, influence of glial cells, 151-152 potassium signals, glial cell metabolic responses, 172-174 pyramidal tract, firing prior to and during voluntary movement, 250 role in myelin basic protein expression in cultured oligodendrocytes, 174-175 myelin protein synthesis, 176 septal, as pacemakers for hippocampal rhythm, 263 serotonergic, in dorsal and median raphe nuclei, firing rate, 311 thalamic low-voltage fast activity after destruction, 291 recruiting response-mediating, cortical activation and, 290 synchronized rhythmical bursting, 288 tyrosine hydroxylase immunoreactive from substantia nigra, morphological differentiation, 156-157 Neuron-specific enolase, identification of neurons with, 171 Neuropeptide-transformingenzymes in cerebrospinal fluid, 104-105 isolation from cerebrospinal fluid, 105 Neurotransmission, astrocyte role, 183-194 Neurotransmitters N-acetylaspartylglutamate, at glutamatergic synapses, 45-46 acidic amino acids, 41-45 glial cell responses. 173 receptors in glial cell ines and cultured astroglia, 192-194
353
INDEX
uptake, neuron-glia interactions y-aminobutyric acid, 188-192 aspartate, 188-192 choline, 187-188 glutamate, 188-192 monoamines, 183-187 Nialamide, effects on atropine-resistant lowvoltage fast activity, 278 Nicotine probe for nicotinic acetylcholine receptors, 4-7 production of slow wave rhythmical slow activity, 264 suppression of hippocampal theta cell activity, 264 targets a-bungarotoxin receptor, 26-27 nicotinic acetylcholine receptor agonist receptor, 21 Nicotinic acetylcholine receptors, 1-3 agonist receptor antibodies and, 19-20 as functioning aceytlcholine receptor, 23-24 as member of receptor gene family, 22-23 as nicotine target, 21 synaptic role, 21-22 targets of nicotinic drugs, 16-17 toxin receptor and, 17 binding proteins, muscle receptor-like genes and, 20 probes antagonists, 7-9 antibodies, 14-15 a-bungarotoxin, 10-12 cDNAs, 15-16 cholinergic ligands, 5-9 curarimimetic protein toxins, 10-14 cytisine, 7 dihydro-P-erythroidine,8-9 ganglionic blockers, 7-8 histrionicotoxin, 8 lophotoxin, 8 neosurugatoxin, 8 neuromuscular junction-specific, 10-16 nicotine, 4-7 snake venom toxins, 13 substance 9 tritiated acetylcholine, 7 tritiated N-methyl-carbamoylcholine,7 D-tubocurarine. 8-9
toxin receptor agonist receptor and, 17 antibodies and. 17-19 cross-reactivity of toxin-binding proteins, 18 Nonneuronal cells effect on primary neuronal growth in culture, 154-155 trophic support of neurons, 158-159 Noradrenalin, role in atropine-resistant rhythmical slow activity, 262 Norepinephrine glial cell response, 173 uptake by astrocytes, 184-185 Nucleotide binding protein inhibitory, 129 stimulatory, 129
0 Olfactory bulbectomy, effect on N-acetylaspartylglutamate levels, 70-71 Oligodendrocytes cell cultures enriched with, 175 2 ',3 '-cyclic-nucleotide 3 '-phosphodiesterase biochemical marker, 168-169 galactocerebroside expression, 171 growth and differentiation, effect of neurons, 170-172 myelin basic protein, 171 myelin basic protein expression, role of neurons and astrocytes, 174-175 neuron -oligodendrocyte relationship, 175 Ontogenesis, neuron - glia interactions during, 150-177 Optic system, neuronal migration, glial cell influence, 161-162 Oxotremorine, rhythmical slow activity production by microinjection into hippocampus, 263
P Pargyline effects on atropine-resistant low-voltage fast activity, 278 restoration of atropine-resistant low-voltage fast activity in resperinized rats, 281 Peripheral nervous system, axonal regeneration, 194-195
354
INDEX
Pertussis toxin, as ADP-ribosylating toxin, 128-129 Pharmacology, botulinum toxin and tetanus toxin, 133-134 pH dependence, diphtheria toxin insertion into endosomal membrane. 127 Phencyclidine behavioral disturbances, blockade of serotonin-dependent hippocampal and neocortical activation and, 304 effect on atropine-resistant rhythmical slow activity, 261 Phenotypic expression, glial cell influence, 151-152 0-Phenylethylamine effect on atropine-resistant low-voltage fast activity, 278 effects on spontaneous neocortical activity in reserpinized rats, 280 Pheochromocytoma, treatment with chimeric drugs, 140-141 Pilocarpine effect on slow waves, 273 production of hippocampal rhythmical slow activity, 260 neocortical low-voltage fast activity, 268 Pimozide, and atropine treatment, neocortical slow wave activity after, 276 Place cells, 237-238 Poly-L-lysine, neuron adherence, 154-155 Potassium signals, glial cell metabolic responses, 172-174 Precursors preproenkephalin A, schematic, 103 preproenkephalin B, schematic, 103 a-preprotachykinin, schematic, 103 Preoptic area, electrical stimulation, 306-307 Preoptic lesions, low-voltage fast activity after, 305 Preproenkephalin A, schematic, 103 Preproenkephalin B, schematic, 103 a-Preprotachykinin, schematic, 103 Probes, nicotinic acetylcholine receptor, 1-3 antagonists, 7-9 antibodies, 14-15 a-bungarotoxin, 10-12 cDNAs, 15-16 cholinergic ligands, 5-9 curarimimetic protein toxins, 10-14
cytisine, 7 dihydro-0-erythroidine, 8-9 ganglionic blockers, 7-8 histrionicotoxin. 8 lophotoxin, 8 neosurugatoxin, 8 neuromuscular junction-specific, 10-16 nicotine, 4-7 snake venom toxins, 13 substance P, 9 tritiated acetylcholine, 7 tritiated N-methyl-carbamoylcholine,7 D-tubocurarine, 8-9 Prohormones, 101-104 Promethazine, effect on low-voltage fast activity, 270-271 Protease inhibitors, effects on '25-labeled dynorphin B conversion to Leuenkephalin-Arg6, table, 113 Proteases-peptidases, role in cerebrospinal fluid, 116-118 Protein toxins, 124-128 ADP-ribosyltransferase toxins, 128-130 channel formation, 127 chimeric, see Chimeric toxins curarimimetic, probes for nicotinic acetylcholine receptors in neuromuscular junction, 10-14 diphtheria toxin, 125-126 import, 143-144 internalization process, 126 magic bullets, 134-138 membrane perturbation, 127 RNA N-glycosidase toxins, 130-131 toxins with unknown mechanism of action, 131-134 Proteolipid protein, 174 Pseudomonas aeruginosa exotoxin, as ADPribosylating toxin, 128 Psychotomimetic drugs, effect on atropineresistant rhythmical slow activity, 261 Psychotomimetic opioids, behavioral disturbances, blockade of serotonindependent hippocampal and neocortical activation and, 304 Pyramidal cells, hippocampal phase-reversed intracellular rhythmical slow activity rhythm, 264 serotonin inhibitory effect, 266 Pyramidal tract neurons, firing prior to and during voluntary movement, 250
INDEX
Q Quinuclidinyl benzilate abolishment of hippocampal rhythmical slow activity, 260 effect on low-voltage fast activity, 270-271
R Radioimmunoassays free N-acetylaspartylglutamate, 48-49 solid-phase, N-acetylaspartylglutamate-like immunoreactivity, 53- 54 Raphe nuclei dorsal effects of localized electrical stimulation, 312 firing rate of serotonergic neurons, 311 5-hydroxytryptamine immunoreactive neurons, morphological differentiation, 156-157 median firing rate of serotonergic neurons, 311 high-frequency stimulation, atropineresistant rhythmical slow activity produced by, 261 lesions, atropine-resistant rhythmical slow activity and, 284 localized electrical stimulation effects, 312 stimulation effects on hippocampal activity, 266 stimulation frequency effects, 312-313 stimulation, serotonin release from ascending serotonergic fibers and, 312 Readiness potential, prior to voluntary movement, 250 Recruiting responses electrocorticogram, 242-243 thalamic neurons mediating, cortical activation and, 290 Regeneration, axonal, role of glial cells, 194-197 Reserpine with atropine or scopolamine behavioral effects, 298 effect on neocortical low-voltage fast activity, 275 effects on atropine-resistant rhythmical slow activity, 261
355
neocortical electrical activity and behavior, 277 slow wave and multiunit activity in somatosensory neocortex, 297 restoration of catecholaminergic function by L-dopa or dopaminergic agonist injection, 298 and scopolamine, effects on neocortical electrical activity, 295 treatment, neocortical slow wave activity after, 276 Reticular activating system, conventional view, 227 Reticular formation, stimulation effect on neocortical acetylcholine, 267 production of hippocampal rhythmical slow activity, 260 Retina, N-acetylaspartylglutamate-like immunoreactivity, 65-69 Rhythmical inhibitory postsynaptic potentials, hippocampal pyramidal cells, 264 Rhythmical slow activity, 315 abolishment by antimuscarinic drugs, 260 anticipatory activity, 230-231 atropine-resistant, 260 p-chlorophenylalanineeffects, 261 cyclazoncine effects, 261 dependence on serotonergic input to hippocampus, 261-262 dopamine role, 262 effect of median raphe nucleus lesions, 284 entorhinal cortex removal effects, 262 hippocampal, 260 lesion effects, 261-262 noradrenalin role, 262 phencyclidine effects, 261 phencylidine effects, 261 produced by high-frequency stimulation of median raphe nucleus, 261 reserpine effects, 261 section of afferent pathways effects, 262 atropine-sensitive, 260 during behavioral immobility, 233 complex spike cell firing rate during, 265 correlation with motor activity, 233-234 dorsal hippocampus, effects of evoked responses produced by dentate hilar CA4 area stimulation, 320 effects of
356
INDEX
anesthetics, 284 central cholinergic and Serotonergic blockage, 297-298 generation of voluntary movements and, 231-233 granule cell firing in dendate gyrus during, 265 local interference, behavioral effects, 317-318 phase-reversed rhythm in hyppocampal pyramidal cells, 264 postural change or locomotion accompanied by, 229-230 production by reticular formation stimulation, 260 relation to behavior, 228-233 relation to mechanisms controlling voluntary motor activity, 231 role in movement control, 316 slow wave sleep and, 237 still alertness and, 237 during Type 1 and Type 2 behaviors, 262-263 waves without rhythmical cholinergic input, 263 Ribosomal biosynthesis, N-acetylaspartylglutamate, 72-75 Ricin chimeric drug, 140 RNA N-glycosidase toxin, 130-131 RNA N-glycosidase toxins, 130-131
S Schwann cells growth and differentiation, influence of neurons, 166-167 processes, morphological relationship with Locwta migratonh motor nerve terminals, 178 Scopolamine abolishment of hippocampal rhythmical slow activity, 260 with @-chlorophenylalanine, behavioral effects, 298-300 dose required to affect cortical activity, 271-272 effect on low-voltage fast activity, 270-271 inactivation by atropine esterase, 271 and reserpine, effects on neocortical electrical activity, 295 Sendai virus, ghosts, 138, 143
Sensory cortex, motor functions, 323 Septa1 nuclei, mechanisms for rhythmical activity, 263 Serotonergic antagonists, lack of effect on blockade of atropine-resistant waveforms, 284 Serotonergic cerebral activating mechanisms, role in engram formation, 303 Serotonergic control hippocampus, 261-262 neocortex, 275-284 Serotonergic function behavioral effects of interference, 301 tests involving training, 301 untrained or instinctive behaviors. 301-302 effect of anesthetics, 284 Serotonergic innervation, dorsal hippocampus, 261 Serotonergic mechanisms, central, effect of anestheics, 266 Serotonergic stimulation, motor excitation produced by, 311 Serotonergic systems and cholinergic systems, behavioral effects of blockade, 296-304 control of cerebral activity, 303-304 sleep occurrence and, 307-314 Serotonin brain penetration ability, 309-310 effects mediated by glutamate interaction, 266 functions during active sleep, 314 inhibitory effect on hippocampal pyramidal cells, 266 intravenous or intra-arterial injections, low-voltage fast activity and slow wave activity after, 309 promotion of slow wave sleep, 307-314 release correlations with motor activity, 312 during sleep-waking cycle, 311-314 role in atropine-resistant activating input to cerebral cortex during active sleep, 314 uptake by astrocytes, 186-187 Serotonin agonists, restoration of low-voltage fast activity in reserpinized rats, 278-280 Serum contents, effects on primary neuronal growth in cultural, 154-155 Sleep active, serotonin functions during, 314
357
INDEX
slow wave, serotonin promotion, 307-314 L-tryptophan ingestion and, 313-314 Sleep center, in basal forebrain, 304-307 Sleep-waking cycle, serotonin release during, 311-312 Sloviter, effects on atropine-resistant low-voltage fast activity, 278 Slow wave activity after intravenous or intra-arterial serotonin injections, 309 large-amplitude, irregular, see Large-amplitude irregular activity neocortical, after reserpine or pimozide plus atropine treatment, 276 somatosensory neocortex, effects of reserpine and atropine sulfate, 297 spindle activity, correlation with rhythmical discharges, 249 thalamus, effect of lesions, 291 Slow waves, hippocampal, 228-235 Slow wave sleep acetylcholine release-associated in neocortex, 268 serotonin promotion, 307-314 Small-amplitude irregular activity, 237 Snake venom toxins a-bungarotoxin, 10-12 curarimimetic, 10-14 neuronally active, identification and use, 13 Sodium ion, astrwcyte membrane permeability, 181-182 Somatosensory cortex, unit response variation with motor activity, 253 Somatosensory neocortex, slow wave and multiunit activity, effects of reserpine and atropine sulfate, 297 Somatostatin, cleavage by substance P-converting enzyme, 109 Spinal cord N-acetylaspartylglutamate colocalization, table, 64 N-acetylaspartylglutamate levels, transection effects, 69-70 N-acetylaspartylglutamate-likeimmunoreactive neurons, distribution (rat), table, 55 N-acetylaspartylglutamate-like immunoreactivity (rodent), 54-58 developing, neuronal migration (mouse), 160-161 Spiroperidol, lack of effects on blockade of atropine-resistant waveforms, 284
Striatum, N-acetylaspartylglutamatelevels, excitoxin lesion effects, 69-70 Substance P a-preprotachykinin, schematic, 103 probe for nicotinic acetylcholine receptors, 9 Substance P-converting endopeptidase in cerebrospinal fluid, 108-110 cleavage of calcitonin gene-related peptide and somatostatin, 109 degradation, 108 properties in human cerebrospinal fluid, table, 109 substrate specificity, 109 Substantia nigra, tyrosine hydroxylase immunoreactive neurons, morphological differentiation, 156-157 Substrata effect on primary neuronal growth in culture, 154-155 glia-enriched, 152-159 Superfunctions, 135-136 Supergenes, 135 Supramodal association cortex, hippocampus as, 322 Surface negativity, slowly developing prior to voluntary movement, 250 Synapses, glutamatergic, N-acetylaspartylglutamate neurotransmission, 45-46 Synaptic contacts, between neuronal processes and glial cells, 177-178 Synaptic density, regulation, role of astrocytes, 180 Synaptic events, neuron-glia interactions and, 177-194 Synaptic function nicotinic acetylcholine receptor agonist receptor, 21-22 role of glial cells, 207-208 Synthetic peptides, effects on lz5-labeled dynorphin B conversion to Leuenkephalin-Arg., table, 113
T Tetanus toxin chimeric drug, 142 chimeric molecule with botulinum neurotoxin, 142 import device for, 144
358
INDEX
mechanism of action, 131-134 pheochromocytoma treatment, 140-141 Thalamocortical discharges, repetitive, Type 1 spindles dependent on, 242 Thalamus lesions, effects on irregular slow wave activity, 291 low-voltage fast activity, after neuron destruction, 291 neurons, synchronized rhythmical bursting, 288 as pacemaker, 287 parallel regulation with neocortex, 294 projection cells, firing patterns, 287-288 role in neocortical activity, 287-296 spindle bursts dependent on, 290 thalamocortical transmission after activation blockade, 291 Theta cells firing characteristics during Type 1 and Type 2 behavior, 235 firing rate and pattern, 235 Theta rhythm, relation to behavior, 228-233 Toxin F, 3, 13 Toxins, protein, 124-128,see also specific toxin Transcallosal evoked response early component, 253-254 effect of
motor activity, 257 passive movement, 256 late component, 253-254 during quiet or slow wave sleep, 254 suppression of late component, 256 variation with concurrent motor activity, 253 with waking behaviors, 254 under various conditions of background electrocortical activity, 255 Tranylcypromine, effects on atropine-resistant low-voltage fast activity, 278 L-Tryptophan, ingestion, sleep and, 313-314 D-Tubocurarine probe for nicotinic acetylcholine receptors,
8-9 target, 16 Tyrosine hydroxylase. immunoreactive neurons from substantia nigra, morphological differentiation, 156-157
W Weaver strain, neuronal migration, glial cell influence, 164 Wicket rhythm, blockade prior to and during voluntary movement, 250
CONTENTS OF RECENT VOLUMES Volume 20
Choline Acetyltransferase: A Review with Special Reference to Its Cellular and Subcellular Localization Jean Rosier
Functional Metabolism of Brain Phospholipids G. Brain Ansell and Sheila Spanner
SUBJECT INDEX
Isolation and Purification of the Nicotine Acetylcholine Receptor and Its Functional Reconstitution into a Membrane Environment Michael S. Briley and Jean-Pierre Changeux Biochemical Aspects of Neurotransmission in the Developing Brain Joseph T Coyle
V O h W 21
T h e Formation, Degradation, and Function of Cyclic Nucleotides in the Nervous System John W . Daly
to the Pathophysiology
Relationship of the Actions of Neuroleptic Drugs Of Tardive Dyskinesia Ross J. Baldessarini and Daniel Tarsy
Soviet Literature o n the Nervous System and Psychobiology of Cetacea Theodore H. Bullock and Vladimir S. Gurevich
Fluctuation Analysis in Neurobiology Louis J . DeFelice Peptides and Behavior Georges Ungar
Binding and Iontopheretic Studies on Centrally Active Amino Acid.-A Search for Physiological Receptors c! DeFeudis
Biochemical Transfer of Acquired Information S. R. Mitchel1,J. M . Beaton, and R.J. Bradley Aminotransferase Activity in Brain M . Benuck and A . Lajtha
Presynaptic Inhibition: Transmitter and Ionic . . Mechanisms R . A . Nicoll and B. E. Alger
The ~ ~Structurelof Acetylcholine ~ and ~ Adrene+ Receptors: All.protein Model J. R . Smythies
Microquantitation of Neurotransmitters in ~ l ~ ~ Specific Areas of the Central Nervous System Juan M . Saauedra
Structural Integration of Neuroprotease Activity Elena Gabrzelescu
Physiology a n d Glia: G l i a l - N e u r o n a l Interactions R. Malcolm Stewart and Roger N . Rosenberg
Lipotropin and the Nervous System W . H . Gispen, J M . wan Ree, and De. de Wied
Molecular Perspectives of Monovalent Cation Selective Transmembrane Channels Dan W. Urry
Tissue Fractionation in Neurobiochemistry: An Analytical Tool or a Source of Artifacts Pierre Laduron
Neuroleptics a n d Brain Self-Stimulation Behavior Albert Wauquier
359
360
CONTENTS OF RECENT VOLUMES
Volume 22 Transport and Metabolism of Glutamate and GABA in Neurons and Glial Cells Arne Schowboe Brain Intermediary Metabolism in Vivo: Changes with Carbon Dioxide, Development, and Seizures Alexander L. Miller
Benzodiazepine Receptors in the Central Nervous System Phil Skolnick and Steven M . Paul Rapid Changes in Phospholipid Metabolism during Secretion and Receptor Activation R I: Crews Glucocorticoid Effects on Central Nervous Excitability and Synaptic Transmission Edward D. Hall
N,N-Dimethyltryptamine: An Endogenous Hallucinogen Steven A . Barker, John A . Monti, and Samuel T Christian
Assessing the Functional Significanceof LesionInduced Neuronal Plasticity Oswald Steward
Neurotransmitter Receptors: Neuroanatomical Localization through Autoradiography L. Charles M u m ' n
Dopamine Receptors in the Central Nervous System Ian Cresse, A . Leslie Morrow, Stuart E. LefJ David R. Sibley, and Mark W Hamblin
Neurotoxins as Tools in Neurobiology E. G. McCeer and E( L. McGeer Mechanisms of Synaptic Modulation William Shain and David 0. Carpenter Anatomical, Physiological, and Behavioral Aspects of Olfactory Bulbectomy in the Rat B: E. Leonard and M . W t e The Deoxyglucose Method for the Measurement of Local Glucose Utilization and the Mapping of Local Functional Activity in the Central Nervous System Louis Sokoloff
Functional Studies of the Central Catecholamines I: W Robbins and B. J Evenjt Studies of Human Growth Hormone Secretion in Sleep and Waking Wallace B. Mendelson Sleep Mechanisms: Biology and Control of REM Sleep DennisJ McCinty and Rend R . Drucker-Colin INDEX
INDEX
Volume 24 Volume 23 Chemically Induced Ion Channels in Nerve Cell Membranes David A . Mathers and Jeffery L. Barker Fluctuation of Na and K Currents in Excitable Membranes Berthold Neumcke Biochemical Studies of the Excitable Membrane Sodium Channel Robert L. Barchi
Antiacetylcholine Receptor Antibodies and Myasthenia Gravis Bernard W Fulpiuc Pharmacology of Barbiturates: Electrophysiological and Neurochemical Studies Max Willow and Graham A . R. Johnston Immunodetection of Endorphins and Enkephalins: A Search for Reliability Alejandro Bayon, William J Shoemaker, Jacqueline R McCinty, and Floyd Bloom
CONTENTS OF RECENT VOLUMES
On the Sacred Disease: The Neurochemistry of Epilepsy 0. Carter Snead 111 Biochemical and Electrophysiological Characteristicsof Mammalian GABA Receptors Salvatore J Enna and Joel f? Gallagher Synaptic Mechanisms and Circuitry Involved in Motorneuron Control during Sleep Michael H . Chase Recent Developments in the Structure and Function of the Acetylcholine Receptor E J Barrantes Characterization of (11,- and wAdrenergic Receptors David B. Bylund and David C. U'Prichard Ontogenesis of the Axolemma and Axoglial Relationships in Myelinated Fibers: Electrophysiological and Freeze-Fracture Correlates of Membrane Plasticity Stephen G. Waxman, Joel A. Black, and Robert E. Foster
361
Opioid Actions on Mammalian Spinal Neurons W. Zieglgans berger Psychobiology of Opioids Albert0 Oliveno, Claudio Castellano, and Stefan0 Publisi-Allegra Hippocampal Damage: Effects on Dopaminergic Systems of the Basal Ganglia Robert L. Isaacson Neurochemical Genetics V. Cscinyi The Neurobiology of Some Dimensions of Personality Marwin Zuckerman, James C. Ballenger, and Robert M . Post INDEX
Volume 26
INDEX
The Endocrinology of the Opioids Mark J. Millan and Albert H e m Multiple Synaptic Receptors for Neuroactive Amino Acid Transmitters -New Vistas Najam A . Shanif
Volume 25 Guanethidine-Induced Destruction Sympathetic Neurons Eugene M . Johnston, J r and Pamela Toy Manning
of
Dental Sensory Receptors Margaret R. Byers Cerebrospinal Fluid Proteins in Neurology A. Lowenthal, R. Crols, E . De Schutter, J. Gheuens, D. Karcher, M . Noppe, and A . Tasnier Muscarinic Receptors in the Central Nervous System Mordechai Sokolousky Peptides and Nociception Daniel Luttinger, Daniel E. Hernandez, Charles B. Nemeroff, and A r t h u r J Prange, Jr
Muscarinic Receptor Subtypes in the Central Nervous System Wayne Hoss and John Ellis Neural Plasticity and Recovery of Function after Brain Injury John E Marshall From Immunoneumlogy to Immunopsychiatry: Neuromodulating Activity of Anti-Brain Antibodies Branislav D. JankoviC Effect of Tremorigenic Agents on the Cerebellum: A Review of Biochemical and Electrophysiological Data V. G. Longo and M . Massotti INDEX
362
CONTENTS OF RECENT VOLUMES
Volume 27 The Nature of the Site of General Anesthesia Keith W Miller
Biological Aspects of Depression: A Review of the Etiology and Mechanisms of Action and Clinical Assessment of Antidepressants S. I. Ankier and B. E. Leonard
The Physiological Role of Adenosine in the Central Nervous System Thomas V . Dunwiddie
Does Receptor-Linked Phosphoinositide Metabolism Provide Messengers Mobilizing Calcium in Nervous Tissue? John N . Hawthorne
Somatostatin, Substance P, Vasoactive Intestinal Polypeptide, and Neuropeptide Y Receptors: Critical Assessment of Biochemical Methodology and Results Anders Unddn, Lou-Lou Peterson, and Tamas Bartfai
Short-Term and Long-Term Plasticity and Physiological Differentiation of Crustacean Motor Synapses H . L. Atwood and J. M . Wojtowicz
Eye Movement Dysfunctions and Psychosis Philip S. Holzman Peptidergic Regulation of Feeding J. E. Morley, Z J. Bartness, B. A. Gosnell, and A . S. Lemne Calcium and Transmitter Release Ira Cohen and William Van der Kloot Excitatory Transmitters and Epilepsy-Related Brain Damage John W Olney Potassium Current in the Squid Giant Axon J o h n R . Clay INDEX
Volume 28 Biology and Structure of Scrapie Prions Michael I? McKinley and Stanley B. Ausiner Different Kinds of Acetylcholine Release from the Motor Nerve S. Thesleff Neuroendocrine-Ontogenetic Mechanism of
Aging: Toward an Integrated Theory of Aging V. M . Dilman, S. Y. Revskoy, and A. G. Golubev The Interpeduncular Nucleus Barbara J . Morley
Immunology and Molecular Biology of the Cholinesterases: Current Results and Prospects Stephen Bn'mijoin and Zoltan Rakonczay INDEX
Volume 29 Molecular Genetics of Duchenne and Becker Muscular Dystrophy Ronald G. Worton and Arthur H . M . Burghes Batrachotoxin: A Window on the Allosteric Nature of the Voltage-Sensitive Sodium Channel George B. Brown Neurotoxin-Binding Site on the Acetylcholine Receptor Thomas L. Lentz and Paul Z Wilson Calcium and Sedative-Hypnotic Drug Actions Peter L. Carlen and Peter H . Wu Pathobiology of Neuronal Storage Disease Steven U. Walkley Thalamic Amnesia: Clinical and Experimental Aspects Stephen G. Waxman Critical Notes on the Specificity of Drugs in the Study of Metabolism and Functions of Brain Monoamines S. Garattini and i? Mennini
CONTENTS OF RECENT VOLUMES
Retinal Transplants and Optic Nerve Bridges: Possible Strategies for Visual Recovery as a Result of Trauma or Disease James E. Turner, Jerry R . Blair, Magdalene Seiler, Robert Aramant, Thomas W Laedtke, E . Thomas Chappell, and Lauren Clarkson
363
Schizophrenia: Instability in Norepinephrine, Serotonin, and y-Aminobutyric Acid Systems Joel Gelernter and Daniel I? van Kammen INDEX
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