International Review of Neurobiology Volume 16

INTERNATIONAL REVIEW OF

Neurobiology VOLUME 16

Associate Editors

w. R. b E Y

SIRJOHN ECCLES

D. BOVET

H. J. EYSENCK

JOSE

DELGADO

C . HEBB

0.ZANGWILL

Consultant Editors Prf. BORNSTEIN

A. LAJTHA

F. BRUCKE

B. A. LEBEDEV

J. ELKES

SIRAUBREY LEWIS

R. HEATH

V. LONGO

B. HOLMSTEDT

D. M. MACKAY

P. JANSSEN

S. MARTENS

S. KETY

F. MORRELL

K. KILLAM

H. OSMOND

C. KORNETSKY

s. SZARA

INTERNATIONAL REVIEW OF

Neurobiolonv U I

Edited

by CARL C. PFEIFFER New jersey Neuropsychiatric lnstitute Princeton, New jersey

JOHN

R. SMYTHIES

Department of Psychiatry University of Edinburgh, Edinburgh, Scotland

VOLUME 16

I974

ACADEMIC PRESS

0

New York San Francisco London

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COPYRIGHT 0 1974, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY A N Y MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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CONTENTS CONTRIBUTORS.

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vii

Model of Molecular Mechanism Able to Generate A Depolarization-Hyperpolarization Cycle

CLARATORDA

I. A Proposed Model of a Depolarization-Hyperpolarization Cycle 11. Depolarization . . . . . . . . . . . 111. Hyperpolarization-A Cyclic AMP-Dependent Form . . IV. V. VI. VII.

Current Concepts of Depolarization and Hyperpolarization General Discussion . . . . . . . . . General Conclusions . . . . . . . . Summary . . . . . . . . . . . References . . . . . . . . . . .

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1 3 32 50 53 58 58 59

Antiacetylcholine Drugs: Chemistry, Stereochemistry, and Pharmacology

T. D. INCH AND R. W. BRIMBLECOMBE

I. Introduction . . . . . . . . . 11. Chemical Evolution of Antiacetylcholine Drugs . . 111. Absolute Stereochemistry and Antiacetylcholine Activity IV. Quantitative Correlation of Chemical Structure and

. . . . . . Antiacetylcholine Activity . . . . . . . . V. Factors That Influence the Time-Activity Profile of Antiacetylcholine Drugs . . . . . . . . . VI. Relative Potencies of Antiacetylcholine Drugs in the Central . . Nervous System and the Peripheral Nervous System . VII. Behavioral Studies . . . . . . . . . VIII. Antiacetylcholine Drugs in the Treatment of Poisoning by Anticholinesterase Agents . , . . . . . . . . . . . . . . , . . IX. Metabolism X. Pharmacological Methods . . . . . . . . . . . . . . . . . . . References

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125 133 137 139

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146 147 151 158 165

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Kryptopyrrole and Other Monopyrroles in Molecular Neurobiology DONALD G. IRVINE

I. Introduction . . . . . Molecular Structures and Context Chemistry and Chromatography . Biochemistry . . . . . . . . . Pharmacology .

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CONTENT3

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Introduction . . . . . . . . . . RNA Content of Brain as Compared to Other Organs . Anatomical and Subcellular Distribution of Brain RNA RNA Changes in Single Cells Produced by Physiological . . . . . . and Electrical Stimulation . . . V . RNA Changes in the Brain during Development . . . . . . . . VI . Types of Brain RNA VII . Brain Function and RNA Metabolism . . . . . VIII . Biogenic Amine Effects on Brain RNA Metabolism . . . . . . I X . Drug Effects on RNA Synthesis . . . X . A Theoretical Model for Information Storage . References . . . . . . . . . .

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VI . Behavioral Pharmacology . . VII . Neurobiology of Other Pyrroles . VIE1. Relationship to Clinical Psychiatry References . . . . .

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168 i70 176 179

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RNA Metabolism in the Brain VICTORE. SHASHOUA

I. I1. I11. IV

183 184 189

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197 199 204 206 217 219 222 228

A Comparison of Cortical Functions in Man and

I . Introduction I1. Anatomy I11. Behavior . IV . Discussion . References

the Other Primates

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R E . PWINGHAM AND G. ETTLINGER

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Porphyria: Theories of Etiology and Treatment H. A. PETERS,D.J . CRIPPS.AND H H. REESE . . . . . . . I . Introduction and Scope I1. Classification of Disturbed Porphyrin Metabolism . . . I11. Clinical Symptoms and Signs in Hepatic Porphyrics . IV . Gastrointestinal Signs . . . . . . . . . . . . . . . . V . Genitourinary Signs . . . . . . . VI . Cardiorespiratory Signs . . . . . . . . . VII . Dermatological Signs . . . . . . . . VIII . Precipitating Factors I X. Pathology . . . . . . . . . . X. Biochemistry . . . . . . . . . . . . . . . . . XI . Pathogenesis of Attacks . . . . . . . . . XI1. Therapy . . . XI11. Discussion . . . . . . . . . . . References . . . . . . . . . .

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233 237 260 290 292

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SUBJECT INDEX .

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CONTENTS OF PREVIOUS VOLUMES

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302 305 308 310 310 310 311 311 316 318 322 327 340 348

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357

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365

CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

R. W . BRIMBLECOMBE,* Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire, England (67) D. J . CRIPPS, Departments of Neurology and Dermatology, University o f Wisconsin Medical School, Madison, Wisconsin (301)

G. ETTLINGER, The Institute of Psychiatry, De Crespigny Park, London, England (233)

T. D. INCH,Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire, England (67) DONALDG. IRvINE,t Saskatchewan Hospital Research Unit, North Battleford, Saskatchewan, Canada (145)

R. E. PASSINGHAM, The Department of Experimental Psychology, South Parks Road, Oxford, England (233) H . A. PETERS, Departments of Neurology and Dermatology, University of Wisconsin Medical School, Madison, Wisconsin (301) H . H . REESE, Departments of Neurology and Dermatology, University of Wisconsin Medical School, Madison, Wisconsin (301) VICTOR E. SHASHOUA, McLean Hospital Research Laboratory, Harvard Medical School, Belmont, Massachusetts ( 183) CLARATORDA, Mount Sinai School of Medicine and Downstate Medical Center, New York, New York (1)

* Present address: The Research Institute, Smith, Kline, and French Laboratories, Welwyn Garden City, Hertfordshire, England. t Present address: The Psychiatric Research Unit, University Hospital, Saskatoon, Saskatchewan, Canada. vii

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MODEL OF MOLECULAR MECHANISM ABLE TO GENERATE A DEPOLARIZATIONHYPERPOLARIZATION CYCLE1 By Clara Torda Mount Sinai School of Medicine and Downstate Medical Center, N e w York. N e w York

I. A Proposed Model of a Depolarization-Hyperpolarization Cycle

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11. Depolarization . . A. Acetylcholine and Receptors (Nicotinic, Muscarinic) . B. Nicotinic Receptor(s) of Acetylcholine . C. Proposed Nicotinic Receptor: The Regulatory Subunit of Triphosphoinositide Phosphomonoesterase. . D. Membrane Depolarization . E. Experimental Observations . 111. Hyperpolarization-A Cyclic AMP-Dependent Form . A. Membrane Hyperpolarization . . B. Specific Receptors for Cyclic AMP: Proposed New Receptor-The Regu. latory Subunit of Diphosphoinositide Kinase C. Experimental Observations . IV. Current Concepts of Depolarization and Hyperpolarization . V. General Discussion . VI. General Conclusions . VII. Summary References

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1. A proposed Model of a Depolarization-Hyperpolarization Cycle

The depolarizafion-hyperpolarization cycle may resemble oscillatory processes. I t seems to depend on preset molecular chain reactions. One of these molecular mechanisms has been identified in the present study (Fig. 1). In cholinergic synapses, postsynaptic depolarization is generated by locally available acetylcholine (ACh). The ACh molecule is encoded with several messages. O n combining with ACh, the postsynaptic nicotinic receptor reads out the message: “depolarize.” The selection results from a particular quality of this specific receptor: on combining with ACh, this receptor acquires the ability to initiate those special processes which are necessary for the execution of depolarization. Presented in part at the Annual Meeting of the Biophysical Society (1972), FASEB (1972), Neurosciences (1972).

I

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CLARA TORDA

DPI K /a

FIG. 1. Schematic representation of the proposed model of a depolarization-hyperpolarization cycle. ACh = acetylcholine; DPI = diphosphoinositide; DPIK = diphosphoinositide kinase; TPI = triphosphoinositide; TPIPM = triphosphoinositide monoesterase.

One of the postsynaptic nicotinic ACh receptors has been identified in vitro and in uivo as the regulatory subunit of triphosphoinositide phosphomonoesterase (Torda, 1972a-g). On combining with ACh, this regulatory subunit ceases to inhibit the enzymatic activity of the catalytic subunit, and, during the transmembrane passage of the ACh-nicotinic receptor complex, triphosphoinositide (TPI) is dephosphorylated to diphosphoinositide (DPI). Concurrent processes quantitatively channel the local electric fields toward depolarization. I t seems, therefore, that activation of triphosphoinositide phosphomonoesterase by ACh may be one of the molecular mechanisms that is able to couple in time and space the formation of the ACh-receptor complex and the depolarization of the postsynaptic neuron. In neurons that accumulate cyclic adenosine monophosphate (CAMP)

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

3

during activity, hyperpolarization may result from the following molecular chain reaction: cAMP forms a complex with one of its specific postsynaptic receptors. This specific receptor has been identified in vitro and in vivo as the regulatory subunit of diphosphoinositide kinase (Torda, 1972a,b,e-g) . O n combining with CAMP,this regulatory subunit ceases to inhibit the enzymatic activity of the catalytic subunit, and DPI is phosphorylated to TPI. Concurrent molecular processes quantitatively channel the local electric field potentials toward hyperpolarization. It seems, therefore, that activation of diphosphoinositide kinase by cAMP may be one of the molecular mechanisms that is able to couple in time and space the formation of the CAMP-specific receptor complex and the hyperpolarization of the postsynaptic neuron (Fig. 1). A comprehensive review of the available literature is beyond the scope of this work. Only the experimental evidence for a molecular chain reaction capable of generating a depolarization-hyperpolarization cycle is here reported : namely, the molecular chain reaction generated through activation of triphosphoinositide phosphomonoesterase by ACh, and activation of diphosphoinositide kinase by CAMP. II. Depolarization

A. ACETYLCHOLINE AND RECEPTORS (NICOTINIC, MUSCARINIC) Two types of specified receptors of ACh have been identified: a nicotinic and a muscarinic. Acetylcholine has 4 pairs of spare electrons: two pairs on the ether 0, and two pairs on the carbonyl 0. Acetylcholine binds on the muscarinic receptor by the two pairs of spare electrons on the ether 0, and on the nicotinic receptor by the two pairs ofspare electrons on the carbony10 (Chothia, 1969; Chothia and Pauling, 1969; Kier, 1967, 1968). Since activation of the muscarinic receptor leads to an increased concentration of tissue cyclic GMP, Ferrendelli et al. (1970) and George et al. (1970) assumed that the muscarinic ACh receptor is part of the guanyl cyclase system. The nicotinic receptor is involved in the generation of fast depolarization. The postsynaptic nicotinic receptor has been identified as the regulatory subunit of triphosphoinositide phosphomonoesterase in the present study (Torda, (1972a-g, 1973a-f).

B. NICOTINIC RECEPTOR(S) OF ACETYLCHOLINE Specific nicotinic receptor(s) have been isolated and purified by various researchers. These receptors are macromolecules that contain protein(s) and phospholipids [e.g., the proteolipid described by De Robertis (1971) and De Robertis et al. (1969, 1971); the phosphoprotein described by O’Brien and

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CLARA TORDA

Gilmour (1969) and O’Brien et al. (1969)l. The purified protein(s) seem to have a molecular weight of 42,000 and 45,000 (De Robertis, 1971; Fulpius etal., 1973; Karlin, 1973; Olsenetal., 1973; Potter, 1973; Waser etal., 1973). Models of this protein molecule have been built by Tasaki (1968) and Smythies (1971). The molecule has a disulfide bond (Karlin and Winnik, 1968) and probably a carboxyl group (Edwards et al., 1970). The third amino acid-base pair ionic dipole (Gill, 1965) seems to form a grid in the membrane (Barlow, 1969). During formation of a complex with ACh, the ionic bonds of this third amino acid base pair become disrupted and the receptor protein assumes conformational changes. The receptor molecule is embedded in discrete area of the postsynaptic membrane. I n this highly dielectric medium, it is held in place by hydrophobic interactions. This receptor is endowed with the ability of stereospecific recognition and rapid binding of ACh. O n combination with ACh, the surface of the receptor becomes more hydrophobic. The complex adopts a tetramic arrangement (Khomorov-Borisov and Michelson, 1966). Because of its hydrophobic surface and elongated shape, the ACh-receptor complex may traverse the postsynaptic membrane (Belleau, 1967; Belleau and Lavoie, 1968; Changeux et al., 1965; Changeux and Podleski, 1968; De Robertis, 1971; Downie, 1970; Ehrenpreis et al., 1969; Eldefrawi et al., 1971 ; Kabachnik et al., 1971; Karlin, 1967a, b, 1969; Khomorov-Borisov and Michelson, 1966; Monod el al., 1965; Silman and Karlin, 1969; Watkins, 1965).

C. PROPOSED NICOTINIC RECEPTOR : THEREGULATORY SUBUNIT OF TRIPHOSPHOINOSITIDE PHOSPHOMONOESTERASE I n the present study (one of) the nicotinic receptor(s) of ACh has been identified as the regulatory subunit of triphosphoinositide phosphomonoesterase (Torda, 1972a-d,g, 1973a-f). This enzyme occurs in a near-inactive form in many resting tissues (Salway et al., 1967, 1968), including the postsynaptic membrane (Torda, 1973f), holding its specific substrate, TPI, in a firm position (Chang and Ballou, 1967; Dawson and Thompson, 1964; Prottey el al., 1968. Triphosphoinositide phosphomonoesterase has been fractionated into a catalytic and regulatory subunit (Torda, 1973a). When united, the regulatory subunit inhibits the enzymatic activity of the catalytic subunit. The regulatory subunit has a far greater affinity for ACh than for the catalytic subunit. O n combining with locally available ACh, the regulatory subunit undergoes conformational changes and ceases to inhibit the enzymatic activity of the catalytic subunit. The direct and indirect consequences of the enzymatic activity of the catalytic subunit seems to cooperate to change the locally available electric field potentials toward depolarization (Torda, 1973b-e).

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

5

D. MEMBRANE DEPOLARIZATION Depolarization results from a special type of channeling of the local electric fields. This type of control may be executed by several molecular mechanisms, including (a) loss of membrane-bound Ca2 alone (Diamond and Wright, 1969; Keynes, 1972; Lecar and Nossal, 1971; Magleby and Stevens, 1972); and (b) cooperation of several processes (e.g., loss of membrane-bound Ca2+',conformational changes, special ionophoresis). At times ionophoresis is sufficient to generate depolarization. According to Hodgkin and Huxley (1952a, b), depolarization is the bioelectric equivalent of the changing sums of the equilibrium potentials of the actually (passively) transported Na and K + ions (calculated following the Nernst equation). Ionophoresis may result from the cooperation of several molecular mechanisms. On combining with ACh, the specific nicotinic receptor acquires the ability to generate the processes that are necessary to execute depolarization. The already identified acquired mechanisms are as follows: the receptor molecule undergoes conformational changes that lead to facilitation of passive ion transport by opening of membrane pores, part of the receptor may serve as a special carrier, and the receptor ceases to inhibit the enzymatic activity of the catalytic subunit. The resulting dephosphorylation of triphosphoinositide concurs with a quantitative release of membrane-bound Ca2+ (see below). Some of these molecular mechanisms are capable of generating depolarization of the postsynaptic neuron. It seems likely, however, that the in uiuo depolarization of the postsynaptic neuron results from the cooperative efforts of several mechanisms, all of them probably being endowed with the ability for mutual interregulation. It seems that the limiting factor of the generated depolarization is the critical membrane potential that coincides with spiking [e.g., an average of 15 mV in the Renshaw cell of the cat, 23 mV in the superior cervical ganglion of the rabbit, and 26 mV in the paravertebral sympathetic chain of the frog (Eccles, 1964)]. The most popular concepts about the nature of the changes generated in the molecule of the nicotinic receptor during combination with ACh are the following. (1) During complex formation, ACh repels the -NH groups of the third amino acid base pair of the receptor protein. During this repulsion of the -NH groups, nucleolipids dissociate from the polypeptide chain of the receptor (Smythies, 1971). This decreased tightening of the packing of the membrane lipids permits ionophoresis. (2) When ACh occupies the anionic and stereophilic sites of the receptor molecule, the interaction of the monomeric unit changes, and the membrane starts to function as though it had open pores (De Robertis, 1971). According to this concept, the same receptor site is equipped to accomplish both the binding of ACh, and the increased ionophoresis. (3) The conformational and other changes of the +

+

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CLARA TORDA

receptor molecule due to combining with ACh terminate the inhibitory effects of the regulatory subunit of triphosphoinositide phosphomonoesterase (the nicotinic receptor) on the enzymatic activity of the catalytic subunit. The direct and indirect consequences of the subsequent enzymatic dephosphorylation of T P I have been only partially identified. Only the effects of the decrease of membrane-bound Ca2 have been quantitatively studied. Due to the lower chelating ability of DPI (Dawson, 1965; Hendrickson and Reinertsen, 1971), dephosphorylation of T P I to DPI concurs with a quantitatively predictable release of membrane-bound Ca2+.This special Ca2 loss may have multiple effects on generation of depolarization: (a) I t seems to be sufficient to channel the local electric fields toward depolarization (Diamond and Wright, 1969; Keynes, 1972 ; Lecar and Nossal, 1971; Magleby and Stevens, 1972); (b) it may contribute to the ionophoretic effects of the changes of the receptor molecule (Baker, 1972; Bangham, 1968; Charnock, 1963; Durrell et al., 1969; Edelman, 1961; Glynn et al., 1965; Krnjevic and Lisievicz, 1972; Mule, 1969; Papahadjopoulos, 1970, 1971; Sheltaway and Dawson, 1966). According to this model described in (3), the binding site of the receptor for ACh (the regulatory subunit of triphosphoinositide phosphomonoesterase) differs from the site that generates the processes that are necessary for the execution of depolarization (the catalytic subunit of triphosphoinositide phosphomonoesterase) . Since depolarization depends not only on the effects of ACh, but also on various local factors, its latency and height may vary. The shortest latency observed in chemically transmitting synapses was 0.2 msec. The rising slope of the postsynaptic depolarization is usually steep, and (depending on the animal) it may reach the critical voltage for spike generation in 1.2 msec (in the frog). The postsynaptic potential decays exponentially and exceeds the membrane time decay by a number of milliseconds, depending on the animal (e.g., with an average of 2.4 msec in the frog). This excess of time suggests that the decay of the postsynaptic potential depends on additional molecular factors, such as the decay of the transmitter. +

+

E. EXPERIMENTAL OBSERVATIONS 1. Biochemicnl (in Vitro) Experiments

Triphosphoinosi tide phosphomonoesterase has been isolated from many tissues, including different membrane material (Hawthorne and Kai, 1970; Hawthorne and Kemp, 1964; Salway et al., 1967, 1968). The triphosphoinositide phosphomonoesterase content of the myelin is high, and of the microsomes (Salway et al., 1967) and the synaptic membrane (Torda, 1973f) low, and of the same order of magnitude. At sites where it is instrumental in performing a fast dephosphorylation that handles small, but quantitatively

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

7

exact, processes, triphosphoinositide phosphomonoesterase seems to occur in small concentrations. At sites, like the myelin, where it participates in gross regulation of trophic processes, it seems to occur in larger concentrations. Similar duality has been observed during the study of other enzymes (Koelle, 1969). The search for a potential postsynaptic nicotinic receptor began by observation of the ability of ACh to increase the activity of various potentially eligible enzymes. Since ACh significantly potentiated the enzymatic activity of triphosphoinositide phosphomonoesterase, attempts were made to 'gain insight into the mechanism through which ACh may activate this enzyme, which was extracted from various types of brains and was purified following the available methods. When further purification remained without effect on the enzymatic activity, enzyme fractionation was attempted by chromatography on special columns. On Sephadex LH-20 the purified triphosphoinositide phosphomonoesterase separated into an enzymatically active and an inactive fraction. The active protein significantly exceeded the enzymatic activity of the purified enzyme. A detailed study revealed that the two fractions had properties of a catalytic (active protein) and a regulatory (inactive protein) subunit. a. Materials Acetyl-l-[14C]ACh (with a specific activity of 9.2 mCi/mmole) was obtained from Radiochemical Centre, Amersham, England; and d-[14C] tubocurarine (with a specific activity of 7.71 mCi/mmole) from the New England Nuclear Co. Bovine serum albumin and chymotrypsin were obtained from Armour and Co., Chicago, Illinois. Sephadex (3-200 and Sephadex LH-20 were obtained from Pharmacia, Stockholm, Sweden. DEAE-cellulose (Whatman DE-52) was obtained from H. Reeve, Angel and Go., London. Triphosphoinositide was prepared from DPI following the combined method of Dawson and Thompson (1964) and Salway et al. (1967). Diphosphoinositide was extracted by the method of Folch (1949). One gram of inositol phosphatide was dissolved in 12 ml of chloroform at 4°C. The impure DPI was precipitated by adding 22 ml of methanol with 30 minutes of shaking. The impure DPI was contained in the precipitate, and represented 20y0 of theinositol phosphatide, or 1 gm/kg of brain tissue (wet weight). Further purification was attempted (Kai et al., 1966a) by suspending the precipitate in 2% (w/v) sodium salt of EDTA at p H 7.0, and dialysis against deionized water overnight. Further purification and separation of DPI and T P I were obtained following the method of Hendrickson and Ballou (1964). By treatment with ethylenediaminetetraacetic acid, the DPI was converted into the sodium form. The solution was passed through a chelating resin,

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CLARA TORDA

DEAE-cellulose (Whatman DE-52), and was eluted with a gradient of 0 to 0.6 M ammonium acetate in chloroform :methanol :water (20: 9 :1). The thus obtained mixed Ca2 and Mg2 chelate of TPI was further purified by chromatography on formaldehyde treated paper. An average of 57.2 pg P was contained in the T P I isolated from 1 gm (wet weight) of cerebral cortex of the ox brain. Formaldehyde-treated paper for chromatography was prepared following the method of Horhammer et al. (1959) and Torda (1973a). +

+

6. Methods ii. Preparation of crude tri@hosphoinositide phosphomonoesterase (method of Thompson and Dawson, 1964). About 50 gm of fresh ox brain from the slaughterhouse was homogenized in a Waring Blendor with 300 ml of acetone (at -10°C). The suspension was filtered by suction through a Buchner funnel a t - 10°C. The residue was washed with 400 ml of acetone and 200 ml of diethyl ether at -10°C. The solid was dried in a rotary evaporator (Buchi: Rotavapor) using a 15-inch Hg vacuum created with a Nalgene vacuum pump, at 4°C collection temperature. The powder was ground in a chilled mortar, and 250 ml of water was gradually added. After standing 45 minutes at 4"C, the supernatant was collected by 20-minute centrifugation ( 104 , , ,g ,v) in a Servall refrigerated centrifuge and was dialyzed against 6 liters of distilled water a t 4°C for 10 hours. ii. Purijication of triphosphoinositide phosphomonoesterase (method of Dawson and Thompson, 1964). Forty milliliters of the extract of acetone-dried brain powder and 20 ml of 0.132 124 tris.HC1 buffer (pH 7.2) were mixed at 0°C. Solid (NH,),SO, (10.64 gm) was slowly added with stirring, and the mixture was left for 30 minutes at 0°C. After 10 minutes of centrifugation a t 0°C a t a speed of lo4 gmaxav,the supernatant was collected and was mixed with 6 gm of new (NH,),SO, until the salt completely dissolved. After standing at 0°C for 30 minutes, the precipitate was recovered by centrifugation at a speed of lo4 g,,, av. The precipitate was taken up in 3 ml of 0.132 i Z i tris . HCl buffer (pH 7.2), and the solution was dialyzed overnight at 4°C against 1 liter of 5 m M dimethylglutaric acid-NaOH buffer (pH 6.4). The yield and activity of this preparation was tested by the method described below. A 2.5- to %fold concentration of activity and 60% yield were obtained. This 25-40z saturated (NH,),S04 fraction was submitted to density gradient electrophoresis, using the LKB column electrophoresis apparatus (Svensson, 1960; Banghatn and Dawson, 1962). The light buffer was 2.5 mhf dimethylglutaric acid-NaOH and the heavy component was the same buffer in 45% (v/v) glycerol. After formation of about 2-3 cm of gradient, the mixture of the enzyme fraction and an appropriate amount of glycerol and buffer was

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

9

inserted. The electrophoresis was carried out for 16-18 hours at 850 V (5 mA) at running tap water temperature. The fractions were recovered from the columr) and were assayed for extinction at 280 nm and for enzymatic activity by the method described below. The fractions constituting the main monophosphoesterase peak were combined and stored at - 15°C. Further attempts at purification (e.g., chromatography on Sephadex G-200 column) did not significantly increase the enzymatic activity of triphosphoinositide phosphomonoesterase. The enzymatic activity was measured by the yield of one of the end products, the inorganic P. Attempts were made to prevent the formation of inorganic P from other sources, e.g., from dephosphorylation of DPI to monophosphoinositide (MPI) by the phosphodiesterase. A large part of phosphodiesterase contamination was removed by density gradient electrophoresis of the ammonium sulfate precipitate of the ox brain (Thompson and Dawson, 1964). The remaining traces of phosphodiesterase were inactivated by preincubation of the triphosphoinositide phosphomonoesterase samples with 4 m M EDTA (sodium salt) for 10 minutes at room temperature before incubation for the assay for the enzymatic activity of the triphosphoinositide phosphomonoesterase (Dawson and Thompson, 1964; Salway et al., 1967). iii. Fractionation of triphosphoinositide phosphomonoesterase into subunits. Since the product of fractionation had comparable enzymatic activity regardless of whether the crude or the purified enzyme was used as starting point, usually the crude triphosphoinositide phosphomonoesterase was used as source material. The protein content of the enzyme preparation was tested by the method of Lowry et al. (1951). Afterward, samples containing about 2.5 mg of protein were extracted with 15 ml of a mixture of chloroform: methanol ('2:1, v/v). After filtration, 7.5 ml of chloroform was added and the mixture was concentrated under nitrogen stream to a volume of 5 ml at room temperature. In order to weaken the suspected bonds of the potentially existing catalytic and regulatory subunits, the concentrate was mixed to [14C]ACh (final concentration of 5 x lo-' M ) at room temperature for 20 minutes. The mixture was chromatographed on a Sephadex LH-20 column [following the method of De Robertis (1971)l. A 2.1 x 18 cm Sephadex LH-20 column was used. The column was previously equilibrated with chloroform (Mokrash, 1967; Soto et al., 1969). The column was eluted with 80 ml of chloroform, followed by a series of mixtures of chloroform: methanol (15:1, 20 ml; lO:l, 20 ml; 6:1, 20 ml; and 4:1, 40-80 ml). The eluate was monitored with an LKB ultraviolet absorptiometer Uvicord at 280 nm, and was collected into 4-ml fractions a t a flow rate of 0.5 ml/min. The phospholipid phosphorus (Chen et d.,1956), the phospholipids (Hess and Lewin, 1965),and the [14C]ACh-dependent radioactivity [tested by Nuclear Chicago scintillation counter (De Robertis et al., 1969)l were measured in each tube.

10

CLARA TORDA

The [14C]ACh-dependent radioactivity was measured on 250-111 samples in triplicate, in standard vials, after the addition of 15 ml of Bray's solution (1960). Counting was conducted from 4 to 10 minutes, background counting for 4 minutes. The maximum deviation between the triplicate samples was 5-1070, The recovery of the bond counts was 80%. Practically all eluted radioactivity was due to protein-bound [14C]ACh. One may assume that the unbound [14C]ACh remained on the Sephadex LH-20 column, because comparable radioactivity was observed in the untreated aliquots of the eluate and in aliquots with removed free [14C]ACh. The free [14C]ACh was removed by repeated washing for 5 minutes with 1 ml of 0.32 M sucrose, and recentrifugation for 30 minutes at room temperature in a Spinco Ultracentrifuge at a speed of lo5 gmaXav.The results were expressed in dpm: 5 x lo-' M [14C]ACh corresponded to 15,000 dpm/ml. The protein-bound [14C]ACh passed into tubes 17-21 (68-83 ml) (fraction ATorpedo) from the extracts of the electric organ of Torpedo, and into tubes 40-43 (156-1 72 ml) (fraction A,) from the extracts of ox brain. The peak radioactivity was from 12 to 13.5 dpm/ml x in the extracts from in the the electric organ of Torpedo, and from 14.4 to 16.2 dpm/ml x extracts from the ox brain. The protein and [I4C]ACh measurements closely resembled the data reported by De Robertis (1971). Fractions A (tubes 17-21, Torpedo; 40-43, ox brain) and fraction B (tubes 1-16 and 22-39, Torpedo; and 44-55, ox brain) were collected and dried separately with a rotary evaporator. Similarly dried material that passed through the Sephadex LH-20 column was added to fraction B. The two fractions were diluted with phosphate or tris.HC1 buffer (pH 7.2)before use. The location of the peaks of triphosphoinositide phosphomonoesterase prepared with or without exposure to ['*C]ACh were comparable on the Sephadex LH-20 column, but the peak lacking ACh contained less organic material (Fig. 2). The specific proteolipid receptor for ACh (De Robertis, 1971) was isolated from the electric organ of Torpedo mannorata, 50-100 mg of lyophilized tissue was extracted with 1 ml of a chloroform :methanol (2:1, v/v) mixture. [l*C]ACh was added in aqueous solution in amounts from 7 x lo-' to 5 x M. After standing for 20 minutes at room temperature, the mixture was passed through a Sephadex LH-20 column. The chloroform eluate of the sharp peak of the Sephadex LH-20 column contained the AChbound specific receptor. The yield was 65 mg per kilogram of tissue. iv. Assay for the enzymatic activity of the triphosphoinositide phosphomonoesterase preparations (combined methods of Dawson and Thompson, 1964; Lee and Huggins, 1968; Salway et al., 1967; Thompson and Dawson, 1 9 6 4 ) . The triphosphoinositide phosphomonoesterase catalyzes the dephosphorylation of 1 mole of its specific substrate, the TPI, to 1 mole of DPI and 1 mole of inorganic P (Chang and Ballou, 1967; Dawson and Thompson, 1964; Prottey et al.,

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

11

Sample number

FIG.2. Chromatogram of triphosphoinositide phosphomonoesterase on Sephadex LH-20 column (ox brain). The protein content of the samples was marked on the vertical axis, the number of tubes on the horizontal axis. Each tube contained about 4 ml of eluate. The peak between the dotted lines represents fraction A. It contained proteolipid bound [14C]ACh. The peak of radioactivity coincided with the protein peak found between the dotted lines. The maximum peak of radioactivity contained 12.6 dpm/ml x (all due to the protein-bound [14C]ACh).

1968). The enzymatic activity was tested by measuring the moles of inorganic P formed per milligram of enzyme protein. A sample of a triphosphoinositide phosphomonoesterase preparation (0.005-0.2 ml), 0.3 ml of a 0.132 M tris.HC1 buffer (pH 7.2), 0.15 ml of MgCI, (to give a final concentration of 15 mM), 0.1 ml of a 0.2 M GSH (sodium salt), 0.1 ml of 40 m M EDTA (sodium salt), and water to give a final volume of 1 ml were incubated for 10 minutes at room temperature. Thereafter, the temperature was raised to 37°C and the ammonium salt of T P I was added (in a final concentration of 43 nmoles). After 30 minutes of incubation at 37°C the reaction was stopped: (1) I n samples selected for measuring the release of inorganic P as an indicator of the enzymatic activity, the reaction was stopped by adding 0.2 ml of bovine albumin (5oJ,, w/v) and 1 ml of trichloroacetic acid (10’7,, w/v) (Chen et al., 1956). (2) In samples selected for the extraction of the lipid-soluble components, the reaction was stopped by adding 0.2 ml of bovine albumin (50/,, w/v) and 10 volumes of chloroform :absolute ethanol (1 :1, v/v) (Lee and Huggins, 1968). The amounts of MPI, DPI, and TPI were measured according to the method of Hendrickson and Ballou (1964). The protein content of each sample was tested by the method of Lowry et al. (1951). The micrograms (or moles) of inorganic P released per minute per milligram of enzyme protein were calculated. Similar incubation of blanks of either the substrate or the enzyme preparations did not release inorganic P. One unit of fraction B was chosen, arbitrarily, as the amount of enzyme that liberated 1 pg of inorganic P per minute. The specific activity was defined as the number of units of enzyme

12

CLARA TORDA

activity per milligram of protein. One unit of fraction A was chosen as twice the amount that inhibits by 50'7, the enzymatic activity of 1 unit of fraction B during incubation of a mixture of fractions A and B at 37°C. Results

c.

Triphosphoinositide phosphomonoesterase removes the phosphate group from the 5 position of the inositol ring of TPI. An obligatory confactor to this enzymatic activity in uitro is MgCI,, with an optimum of 2 m M (Table I). Abrupt changes of enzymatic activity occurred in presence of MgCI, in concentrations between 1.8 and 2.0 mM. Therefore, the mechanics of the activity of this enzyme can be more profitably studied in the presence of MgC1, in concentrations of 1.5 mM. Minor changes in the concentration of MgCl, around 1.5 m M will cause only a gradual, not sudden, change. The rate of activity ofthe enzyme remained near constant for about 60 minutes. The optimum pH range was from 6.8 to 7.5 (in phosphate buffer), and 7.2 to 7.6 TABLE I EFFECT OF VARIOUS CONCENTRATIONS OF Mga , Na , K +,and ACETYLCHOLINE ON THE ACTIVITYOF CRUDEAND OF PURIFIEDTRIPHOSPHOINOSITIDE PHOSPHOMONOESTERASE B (TPIPM) AND FRACTION +

Substance Mga+

Na+

K+O

ACha

a

Conc.

0.5 m M 1.0 m M 1.5 mM 2.0 m M 2.5 m M 3.0 m M 50mM 80 m M 100 m M 125 m M 50mM 100 m M 150 m M 200 m M 10-la M lo-" M 10-lo M M M

No. of experiments

10 10 10 10 10 10 8 6 10 8

a 10 10 8

10 10 10 10 10

+

Released P,/min/mg of protein (pg P f SE) Crude TPIPM

0.020 0.043 0.070 0.550 0.612 0.641 0.106 0.163 0.141 0.118 0.156 0.300 0.326 0.297 0.120 0.154 0.175 0.191 0.199

f 0.009 f 0.023 f 0.014 f 0.090 f 0.080

f 0.090 f 0.019

f 0.022 f 0.024 & 0.018 & 0.017 f 0.019 f 0.029 f 0.025 f 0.018 f 0.014 f 0.020 f 0.021 f 0.016

Purified TPIPM

0.062 0.126 0.222 1.649 1.836 1.923 0.222 0.222 0.222 0.222 0.222 0.222 0.222 0.222 0.380 0.486 0.552 0.607 0.632

f 0.022 f 0.030

f 0.022

Fraction B

3.108 f 0.112 6.300 0.123 11.1 f 1.3

f 0.023 f 0.043 f 0.040 f 0.02 f 0.02

-+ 0.02 f 0.02

& 0.02 f .002 f 0.02 f 0.02

f 0.025 f 0.041 f 0.058 f 0.060 f 0.052

Each incubation mixture contained MgCI, in 1.5 m M conc.

11.1 & 11.1 f 11.1 +11.1 f 11.1 f 11.1 11.1 & 11.1 f 11.1 f 11.1 f 11.1 f 11.1 & 11.1 &

1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

13

(in tris-HCI buffer). The Michaelis ( K J constant for the substrate (in the presence of MgCI, at a concentration of 1.5 mM), was calculated to be 4.8 x M. The activity of the crude triphosphoinositide phosphomonoesterase significantly increased in the presence of Na+ or K + , with an optimum of 0.1-0.15 m M (Table I). Na+ or K + did not affect the enzymatic activity of the purified triphosphoinositide phosphomonoesteraseor fraction B (concentrationsfrom 0 to 0.3 m M were tested). The yield of micromoles of inorganic P per minute per milligram of enzyme protein of crude and of purified triphosphoinositide phosphomonoesterase, fraction B, fraction A, and the specific ACh-receptor isolated from the Torpedo was 2.3, 7.2, 358, 0, and 0, respectively. ACh significantly increased the activity of crude and of purified triphosphoinositide phosphomonoesterase, but did not affect the activity of fraction B (Tables 1-111). The enzymatic activity of fraction B changed with various compositions of the incubation mixtures following a pattern that suggests that fraction B functions as the catalytic, and fraction A as the regulatory, subunit of triphosphoinositide phosphomonoesterase (Tables I1 and 111): (1) Fraction A,, and fraction &orpado lacked enzymatic activity even at high concentrations. (2) Fraction B was about 50 times more potent an enzyme than the purified triphosphoinositide phosphomonoesterase preparation. (3) Incubation with fraction A (prepared without admixture of ACh) inhibited the enzymatic activity of fraction B. Keeping the amounts of fraction B fixed, the inhibition appeared to be proportional with the amounts of added fraction A. (4) Further incubation of the mixture of fractions A and B with ACh reversed the inhibition of the enzymatic activity of fraction B by fraction A. The reversal was proportional with the amounts of fraction A that formed a compound with the newly added ACh. In these experiments, labeled ACh was added to the already incubated mixture of fractions A and B, and the amounts of ACh combined with fraction A was studied at the end of the incubation period (measured by rechromatography on Sephadex LH-20). The [14C]ACh content of the eluates was measured by means of scintillation counting. The unbound [14C]ACh was retained in the Sephadex LH-20 column (De Robertis, 1971). Since the ACh binds to fraction A, by comparing the values obtained for the enzymatic activity and the values of the bound added ACh, one can ascertain the amounts of the gain of the enzymatic activity of fraction B. The collected data suggest (Tables I1 and 111) that ACh increases the enzymatic activity only in mixtures where fraction A inhibits the activity fraction B (e.g., crude and purified enzyme preparations, and mixtures of fractions B and A (prepared without ACh)). Acetylcholine did not affect the enzymatic activity of fraction B (Tables I1 and 111).

ic.

ENZYMATIC ACTIVITY OF

No. of

Incuba-

THE

TABLE I1 VARIOUS TRIPHOSPHOINOSITIDE PREPARATIONS AND ITS CHANGES (VARIOUS INTERVENTIONS)

Specific enzymatic activity

experi- tion time pg Plminlmg protein ments (min) & SE of the means 20 20 20 20 20

20 20 20 30 25 20 20 30 27 27 25

30 30 30 30 30 30 30 30 30 30 30 30 30

0.222 f 0.020 0.248 f 0.024 0.682 f 0.037 0.290 f 0.024 0 0 0 0 11.100 f 1.3 10.700 f 1.0 9.100 f 0.9 3.400 f 0.8 9.200 f 0.7 30 3.700 f 0.8 30 3.700 f 0.8 15 1.700 f 1.1 15 Contin. 7.200 f 1.3

yoof control 100 112 307 136

Purified TPIPMQ TPIPM TPIPM TPIPM TPIPM

Fraction B ox brain (units)

Fraction Ab (units)

Acetylcholine d-Tubocurarine (moles) (moles)

1 x 10-8 1 x 10-6

-

5000

-

-

I

-

100 96 82 31 82 33 33 15 15

-

-

100 405

ACh

-

TPIPM = triphosphoinositide phosphomonoesterase. (1) Fraction A,, from ox brain obtained without first exposing the brain to acetylcholine to loosen bonds between the subunits before chromatography on Sephadex LH-20 column. (2) Fraction A,, mixed with acetylcholine before chromatography on Sephadex LH-20 column. (3) Fraction ATorpsaofrom the electric organ of Torpedo marmorata, prepared without exposure to acetylocholine before chromatography on Sephadex LH-20. (4) Fraction ATorpeao from the electric organ of Torpedo marmoratu, exposed to acetylcholine before chromatography on Sephadex LH-20.

cp

15

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

TABLE I11 EFFECTOF FRACTIONA AND/OR ACETYLCHOLINE ON THE ENZYMATIC ACTIVITY OF FRACTION Ba

Composition of the incubation mixtures No. of Fraction Fraction experiB (units) A (units) ments

P1released in 15 min (PI31

ACh added after 15 min incubation (10-'W

1 1 1 1 1

50 10 10 10 10

15.00 f O.lOb 0 11.15 k 0.12 7.32 k 0.16 3.70 k 0.14

0

1 1 1 1 1

50 10 10 10 10

15.00 f 0.10 15.00 f 0.10 7.32 f 0.16 7.32 f 0.16 7.32 f 0.16

0 12x 0 3x 6x

0 0 0 0

Pi released by the end of 30 min (Pd

PI released by the end of 45 min

0.25

45.00 f 0.31 0 33.75 f 0.29 22.79 0.27 11.00 k 0.16

30.00 f 0 22.27 f 15.19 k 7.56 f

0.19 0.22 0.18

30.00 f 0.25 29.08 5 0.38 15.19 f 0.22 16.50 0.09 18.17 _+ 0.20

45.00 44.01 22.79 24.70 27.25

f 0.31

k 0.45 & 0.27 f 0.23 f 0.31

aCf. Fig. 1 and the text. Average value, followed by standard error of the means.

Attempts were also made to ascertain the amount of ACh required to create saturation of 1 pg of the receptor protein. Sets of mixtures were chromatographed containing the same fixed amount of receptor protein. [14C]ACh was added in increasing concentrations. When the [14C]ACh content of the eluate of the Sephadex LH-20 column remained fixed during further increase of the added [14C]ACh, the saturation point of the receptor protein was reached. At full saturation 4950 to 5050 dpm (due to [14C]ACh) were bound to 1 pg of protein; 4950 dpm corresponded to 1.65 x lo-" M of [14C]ACh per milliliter. These observations confirm the findings of De Robertis et al. (1969, 1971; La Torre et al., 1970). Further extrapolation of the saturation curves also confirms the suggestion of De Robertis et al. (1971) that the receptor protein has multiple binding sites for ACh. De Robertis (1971) succeeded in ascertaining the dissociation constants for two types of M. the binding sites: (1) K1 = 1 x lo-' M , and (2) K 2 = 1 x The enzymatic activity of fraction B from ox brain was inhibited by a comparable amount by fraction A,, and by the specific acetylcholine receptor isolated from the electric organ of the Torpedo marmorata (fraction ATorpodo). This observation substantiates the assumption that the regulatory subunit of triphosphoinositide phosphomonoesterase (fraction Ao,) is the specific receptor (nicotinic) of acetylcholine.

16

CLARA TORDA

The nicotinic transmission of cholinergic synapses is blocked by curare (Curtis and Ryall, 1966; Del Castillo and Katz, 1957; Eccles et al., 1954, 1956; Langley, 1918; Longo el al., 1960; Unna et ul., 1944). I t seemed appropriate, therefore, to observe the effect of d-tubocurarine on the enzymatic activity of triphosphoinositide phosphomonoesterase. A4 to the purified [14C]Tubocurarine, added in amounts of 1 x enzyme before chromatography, for 20 minutes, appeared on the Sephadex LH-20 column bound to the receptor of ACh (fraction A) (see also Azcurra and De Robertis, 1967; De Robertis, 1971). The affinity of the receptor is significantly greater for d-tubocurarine than for ACh. Furthermore, on combining with d-turbocurarine, the regulatory subunit (fraction A) continues to inhibit the enzymatic activity of the catalytic subunit (fraction B) (Torda, 1973a) (Table 11). This lack of activation was attributed to the larger size of the d-tubocurarine molecule (Smythies, 1971), and seems to result in the curare block of the nicotinic transmission. Because of the differences in activation of fraction B, the differences of the affinity of acetylcholine and d-tubocurarine to the receptor protein (fraction A) is easy to test. I n proportion with the added fraction A, the enzymatic activity of fraction B is inhibited during incubation of a mixture of fractions A and B. The inhibition continued in the presence of d-tubocurarine. Acetylcholine added in amounts that were sufficient to displace d-tubocurarine, suddenly increased the enzymatic activity of the fractions A and B mixture.

d. Discussion Isolation of the nicotinic receptor(s) is a difficult task (Beychock, 1966). T h e nicotinic receptor(s) isolated by various researchers seem(s) to contain proteins of a small molecular weight (42,000, 45,000) and a t least one protein with a larger molecular weight (near 300,000) (Changeux, 1972; De Robertis, 1971; Fulpius et al., 1972; Karlin, 1972; Potter, 1972). The molecular weight of fraction A,, seemed to lie between 42,000 and 45,000, and that of the enzymatically active component of fraction B was near 300,000. Triphosphoinositide phosphomonoesterase seemed to contain a catalytic and a regulatory subunit because (1) the catalytic activity of fraction B exceeded that of the purified enzyme; (2) incubation with fraction A decreased the enzymatic activity of fraction B. The regulatory subunit (fraction A) had a greater affinity for ACh than for the catalytic subunit (fraction B constituent) : (a) added ACh significantly facilitated the fractionation of the enzyme; (b) ACh reversed the inhibition of the enzymatic activity of the catalytic subunit by the regulatory subunit.

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

17

The identity of the nicotinic receptor and the regulatory subunit is suggested by the observations that (1) the enzymatic activity of the catalytic subunit (contained in fraction Box)was inhibited to a comparable degree by the regulatory subunit (fraction A,) and by the nicotinic receptor isolated and tested by De Robertis (1971) and De Robertis et al. (1'969, 1971) ; (2) the ACh binding properties of fraction A,, and (fraction ATorpedo) fraction ATorpeao were very close; (3) the d-tubocurarine-binding properties of fraction A,, (Torda, 1973a) and fraction &orp&, (De Robertis, 1971) were very close. The activation of triphosphoinositide phosphoAonoesterase by ACh and the dephosphorylation of T P I to DPI seem to occur with a speed that meets the time requirements of synaptic transmission. The shortest measured delay in chemically transmitting synapses was 0.2 msec. Since it depends, essentially, on the rate of diffusion of ACh, the ACh-receptor complex may be formed in less than 0.1 msec (Ehrenpreis et al., 1969; Kabachnik et al., 1971). Activation of the enzyme does not seem to require added time, since the regulatory subunit ceases to inhibit the enzymatic activity of the catalytic subunit during binding on ACh. The maximum turnover rate of T P I was measured at 14/msec. e.

Conclusions

The biochemical experiments led to the conclusion that one of the postsynaptic nicotinic receptors of ACh is the regulatory subunit of triphosphoinositide phosphomonoesterase. When united, the regulatory subunit inhibits the enzymatic activity of the catalytic subunit. Because of great affinity of ACh, the regulatory subunit combines with locally available ACh and ceases to inhibit the enzymatic activity of the catalytic subunit. The catalytic subunit speeds the dephosphorylation of triphosphoinositide to diphosphoinositide, a process that concurs with generation of a molecular chain reaction that leads to depolarization of the postsynaptic neuron (see following sections).

2. Bioelectric (in Vivo) Measurements The possibility of a casual relationship between changes in the activity of triphosphoinositide phosphomonoesterase and generation of membrane depolarization was tested on the sympathetic ganglia of the frog and the rabbit, and the Renshaw cell of the cat. The results seem to validate the assumption that activation of triphosphoinositide phosphomonoesterase by ACh may be one of the molecular mechanisms that is able to couple in time and space the formation of the ACh-specific receptor complex and depolarization of the postsynaptic neuron.

18

CLARA TORDA

a . Materials Acetylcholine, d-tubocurarine, dehydro-/3-erythroidine, and physostigmine salicylate were commercially obtained. They were administered in concentrations of 1 x 10-5 to 1 x lo-" M. I n order to protect acetylcholine from hydrolysis, administration of physostigmine in about 5 times higher concentrations preceded the administration of ACh. Preparation of the purified triphosphoinositide phosphomonoesterase and its catalytic and regulatory subunits were described in the previous chapter (Torda, 1973a). Usually, less than 5 units sufficed to generate a bioelectiic response. 6. Methods i . Preparation of the superior cervical ganglion of the rabbit (combined methods of Eccles, 1952, 1955; Eccles and Libet, 1961; Libet, 1970; Libet and Tosaka, 1 9 6 8 ) . Rabbits of both sexes, of 4-5 kg body weight, were anesthetized with urethan (100 mg/kg, i.v.). The presynaptic trunk was severed a t the neck, and the postsynaptic trunk at the skull. The two trunks were fastened to suction pipettes mounted in a special chamber containing 30 ml of solution at 37°C. or occasionally at 20°C. The bathing solution contained NaCl (136 gm), KC1 (5.6 gm), NaHCO, (20 gm), NaH,PO, (1.2 gm), and glucose (5.5 gm) per liter at p H 7.2. A gas mixture (950/, of 0, and 5% of CO,) was bubbled through a side-tube providing both an adequate circulation and oxygenation of the immersion fluid. The chamber was suspended in a large box containing water and a heating element. The temperature was monitored with a thermistor probe that was suspended near the ganglion. The presynaptic trunk was immersed in mineral oil. Only B fibers were used. The B fibers were selected by their conduction velocity averaging 20 f. 1.5 msec. ii. Preparation of the paravertebral sympathetic chain of the bullfrog (combined methods of Blackman et al., 1963; Koketsu et al., 1959, 1968; Nishi and Koketsu, 1960, 1967, 1968; Nishi et al., 1967; Riker, 1964; Weight, 1 9 7 2 ) . The 6th ramus communicans (containing only B fibers) was used for preganglionic stimulation, the 10th spinal nerve was used as the postganglionic neuron. After removal of connective tissue under the dissecting microscope, the preparation was transferred into the main compartment of the recording Lucite chamber. The reservoir compartment contained the bathing solution at 24-26°C. The solution contained NaCl (1 12 gm), KC1 (2 gm), CaC1, (1.8 gm), NaHCO, (2.4 gm), glucose (2 gm) per liter at a p H of 7.2. It was oxygenated and circulated by a gas mixture containing 5'7, of 0, and 95y0 of CO,. A strip (3 x 7.5 mm) of filter paper was placed beneath the sluice gate in the front wall of the chamber. This carried the bathing fluid from the reservoir to the main compartment. A small hole was punched with an 18-gauge needle in the filter paper midway in its traverse of the main compartment, and the 8th ganglion was positioned beneath the hole. T h e

A

DEPOLARIZATION-HYPERPOLARIZATION CYCLE

19

preganglionic chain and the spinal nerve extended into mineral oil on opposite sides of the strip of filter paper. Darkfield illumination of the ganglion and high magnification of the dissecting microscope permitted visualization of individual cells for placement of the microelectrode tip prior to attempted impalement. iii. Preparation of the Renshaw cell of the cat (com6ined methods of Curtis and Ryall, 1966; Eccles, 1969; Haase and Meulen, 1961; Long0 et al., 1960; Ryall, 1970; Schibel and Schibel, 1966; Weight and Salmoiraghi, 1966a,b). Cats of both sexes, of 2 4 kg body weight, were anesthetized by pentobarbitone sodium (35 mg/kg, i.p.), or chloralose (50 mg/kg i.v.). Most cats were immobilized by gallamine trichloride to reduce movement artifacts. Gallamine had no obvious effect on the evoked firing or the spontaneous activity of the Renshaw cell. The cats were artificially ventilated through a trachael cannula by a mixture of 5'7, of 0, and 957, of CO,. End-tidal CO, was monitored on a Beckman CO, analyzer and was maintained at about 4'7,. The blood pressure was monitored with a Statham gauge transducer (P 23 Db) through a Teflon cannula inserted into the right common carotid artery. The spinal cord was exposed by laminectomy including the L, to L, vertebrae, and was transected at the level of L, and below S,. The left dorsal roots were severed up to L,, resulting in a complete deafferentation of the limb. The exposed spinal cord and nerves were covered by paraffin oil at 37°C. The body temperature was maintained a t 37.5 +_ 0.5"C by heating paths and a n infrared lamp controlled by a thermistor inserted intrarectally. Antidromic stimulation of the median gastrocnemius nerve corresponded to presynaptic stimulation. Stimulation. The presynaptic neuron was stimulated with bipolar platinum wire electrodes. Rectangular pulses of 0.5-0.8 msec duration, an intensity ranging from subthreshold to supramaximal, and a frequency of I /minute were generated by Tektronix Instruments (Series 160, 161, 162), and were delivered through an isolation unit (Tektronix) . To measure membrane resistance, and to polarize the cell membrane, pulses of constant current were delivered through the recording microelectrode from a bridge arrangement (Araki and Otani, 1959; Ginsborg and Guerrero, 1964). With an electrode tip in the extracellular space before penetrating the cell, the bridge was balanced so that the applied currents produced only a minimum deflection of the base line. The degree of balance was rechecked after withdrawing the electrode at the end of the experiment. The electric constants of the resting postsynaptic membrane were measured by applying depolarizing and hyperpolarizing square pulses of various intensities through the intracellular recording electrodes. 5 x 10 -lo A applied current usually generated depolarizing and hyperpolarizing potentials of a similar magnitude and time course (Coombs et al., 1955; Cole, 1968; Frank, 1961; Frank and

20

CLARA TORDA

Fourtes, 1955, 1956; Furshpan and Potter, 1959; Nelson and Frank, 1967).

Recording. Extracellular recordings were made through platinum microelectrodes prepared by the method of Wolbarsht et al. (1960). Intracellular recordings were made through glass microelectrodes (of 0.5 pm tip diameter, from 10 to 20 megohm tip resistance, filled with 3 M KC1 for the frog and 4 M KC1 for the rabbit and cat). The extracellular electrodes were connected directly, the intracellular ones through a cathode follower to an oscilloscope (Tektronix 561,3A3). T h e final position of the electrode tips were ascertained in preparations selected a t random by histological tests (Thomas and Wilson, 1966). I n the frog and rabbit, the cells were located by lowering the electrodes by micromanipulators. I n the cat a group of Renshaw cells was first located by extracellular electrodes. The evoked potentials were monopolarly recorded from the surface of the spinal cord against an indifferent electrode, during antidromic stimulation of the median gastroenemius nerve. A site of the surface of the spinal cord was located where the repetitive surface potential (Renshaw ripple) was the largest. The Renshaw ripple is characteristic for the synchronous discharge of a larger population of Renshaw cells (Eccles et al., 1961; Renshaw, 1941, 1946; Wilson, 1959; Wilson et al., 1962, 1964). The postsynaptic potentials were followed by intracellular recordings. The resting membrane potential was measured between the intracellular recording microelectrode and an indifferent electrode in contact with the extracellular fluid. The electric constants of the resting membrane were ascertained by applying depolarizing and hyperpolarizing square pulses of various intensities through the intracellular electrode. About 10 mV depolarization was generated by a current of 5 x A, Weaker currents generated depolarizing and hyperpolarizing potentials of similar magnitude and time course (Coombs et al., 1955; Cole, 1968; Nelson and Frank, 1967). The equilibrium (reversal) potential was ascertained by applying a stimulus of the same intensity to a cell with changing resting membrane potential. At the equilibrium potential, the stimulus did not elicit any response. The stimulus elicited response of opposite directions when the membrane potential was changed both ways. Microinjections. The various substances were injected near the synapse through glass micropipettes (about 1 pm of tip diameter) by the combined use of pressure and electrophoresis (Curtis, 1964; Krnjevic et al., 1963; Salmoiraghi and Stefanis, 1967). The electrodes were filled according to the method of Ito et al. (1962) and of Nastuk (1953). Pressure was applied by connecting the upper end of the pipette to a source of compressed air and measuring the pressure with a mercury manometer. The ejected volumes were calibrated by examining the emerging drop under oil. Electrophoresis required passing

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

21

a current (e.g., 30 nA, 3-30 msec) between the solution and an indifferent nonpolarizable electrode connected to the external medium. During the flow of the current, the potential difference was maintained near-constant by connecting the drug barrel to the potential applying polarizer through a resistance of either 100 or 500 megohms, located close to the electrode. The circuit diagram of Curtis (1964, p. 164) was used. The immersion fluids and solvents injected by the same method did not generate measurable changes in the bioelectric processes of the cells under the experimental conditions used.

Experimental procedure. CONTROLS. The presynaptic neuron was stimulated by supramaximal rectangular pulses of 1 per minute frequency. The postsynaptic response that remained near-constant for at least 5 minutes was taken as control. Since spontaneous recovery followed every experimental intervention in less than 30 minutes, each preparation served as its own control. EXPERIMENTS. The bioelectric responses of the postsynaptic neuron were observed during the following interventions: (1) presynaptic stimulation with rectangular pulses of decreasing intensity, (2) near-synaptic microinjections of (a) ACh, (b) d-tubocurarine; (3) dehydro-p-erythroidine; (4) catalytic subunit of triphosphoinositidephosphomonoesterase; (5) regulatory subunit; (6) the substances in various combinations; (7) presynaptic stimulation and the substances in various combinations; ( 8 ) various combinations of subthreshold amounts of presynaptic pulses, ACh, and the catalytic and regulatory subunits. Each type of experiment was observed in over 10 separate preparations. The elicited postsynaptic bioelectricresponse was measured and was compared with the value obtained in the control. c. Results

i. Bioalectric observations obtained from postsynaptic neurons (controls) ( Tables ZV-WZ, Fig. 3). In the frog the resting membrane potential averaged -65 f 1.7 mV, the time constant: 10.6 f 0.5 msec. The effective membrane resistance was calculated as 29 f 1.5 megohm. The equilibrium potential averaged -10 mV. In most cells the voltage vs current curve yielded a linear relationship with a slope near 26 megohms. Presynaptic pulses of supramaximal intensity and 1 per minute frequency generated with an average latency of 1 f 0.3 msec (minimum of 0.6 msec) a postsynaptic activity consisting of postsynaptic depolarization (EPSP) and spiking. The summit of the EPSP averaged 26 mV and was reached in about 3 msec. The maximum rate of rise was from 25 to 32 V fier second. The decay approximated an exponential curve, with the time constant averaging 12.1 f 1.O msec. The time constant of the decay of postsynaptic depolarization exceeded by 2.4 f 0.7 msec the decay of the membrance potential

22

CLARA TORDA

u

Y

> +A-A *Lwrrc --_

A

_._ /_ _

/ 4

-- -- - -

I&--

.._--_-_ -_------

I

----/

- -- -

u

- - - -- - -

Jd---A:-JL JC

-JL_ 1401&c

l-

s--

FIG.3. Effects of changes of the activity of triphosphoinositide phosphomonoesterase (TPIPM) on depolarization of the postsynaptic neuron (intracellular recordings). (A) Superior cervical ganglion of rabbit. Column I : Presynaptic stimulation of various intensity generated postsynaptic responses-additive effects of two types of subthreshold stimuli. Row 1 : Postsynaptic response to supramaximal presynaptic pulse. Rows 2 and 3: Arrow: Decrease of stimulus intensity resulted a decrease of response. (Changes occurred in order: slope of EPSP decreased; latency of spike increased-EPSP remained below threshold of spike generation; no spiking-EPSP further decreased.) Row 4: Arrow: Injection of subthreshold amounts of catalytic subunit of TPIPM. Postsynaptic depolarization returned as if supramaximal stimuli were delivered to presynaptic neuron. of postsynaptic Column 11: Effect of catalytic subunit of TPIPM-Generation depolarization. Row 1 : Postsynaptic response to supramaximal presynaptic pulses. Arrow: Suspension of presynaptic pulses. Injection of the catalytic subunit in increasing concentrations. Rows 2-4: Postsynaptic response gradually increased, and with doses even below 5 units full postsynaptic depolarization was generated. Column 111: Inhibition by the regulatory subunit of the depolarization generated by the catalytic subunit. Row 1: Arrow: Postsynaptic depolarization generated by injection of the catalytic subunit. Double Arrow: Injection of regulatory subunit in increasing concentrations. Rows 2 and 3: Decrease of postsynaptic response to the catalytic subunit in the presence of the regulatory subunit. Dashed line: 30-Minute recovery period. Row 4: Spontaneous recovery occurred : Catalytic subunit generated full postsynaptic depolarization. Therefore, the preparation was still in good functioning condition.

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

23

generated by a current pulse, suggesting the involvement of added molecular processes (e.g., transmitter decay). The depolarization of the postsynaptic membrane was usually completed in 20 msec and was not followed by an afterpotential. The threshold for spike generation averaged 25.5 f 1.3 mV. The peak was completed in 1.2 & 0.4 msec. The spike duration averaged 3.7 f 0.9 msec. The spike potential overshoot averaged 15 mV. The height depended on the resting membrane potential, and averaged 82 f 2.1 mV (min, 75; max, 108 mV), with a membrane potential of -68 mV. The spike was followed by an afterpotential (average height: 6 mV). Presynaptic stimulation with pulses of decreasing intensity generated an EPSP that remained below 25.5 mV, the critical amount for spike generation. As long as spikes were generated they remained unaltered, except for an increase of the latency during stimulation with weaker pulses. Column IV: Inhibition by the regulatory subunit of postsynaptic depolarization by presynaptic supramaximal pulses. Row 1: Postsynaptic depolarization generated by presynaptic supramaximal pulses. Double arrow: Injection of the regulatory subunit in increasing amounts (up to 5 units). Rows 2 and 3: Inhibition of postsynaptic depolarization. Dashed line: 30 Minute recovery period. Row 4: Full postsynaptic depolarization recurred, suggesting that the preparation was in good functioning condition. (B) Renshaw cell of cat. Column I : Effects of decreased presynaptic pulses-summation of subthreshold stimuli. Rows 1-3 : Presynaptic pulse intensity decreased from supramaximal to nearthreshold. Postsynaptic response decreased. Row 4: Arrow: Injection of subthreshold amounts of catalytic subunit: Full depolarization. Column 11: Depolarization by catalytic subunit. Row 1 : Supramaximal pulses to presynaptic neuron. Presynaptic stimulation was suspended. Row 2: Arrow: Injection of catalytic subunit: Full response. Row 3: Double Arrow: Injection of regulatory subunit with catalytic subunit: Inhibition. Row 4: Dashed line: 30-Minute recovery. Presynaptic stimulus elicited full depolarization. Column I11 : Inhibition by regulatory subunit of postsynaptic depolarization by presynaptic pulses (supramaximal intensity). Row 1: Postsynaptic depolarization by presynaptic supramaximal pulse. Rows 2 and 3: Double arrow: Injection of regulatory subunit in increasing concentrations: Inhibition of postsynaptic depolarization. Row 4: After 30 minutes recovery (dashed line) full postsynaptic depolarization occurred.

24

CLARA TORDA

-

I n the rabbit, the resting potential averaged - 80 i: 1.8 mV (min, 65; max, -90 mV). The voltage vs current relationship was linear, with a slope of22 megohms. The time constant averaged 30 msec. The equilibrium potential averaged - 7 mV. Presynaptic stimulation with supramaximal rectangular pulses of 1/minute frequency generated postsynaptic activity with an average latency 2.8 f 1.2 msec (minimum of 1.8 msec). Postsynaptic depolarization reached a summit of 23 _+ 2.4 mV in about 4.4 a 1.3msec. The decay approximated an exponential curve, with a time constant averaging 14.3 5 1.8 msec. The depolarization was completed in less than 30 msec. It was not followed by an afterpotential. With weaker stimuli the summit remained below 23 mV, the critical value of spike generation. The rising slope was less steep, and the latency increased. The threshold for spike generation was 23 5 2.1 mV. The height of the spike averaged 100 f 2.6 mV (min, 75; max, 110 mV). The overshoot averaged 13 mV. The peak was

I0

20

FIG.4. Extracellular records from Renshaw cell. Effects of single stimuli of various intensities in noncurarized cat. Observations on the effect of curare. Graph: Analysis of the response of the Renshaw cell to a stimulus of supramaximal intensity. Ordinate: Number of spikes that have the same frequency. Abscissa: Frequency of each spike. Column I : Single presynaptic pulse of increasing intensity generated a response with increasing number of spikes: From Row 1 (low intensity) to Row 4 (higher intensity). Column 11: Effect of dehydro-@-erythroidine on the response of the Renshaw cell to various stimuli (0.1 mg/kg of dehydro-@-erythroidine,i.v.). Control: Noncurarized cell would respond as recorded on Column I, Row 4. Row 1: Curarized cell, injection of acetylcholine (1 x 10-g M). Row 2: Curarized cell, injected with acetylcholine (1 x lo-' M). Row 3: Curarized cell. Presynaptic neuron stimulated with pulses of intensity recorded in Column I, Row 3. Row 4: Same preparation, injected with the catalytic subunit of triphosphoinositide phosphomonoesterase. Cell responded in full as though it were not curarized.

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

25

reached in 3 msec. Spike duration averaged 6.1 1.2 msec (min, 4; max, 7 msec). The height of the spike depended on the resting membrane potential. The spike was followed by an afterpotential with an average height of 5 mV (max, 15 mV). In the Renshaw cell of the cat, the bioelectric processes recorded with extracellular microelectrodes resembled the observations of past researchers (McLennan, 1970).A single antidromic volley (Fig. 4) generated a repetitive response: a brief intense phase of spikes (up to 2 msec), followed by a residual potential from 50 to 80 msec in length. The latency of the first spike was 0.7 to 0.9 msec. The firing rate of the first wlses averaged 1500 per second. The first spike was followed by 1-20 spikes, spaced into a regular pattern at progressively increasing intervals. The first spike was tested to be a postsynaptic response (Eccles, 1969; Ryall, 1970). A gradual decrease of the stimulus intensity increased the latency. The firing rate of the peak became 4 msec. This early response of the Renshaw cell involved nicotinic receptors (Curtis and Ryall, 1966). The early response of the Renshaw cell became significantly attenuated after administration of low doses of dehydro-p-erythroidine (from 0.05 to 0.2 mg/kg i.v.), or near-synaptic microinjections of small amounts of dehydro-/3M). Larger amounts of dehydro-jl-erythroidine erythroidine (e.g., 1 x (e.g., 0.7 mg/kg i.v.) reduced the number of spikes to about one-third of the original response; 2 mg/kg i.v. abolished all spikes, except for the first one. The basic values of the bioelectrical processes obtained through intracellular recordings were as follows : the resting membrane potential averaged - 66 mV (min, - 53; m a , - 75 mV). The time constant averaged 3.6 msec. The equilibrium potential averaged -1 mV. In most cells the voltage vs current curve yielded a linear relationship with a slope of 22 megohms. The threshold for spike generation averaged 15 mV. The spike duration averaged 3.5 msec. The height depended on the resting potential. The overshoot averaged 14 mV. The spike was followed by afterpotential (average of 5 mV). The summit of the PSP averaged 15 mV and was reached in about 1.2 msec. The maximum rate of rise was 32 per second. The decay approximated an exponential curve, with a time constant of half decay averaging 3.5 msec. The residue usually lasted for a few milliseconds and was probably the reason for the repetitive discharge of the Renshaw cell. It was not followed by an afterpotential. Antidromic pulses of supramaximal intensity (endogenous ACh) or near-synaptic microinjections of acetylcholine (average of 1 x M) generated with a latency of 0.7 to 0.9 msec a postsynaptic response consisting of EPSP and spiking generated at a critical value of 15 mV. A gradual decrease of the stimulus intensity generated a gradual increase of the EPSP period, with a slower slope of the rising phase of the EPSP, and a prolonged

26

CLARA TORDA

latency of the spike. A further decrease of the stimulus intensity generated only EPSP with a summit below 15 mV; this was insufficient to reach the critical value for spike formation. ii. Efects of the catalytic subunit on postsynaptic bioelectric processes (Tables IV- Vrl, Fig. 3 ) . Near-synaptic microinjections of the various substances generated comparable changes in the postsynaptic bioelectric processes in all three preparations (the sympathetic ganglia of frog and rabbit, and the Renshaw cell of the cat). Microinjections of the purified triphosphoinositide phosphomonoesterase and the catalytic and regulatory subunits did not generate measurable changes in the bioelectric characteristics of the resting membrane (Table IV), except for some decrease of the membrane resistance in presence of the catalytic subunit in near threshold concentrations. Comparable postsynaptic bioelectric processes were generated by presynaptic TABLE IV EFFECTS OF NEAR-SYNAPTIC MICROINJECTIONS OF THE VARIOUS ENZYME PREPARATIONS ON SOMEBIOHI.ECTRIC PROPERTIES OF THE RESTING MEMBRANE OF FROG (PARAVERTEBRAL SYMPATHETIC GANGLIA) AND CAT ( RENSHAW CELL)^ Enzyme preparationb Animaf

P

C

R

Resting mem- Resting memNo. of experiments brane potential brane resistance (mV) (megohms) Preparations Cells

-

Triphosphoinositide phosphomonoesterase Frog oc 0 0 -65 f 1.7' + o o -68 f 3.2 o + o -63 f 3.1 o o + -66 f 2.9 Cat OC 0 0 -66 f 1.9 + o o -65 f 2.6 o + o -62 3.0 o o + -67 f 2.8 Diphosphoinositide kinase Frog oc 0 0 + O O

Cat

o

+

o

0

0 0 0

0 0

oc

4

o

+

0

0

+ o

+

-65 -68 -69 -67 -66 -64 -69 -63

+-

1.7

f 2.9 f 2.5 f 2.0

& 1.9 2.7 f 2.0 f 2.6

+

29 27 22 28 3.4 3.2 2.6 3.5 29 27 34 28 3.4 3.3 4.2 3.5

f 1.4

+ 2.8

f 3.1

+ 2.7

f 0.18 f 0.22 f 0.25 f 0.19 f 1.4

& 1.9 f 3.0 f 2.2 f 0.18 f 0.20 f 0.19 f 0.18

40 19 24 23 36 12 12 12

212 100 118 108 109 40 48 48

30 20 24 23 30 12 12 12

120 96 110 80 98 50 46 47

First the amounts were established that generated a measurable bioelectric effect (threshold concentration) ;afterward subthreshold amounts were used in theseexperiments. P: purified enzyme; C: fraction B: catalytic subunit; R : fraction A: regulatory subunit. Controls. Average followed by the standard error of the mean.

TABLE V OF CHANGES OF RESTING MEMBRANE POTENTIAL ON EPSP AND SPIKE-THE EQurLmRxm POTENTIAL EFFECTS Presynaptic pulses of supramaximal intensity

No.of Membrane Animal Frog

Rabbit Cat

potential" (mV) -65 -70 -30 -15 -12 -2 +15 +30 -80 -8 -66 -30 -20 -1 +1 +15 +30

f 1.7' f 1.9 f 2.0

4 0.8 f 0.4'

f 0.8

+, 1.3 f 1.5 f 3.0 f 0.5d

f 1.9

f 0.7 f 0.8

+, 1.0' f 0.6 f 1.1 f 1.3

experiments Preparations 25 15 12 10 16 12 12 12 25 25 25 15 15 25 15 15 15

Cells 100 68 50 49 74 50 50 50 110 80 100 66 70 125 72

70 76

TPIPM: Injected catalytic subunitb

No. of experiments EPSP summit (mV) 26 f 2.8 26 f 2.8 14 &- 1.9 6 f 1.1 0 f 0.7 -4 f 1.0 -12 f 1.2 -30 f 0.9 23 f 2.4 0 f 0.4 16 f 2.1 8 f 1.0 5 f 1.2 0 f 0.6 -3 f 0.7 -7 & 1.0 -14 f 1.8

Spike peak (mV) 82 f 82 f 0 0 0 0 0 0 100 f 0 80 f 0 0 0 0 0 0

2.1 2.1

2.6 2.8

Preparations 25 16

14 11 20 12 12 12 26 26 27 13 12 25 14 14 14

Cells 110 60 50 50

80 48 48 46 70 71 96 50 50 81 46 48 50

9

ESP summit (mV) 26 f 2.0 26 f 2.0 16 f 1.5 5 f 1.0 0 & 0.7 -5 &- 0.8 -14 2 1.4 -28 f 1.6 23 f 5.5 0 16 f 2.6 8 f 2.2 4 f 1.3 0 f 0.6 -2 f 0.9 -7 f 1.4 -14 f 2.0

Spike peak (mv) 85 85 30 0 0 0 0 0 90 0 80 0 0 0 0 0 0

f 2.4 f 2.4 f 1.2

w

$ ti Ki

9

=!

7 X

5m

f 4.2

0

+, 3.5

Li

FN 9

=!

i2

n

Ic n P

m

" Changes of resting membrane potential were generated through constant current through intracellular electrode. c

TPIPM = Triphosphoinositide phosphomonoesterase. Average followed by the standard error of means. Equilibrium potential.

N

v

28

CLARA TORDA

stimuli, injectionsof ACh and the catalytic subunit in supramaximal amounts. The equilibrium potentials obtained for the three depolarizing agents were comparable. Subthreshold amounts of any two of the three depolarizing agents were additive in generating a postsynaptic response (Tables V-VII, Fig. 3 ) . iii. Effects of the catalytic subunit on denervated preparations. The presynaptic chain leading to the 10th parasympathetic ganglia of the bullfrog was removed 2-5 days before the experiments. Nearsynaptic microinjections of the catalytic subunit generated postsynaptic depolarization in a comparable manner to the depolarization in intact ganglia. Therefore, the catalytic subunit has a direct effect on the postsynaptic neuron. Conclusive evidence is lacking regarding the possible presynaptic effects of the catalytic subunit. iu. Dzrerences of the postsynaptic bioelectric processes generated by the catalytic subunit, acetylcholine, or presynaptic pulses. Postsynaptic depolarization and spike generated by any one of the three depolarizing agents were comparable or identical. At times the EPSP generated by the catalytic subunit had a longer latency and prolonged duration. This difference was attributed to the several uncontrollable factors innate in the experimental technique ; e.g., the catalytic subunit may have been injected at a distance from the site of action, its delivery rate could not be restricted to amounts that would correspond to quanta, one could not terminate an ongoing diffusion process, etc. u. Effects of the regulatory subunit on postsynaptic bioelectric processes. The regulatory subunit of the triphosphoinositide phosphomonoesterase did not affect the bioelectric characteristics of the resting membrane, if injected at subthreshold concentrations (Table IV). Microinjections of the regulatory subunit (tried up to 15 units) did not generate postsynaptic depolarization or spiking. If injected during presynaptic pulses, or administration of ACh, or the catalytic subunit, the regulatory subunit inhibited the postsynaptic response generated by one of the three depolarizing agents to a comparable degree (Tables V-VII, Fig. 3). First the latency of spike generation increased, then spiking disappeared, and finally the EPSP decreased to zero. vi. Effect of curarizing agents (Tables II and VZI, Fig. 4 ) : Curare blocks the nicotinic effects of ACh (Curtis and Ryall, 1966; Eccles et al., 1954, 1956, 1961; Longo et al., 1960). This block may be overcome by administration of several multiples of ACh. This effect of curare results (1) from the greater affinity of the common receptor to curare than to ACh and (2) because, on combining with curare, the regulatory subunit of triphosphoinositide phosphomonoesterase continues to inhibit the catalytic subunit, and the molecular chain reaction leading to postsynaptic depolarization cannot start (Torda, 1973a). The validity of this assumption would be substantiated if activated catalytic subunit could overcome the curare block. In the control experiments d-tubocurarine was administered to the

.

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

29

sympathetic ganglion of frog and rabbit, and dehydro-p-erythroidine to the cat. The nicotinic transmission became blocked. Microinjections of ACh in relatively large doses overcame, at times, the curare block. Microinjections of the catalytic subunit generated comparable postsynaptic depolarization in both intact and curarized preparations (Table VII, Fig. 4). I t seems, therefore, that the curare block is the bioelectric expression of the assumption that activation of the catalytic subunit may be necessary for the generation of postsynaptic depolarization.

d. Discussion Even though the accumulated evidence does not offer conclusive proof, it fully substantiates the assumption that depolarization of the postsynaptic neuron may depend on the activation by acetylcholine of the enzyme triphosphoinositidephosphomonoesterase. (1) The equilibrium potential for ACh, presynaptic supramaximal pulses, and catalytic subunit were almost identical. (2) Comparable depolarization of the postsynaptic neuron was generated by the depolarizing agents used. (3) The postsynaptic effects of any two of the three depolarizing agents (subthreshold amounts) were additive. (4) Inhibition of the activity of triphosphoinositide phosphomonoesterase (e.g., by the regulatory subunit) inhibited in a comparable manner the postsynaptic response to one of the three depolarizing agents. ( 5 ) The curare block of nicotinic transmission was overcome by administration of the active catalytic subunit. This suggests that the catalytic subunit-dependent processes are prevented by curare. ( 6 ) Triphosphoinositide phosphomonoesterase has been isolated from the postsynaptic membrane (Torda, 1973f). These in vivo experiments substantiate the in vitro observations (Torda, 1973a) that ACh may use the regulatory subunit as its postsynaptic nicotinic receptor. (1) From all enzymes tested, only triphosphoinositide phosphomonoesterase was activated by ACh to a significant degree. (2) The regulatory subunit has a greater affinity for ACh than for the catalytic subunit.

e. Conclusions The biochemical and bioelectric observations suggest that ACh may use the regulatory subunit of triphosphoinositide phosphomonoesterase as its postsynaptic specific receptor. On combining with ACh, this regulatory subunit ceases to inhibit the catalytic subunit and TPI is dephosphorylated to DPI. This concurs with release of some membrane-bound Ca2+,an event that channels the local field potentials toward depolarization (Torda, 1973e). This channeling may result from the cooperative efforts of several processes : e.g., release of membrane-bound Ca2 , conformational changes, special ionophoresis (passive transport of Na+ and K +). Loss of membrane-bound +

TABLE VI : DEWLARIZATION-EFFECTS OF VARIOUS AGENTS O N GENERATION OF POSTSYNAPTIC BIOELE~TRIC PROCESSES Frog (B fibers, paravertebral sympathetic chain)

Experimental procedure

No. of expt. Procedure (agent)

Amount

Acetylcholine 1x 1 0 - 7 ~ Presynaptic pulses Supramaximal Presynaptic pulses Near-threshold Catalytic subunit (TPIPM) 0.5 Unit Catalytic subunit 2 Units Catalytic subunit 5 Units Presynaptic pulses Near-threshold Presynaptic pulses and Near-threshold and catalytic subunits 1.5 Units M Acetylcholine (ACh) 1 x ACh and catalytic 1 x lo-" M a n d subunits 1.5 Units Denervation and catalytic subunits 1-5 Units Regulatory subunit of TPIPMb 1-15 Units Presynaptic pulses Supramaximal Presynaptic pulses and Supramaximal and regulatory subunit 5 Units Catalytic subunit 5 Units Catalytic subunit and regulatory subunit 5 Units each ACh 1 x lO-6M ACh and regulatory 1x Mand subunit 5 Units a

b

Preparation

Cells

EPSP (summit) (mV)

20 16 12 12 12 20 I2

78 102 54 60 56 92 54

26 f. 0.2O 26 0.3 15 & 0.9 12 & 0.8 20 & 0.6 26 f. 0.3 15 & 0.9

12 12

84 70

12

Spike

w

0

Cat (Renshaw cell) No. of expt.

ESPS (summit) (mV)

Spike (peak) (mv)

70 100 46 50 60 100 48

15 f 0.8 15 f 0.8 5 & 1.6 10 f. 2.1 15 2.2 6 k 1.2

82 & 3.0 80 f 1.5 Not reached Not reached Not reached 81 f. 1.8 Not reached

12

58 52

16 & 2.2 7 & 0.9

a3 2.5 Notreached

12

56

15 f 1.5

15 20

60 100

Notreached 75-115

15 20

80 100

3 & 1.6 15 f 2.2

Not reached 81 & 1.8

4 f 0.5 26 f 0.2

Notreached 75-115

15 12

72 48

5 f. 1.2 15 k 0.8

Not reached 83 k 3.7

5 f 1.0

Notreached

12

54

4 f 1.8

Not reached

(peak) (mV)

Preparation

Cells

75-115 75-115 Notreached Notreached Notreached 75-115 Not reached

15 20 10 10 14 20 12

26 f. 0.3 12 f. 1.0

75-115 Notreached

12

84

26 f. 0.2

75-115

10

34

26 f. 0.5

75-115

12 15

105 105

15 15

112 105

6 f. 0.7 26 f 0.2

15 20

112 122

15

140

No response No response 26 f 0.2 75-115

Average, followed by the standard error of the mean. Catalytic and regulatory subunits of triphosphoinositide phosphomonoesterase (TPIPM).

80 & 3.2

No response No response 15 f. 0.5 80 k 1.5

~

8*

TABLE V I I COMBINED EFFECTS OF CURARE [~-TUBOCURARINE (DMTC), DEHYDRO-&ERYTHORIODINE (DHPE)], ACETYLCHOLINE, AND THE CATALYTIC SUBUNIT OF TRIPHOSPHOINOSITIDE PHOSPHOMONOESTERASE (TPIPM) ON POSTSYNAPTIC BIOELECTRIC PROCESSES

2!+d

No. of Presynaptic

DMTC or DHBE

Acetylcholine

Catalytic subunit

pulses

(mole)

(mole)

(unit)

ration

Cell

0 0

0 1 x 10-7

20 20

I00 103

1 x 10-7 1 x ro-7 1 x 10-7 1 x 10-7 0 1 x 10-7 1 x 10-7

0 1 x 10-7 1 x 10-8 1 x 100 0

0 0 0 0 0

20 10 10 10 15 20 20

101

+ 0 +0 0

0 0

+ 0

0

0 5 0 50

experiments Prepa-

68 72 70 80 101 104

* h

Frog (DMTC given)

CAT(DHPE given)

EPSF (summit) Spike (peak) EPSP (summit) Spike (peak) (mV) 25.2 f 1.3” 26.0 & 2.4 Blocked Blocked 20.7 f 2.5 25.5 f 1.3 24.9 f 2.6 Blocked 26.0 2.0

(mV)

(mv)

(mV)

82 f 2.1 83 f 2.8 Blocked Blocked

15 f 0.5 15 & 0.8 Blocked Blocked 10 1.0 15 & 1.7 15 2.2 Blocked 16 & 4.3

80 k 1.5 82 f 3.6 Blocked Blocked Notreached 78*+ 4.2 81 f 1.8 Blocked 80 f 4.3

No 83 f 2.9 82 f 3.5 Blocked 82 f 3.4

*

The average, followed by the standard error of the mean. The indicated concentrations of acetylcholine may not correspond to the amount of acetylcholine that reached the effective sites of the synaptic membrane. Longer exposure to less acetylcholine is more effective. At times somewhat higher concentrations of the catalytic subunit of triphosphoinositide phosphomonoesterase were required to generate comparable postsynaptic bioelectric responses from the curarized and the not curarized ganglion.

F

t

5

3i1: ki

2m

f

?J

z9

d 0

1

2m

F

32

CLARA TORDA

Ca2 may affect depolarization either directly or indirectly. Therefore, activation of triphosphoinositidephosphomonoesterase is the coupling mechanism of depolarization. +

111. Hyperpolarization-A

Cyclic AMP-Dependent Form

A. MEMBRANE HYPERPOLARIZATION According to the available literature, the local electric fields can be channeled toward hyperpolarization by (a) an increase of membrane-bound and (b) special ionophoresis (Keynes and Lewis, 1951 ; Magleby and Ca2+, Stevens, 1972). Hyperpolarization can be generated by intraneuronal accumulation of cAMP (reviewed by Robison et al., 1971; Torda, 1971). Therefore, the special postsynaptic receptor for selecting the message “hyperpolarize” from all the messages encoded in the cAMP molecule, is a molecule that is able to generate during binding cAMP either an increase of membrane-bound Ca2+,or special ionophoresis, or similar processes.

B. SPECIFIC RECEPTORS FOR CYCLIC AMP: PROPOSED NEWRECEPTOR-THE OF DIPHOSPHOINOSITIDE KINASE REGULATORY SUBUNIT Several protein kinases may serve in the various tissues as specific receptor for CAMP. On combining with CAMP, these protein kinases phosphorylate key cellular proteins (Greengard et al., 1972; Miyamoto et al., 1969; Walsh et al., 1968, 1971 ; Weller and Rodnight, 1971). Several of these protein kinases seem to function according to the model suggested by Monod ct al. (1965), namely: CR + X = C + XR (C is the catalytic, R the regulatory subunit, and X is CAMP). The CR form of the enzyme is near inactive. The regulatory subunit has a greater affinity for cAMP than for the catalytic subunit. On combining with CAMP, the regulatory subunit ceases to inhibit the enzymatic activity of the catalytic subunit (Erlichman et al., 1971; Gill and Garren, 1970; Reimann et al., 1971a,b; Tao et al., 1970). One of the specific postsynaptic receptors of cAMP has been identified as the regulatory subunit of diphosphoinositide kinase (Torda, 1972a-g) . On combining with CAMP, this regulatory subunit ceases to inhibit the catalytic subunit, and diphosphoinositide is phosphorylated to triphosphoinositide. Since this phosphorylation coincides with a quantitatively predeterminated increase of membrane-bound Ca2+,and probably also special ionophoresis (see below), the regulatory subunit of diphosphoinositide kinase seems to qualify as the specific postsynaptic receptor for cAMP that mediates hyperpolarization. Diphosphoinositide kinase has been isolated from several tissues in a near-active form (Hawthorne and Kai, 1970; Hawthorne and Kemp, 1964; Salway et al., 1967),including the postsynaptic neuron (Torda, 1972g, 1973f).

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

33

The postsynaptic neuron, however, lacks DPI, the specific substrate. Free DPI is delivered during depolarization into the postsynaptic neuron (Torda 19728). For this DPI to be handled, diphosphoinositide kinase must first be activated. The only identified activator is cAMP (Torda, 1972c-g). Should other hyperpolarizing agents be able to activate diphosphoinositide kinase, the model for hyperpolarization proposed in this study will become valid for a fay wider range of events. According to this model, hyperpolarization is the bioelectric by-product of the phosphorylation of DPI to TPI.

OBSERVATIONS C. EXPERIMENTAL 1. Biochemical (in Vivo) Experiments During the search for a specific postsynaptic receptor, the effects of cAMP on various enzyme systems were assayed. Incubation with cAMP significantly increased the activity of diphosphoinositide kinase. Therefore, attempts were made to gain an insight into the mechanism through which cAMP may activate this enzyme. Diphosphoinositide kinase was extracted from various types of brains and purified. When further purification failed to increase the enzymatic activity, fractionation was attempted by chromatography. Diphosphoinositide kinase seemed to yield a catalytic and a regulatory subunit. The regulatory subunit seemed to have a greater affinity for cAMP than for the catalytic subunit. Therefore, CAMP may serve as the activator of diphosphoinositidekinase, and part of diphosphoinositide kinase may serve as a postsynaptic receptor for CAMP.

Materials Sephadex G-200, carboxymethyl (CM)-Sephadex (3-50, and Sepharose 4B were purchased from Pharmacia, Stockholm, Sweden ; DEAE-cellulose (Whatman DE-52) from H. Reeve, Angel and Co., London; Dowex resin from Dow Chemical Co. (Midland, Michigan) ;Triton X-100from Lenning Chem. Ltd., Durham, England; and [3H)cAMP from Schwarz BioResearch, Orangeburg, New York. Terminally labeled [3aP]ATP was prepared following the method of count per 100 seconds per Glynn and Chappell (1964). It contained nanomole in a 6% efficient liquid Geiger counter. Diphosphoinositide was prepared by the method of Kai at al. (1966a,b, 1968). a,

6 . Methods i. Preparation of crude diphosphoinositide kinase (method of K a i et al., 1966a,b, 1968). Brain was obtained from albino rats of both sexes, about 250 gm body weight, within a few minutes after death. The brain was homogenized on 0.32 M sucrose by the use of a Teflon-glass homogenizer with a radial

34

CLARA TORDA

clearance of 0.1-0.15 mm and a pestle rotating at 600 rpm at 4°C. The subcellular fraction was obtained according to the method of Nyman and Whittaker (1963). After repeated centrifugation (1 1 minutes, lo3 gmax and repeated washing, the fraction that contained the nerve endings was obtained by density-gradient centrifugation for 45 minutes at lo5 g,, Equal volumes of 0.8 and 1.2 M sucrose were used. The preparation was dialyzed at 4°C against 100 volumes of 20 m M tris. HCl buffer at pH 7.4 for 20 hours. ii. Purification of diphosphoinositide kinaJe (method of Kai et al., 1968). The crude preparation was treated with ammonium sulfate (20-4070, w/v), and the precipitate was chromatographed first on a Sephadex G-200 column, then on a CM-Sephadex C-50 column. The Sephadex G-200 column (3.4 x 36 cm) was first equilibrated with 20 m M sodium phosphate buffer at pH 6.0. A kinase sample containing about 15.5 mg of protein was applied to the column in 15 mi of 20 m M tris-HC1 buffer at pH 7.4 and eluted with phosphate buffer at a rate of 2.0 ml/cma of column per hour. To the CMSephadex (2-50 column (1.4 x 19 em), about 9.5 mg of protein was applied. The kinase was removed by gradient elution with 20 m M sodium phosphate buffer containing 0.4 M sodium chloride. The gradient was obtained by having 80 ml of phosphate buffer in the mixing chamber and 80 ml of the same buffer containing 0.1 m M sodium chloride in the reservoir. The active fractions were combined and dialyzed against 20 m M tris .HCl buffer at pH 7.4. This procedure reduced the contamination by triphosphoinocitide phosphomonoesterase practically to traces. The active fractions obtained from the two columns were concentrated by means of a rotary evaporator (Buchi Rotavapor with a Nalgene vacuum pump) at a bath and collection temperature of 4°C. iii. Attempted fractionation of the puriJied diphosphoinositide kinase. In order to weaken the bonds between potentially existing subunits of diphosphoinositide kinase, enzyme samples with about 7 mg of protein content were M of [3H]cAMP at room temperamixed and incubated with 0.1 x ture for 10 minutes. The mixture was chromatographed on a Sepharose 4B column (Cuatrecasas, 1970; Reimann et d., 1971a,b). The column was prepared by equilibrating it (0.9 x 7 cm) with 10 ml of a 3-(N-morpholinoethane)sulfonic acid buffer (pH 6.0) containing 2 m M EDTA (sodium salt), M [3H]cAMP, followed by 7.2 ml of buffer, containing 0.1 ml of 7.5 x and by 3 ml of buffer. The column was eluted with 190 ml of a linear gradient of NaCl in a 2-(N-morpholinoethane)sulfonicacid buffer (pH 6.0). 0.05-ml samples of the eluate were analyzed for 3H in a 10 ml of dioxane-based scintillant, and 3.5-ml samples were tested for absorbance at 280 nm. The protein-bound [3H]cAMP passed into 9-26 ml of the eluate. Practically all the eluted

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

c 0

9

35

2ol 0

Sample number

FIG. 5. Chromatography of diphosphoinositide kinase on Sepharose 43 column. Each sample contained 3.5 ml of eluate. The number of the samples is indicated on the horizontal axis. The protein contents of the samples are indicated on the vertical axis. All 3H content appeared between the dashed lines and was protein bound.

radioactivity was due to protein-bound r3H]cAMP. This assumption was based on the observation that the 3H content of the 9-26 ml of the eluate did not decrease after an attempt to remove free CAMP through centrifugation ( lo5 g,, av) in a Spinco ultracentrifuge for 30 minutes at room temperature. The first 26 ml of the eluate (fraction A) and 27-190 ml of the eluate (fraction B) were collected separately, then concentrated by means of a rotary evaporator (Fig. 5). The eluate contained usually over 80%, but never less than SO%, of the original diphosphoinositidekinase activity. iv. Assay f o r the enzymatic activity of the various diphosphoinositide kinase preparations (method of K a i et al., 1968; Sdway, et al. 1967). Diphosphoinositide kinase phosphorylates the 5 position on the inositol ring of its specific substrate, DPI. Therefore, the enzymatic activity was ascertained by measuring the amounts of [32P]TPI formed from DPI and r3'P]ATP during incubation at 37°C for 5 minutes. The basal incubation mixtures contained, in a volume of 5 ml, 5 m M [32P]ATP,30 m M MgCI,, 10 m M GSH (sodium salt), 20 m M tris.HC1 buffer (pH 7.4), one of the enzyme preparations (0.1-0.2 mg of protein) and 4 m M EDTA (sodium salt). The substances were mixed at room temperature for 10 minutes in order to permit the EDTA to inhibit the possible contaminating traces of phosphodiesterase The reaction was started by adding the DPI (sodium salt) 0.9 mM. At the end of the 5-minute incubation period, the reaction was terminated by adding a chloroform :methanol (1 :2, v/v) mixture. The various phosphoinositides were extracted, then were separated by two methods (Galliard st al., 1965): (a) descending chromatography on formaldehyde-treated paper (Horhammer et al., 1959);and (b) chromatography of the hydrolyzates on Dowex I-Xlo resin (Ellis et al., 1963). The [32P]triphosphoinositidewas measured after separation from DPI by a liquid Geiger-Muller counter. The protein

36

CLARA TORDA

contents were ascertained by the method of Lowry et al. (1951). The diphosphoinositide kinase activity of the various samples was calculated as the amount of [32PljTPI formed in nanomoles per minute per milligram of protein. [32P]TPI was not formed in the blanks. Attempts were made to ascertain whether the measured [3aP]TPI corresponded to the actual yield of the TPI by diphosphoinositidekinase. The difference between the actually produced and the measured TPI could have resulted mainly from two sources: (a) dephosphorylation of the newly formed TPI by triphosphoinositide phosphomonoesterase or phosphodiesterase; and (b) synthesis of TPI by enzymes other than diphosphoinositide kinase (e.g., diglyceride kinase, phosphatidylinositol kinase) .( Attempts were made to minimize the enzymes that dephosphorylate : triphosphoinositide phosphomonoesterase contamination was significantly decreased by chromatography on Sephadex G-200 and CM-Sephadex-50 column (Kai et al., 1968). Phosphodiesterase contamination was significantly decreased by precipitation with ammonium sulfate (25-40Oj,, w/v). The remaining activity was inhibited by preincubation of the enzyme preparations with 4 m M EDTA (Salway et al., 1967) at room temperature for 10 minutes. Random samples of the diphosphoinositide kinase preparations were tested for contamination by these four enzymes. Triphosphoinositide phosphomonoesterase was tested by the method of Dawson and Thompson (1964), phosphodiesterase by the method of Thompson and Dawson (1964), diglyceride kinase and phosphatidylinositolkinase by the method of Kai et al., (1 966a). The absence of phosphatidylinositol kinase was also ascertained by observing the effects of Triton X-100 and sodium deoxycholate on the yield of TPI. The activity of diphosphoinositide kinase is known to significantly decrease in the presence of Triton X-100, and is unaffected by sodium deoxycholate. Both Triton X- 100 and sodium deoxycholate significantly increase the yield of TPI by phosphatidylonisitol kinase (Kai et al., 1968). Purified diphosphoinositide kinase and fraction B seemed to lack all four enzymes: triphosphoinositide phosphomonoesterase, phosphodiesterase, diglyceride kinase, and phosphatidylinositol kinase (Table VIII) . Results ATP and Mg2+ (with an optimum of 20 mM) are obligatory cofactors of diphosphoinositide kinase. Maximum enzymatic activity occurred in the presence of 0.6-2 m M DPI and 5-8 m M ATP. Under these optimum concentrations of DPI and ATP, and in the presence of 2-4 m M EDTA and 20 m M MgCI,, the enzymatic activity of diphosphoinositide kinase was proportional to the protein concentrations in the range of 0-0.4 mg of protein per milliliter, and the rate of reaction was constant for 10 minutes. Theoptimum pH was between 7.0 and 7.5. c.

9 U

m

ENZYME IMPURITIES CONTAINED IN

THE

TABLE VIII VARIOUS DIPHOSPHOINOSXTIDE KINASE(DPIK) PREPARATIONS

2

Activity (nmoles productlmg enzyme proteinlmin of incubation at 37OC Type of DPIK preparation Dialyzed brain homogenate Sephadex G-200 eluate Sephadex C-50 eluate Sepharose 4B eluate, fraction B

DPIK 2.2 f 0.07' 6.6 & 0.12 6.5 k 0.15 111.0 0.15

Triphosphoinositide Phosphatidylinositol phosphomonocsterase kinase

0.92 k 0.05 0.14 f 0.01

0.03 f 0.01 0.00

0.00

0.00

0.00

0.00

The average, followed by the standard error of the mean.

Diglyceride kinase

0 0 0 0

No. of

?m

experiments

2m

20 20

0

20 20

k

F F

=I

0

z n .e n r R

w

38

CLARA TORDA

The crude enzyme preparation yielded an average of 2.2 nmoles of TPI per minute per milligram of enzyme preparation. The enzymatic activity depended on an active -SH group and significantlyincreased in the presence of cyclic .4MP (Table IX). It was inhibited by Ca2+. From 30 to 60y0 inhibition was generated by NaCl (150 mM), KCl (50-100 mM), or a mixture of NaCl (120 mM) and KCl (30 mM). It was heavily contaminated by triphosphoinositide phosphomonoesterase, slightly by phosphatidylinositol kinase and lacked diglyceride kinase activity (Table VIII). The purified enzyme yielded an average of 6.6 nmoles of TPI per minute per milligram of enzyme protein. The purified diphosphoinositide kinase did not depend on active -SH groups. Its enzymatic activity was not affected by Na+, K + , or C1-. The activity of the diphosphoinositide kinase decreased in the presence of an excess of ATP, ADP, and TPI and increased in the presence of cAMP (Table IX). Further purification did not increase the enzymatic activity of diphosphoinositide kinase. Fraction A (prepared with or without added CAMP) lacked diphosphoinositide kinase activity. Fraction B contained a highly active form of the enzyme. It yielded an average of 111 nmoles of [32P]TPI per minute per milligram of enzyme protein (Table IX). The catalytic activity of fraction B did not depend on an active -SH group, and was not affected by Na+, K + , C1-, and CAMP. Both ATP and MgCl, remained obligatory cofactors. In M the presence of 15 m M MgC12, the K,,, was calculated to be 1.43 x for DPI, and 2.5 x M for ATP. Fraction B lacked enzymatically active impurities, e.g., triphosphoinositide phosphomonoesterase, phosphodiesterase, diglyceride kinase, phosphatidylinositol kinase (Table IX). Fraction A seemed to function as the regulatory subunit of diphosphoinositide kinase, and fraction B as the catalytic subunit. Fraction A lacked enzymatic activity and contained the protein-bound [3H]cAMP. Fraction A (prepared without CAMP) significantly inhibited the activity of fraction B during the 5-minute incubation period. The inhibition was proportional with the concentrations of fraction A, given a fixed amount of fraction B. Further incubation of the mixture of fractions A and B with added cAMP reversed the inhibition. Rechromatography at the end of the incubation period of the mixture of fractions A and B and cAMP revealed that the added cAMP was bound to fraction A. The reversal of the inhibition generated by fraction A was proportional with the amount of fraction A that became bound to CAMP. In this set of experiments [3H]cAMP was used. Cyclic AMP increased the enzymatic activity only of those preparations which contained fraction B bound either partially or completely to fraction A, e.g., purified diphosphoinositidekinase, the crude enzyme, and mixtures of fractions A and B. Cyclic AMP did not affect the enzymatic activity of fraction B. Fraction A saturated with cAMP did not inhibit the enzymatic

TABLE IX: OBSERVATIONS ON THE DIPHOSPHOSNOSITIDE KINASE(DPIK) ACTIVITY OF

Cyclic AMP (mole)

1x 1x 1x 1x 1x

Enzyme preparations and their fractions ~

~

.

.

Method used to prepare DPIK

-

.

Fraction B

.

..

Fraction A" (1) (2)

Dialyzed homogenate 10-8 Dialyzed homogenate lo-' 10-6

10-6 10-4

Sephadex G-200 Sephadex C-50

(1) (2) 1 x 10-7

1 x 10-3

1 x 10-6. 1 x 10-7

1 x 10-8

B B B B B B B B B B

(2) 4 Unitsd (2) 2 Units

c2) (2)

No. of experiments

Incubation time (min)

25 10 10 10 10 10 25 25 26 10 30

5 5 5 5 5 5 5 5 5 5 5

25 25 25 27 25

THE

VARIOUS ENZYME PREPARATIONS Enzymatic activity Nmoles/min/mg protein of [3aPJTPIformed

2.2 f 0.07b 3.0 f 0.10 11.0 f 0.12 14.1 k 0.13 15.9 f 0.16 16.7 f 0.08 6.6 k 0.12 6.5 f 0.15

0 0 111.0 f 0.15 109.7 k 0.16 113.1 f 0.17 5 92.8 k 0.12 5 21.7 k 0.09 5 42.0 +_ 0.20 5 21.7 & 0.09 2.5Contin. 11.1 k 0.08 +2.5 75.0 f 0.17 +2.5 55.2 f 0.14 2.5 18.0 f 0.10

+

yoof Control 100 150 500 640 725 759 300 295 0 0 5454

100 99 100 83 20 100 38 194 20 10 100 68 680 50 500 16 160

Fraction A: (1) prepared adding cyclic AMP before chromatography; (2) prepared without added cyclic AMP. Average, followed by the standard error of the mean. c The slight inhibition resulted from redistribution of fraction A between fraction B and cyclic AMP during incubation. d The inhibition seemed to be proportional with the amount of the added fraction A (incubation of a fraction A and B mixture). 6 The reversal of inhibition was proportional with the amount of cyclic AMP that became bound on fraction A. b

40

CLARA TORDA

activity of fraction B during the incubation period. When fraction A became redistributed between cAMP and fraction B according to their relative affinity during the 5 minutes of incubation, some inhibition became measurable (Table IX).

d. Discussion The near-inactive form of diphosphoinositide kinase has been fractionated onto a n enzymatically active (catalytic) and an enzymatically inactive (regulatory) subunit. The catalytic subunit phosphorylated the fifth position on the inositol ring of DPI yielding TPI. When combined, the regulatory subunit inhibited the enzymatic activity of the catalytic subunit. This inhibitory effect was suggested by the observations that (1) the enzymatic activity of fraction B significantly exceeded that of the purified diphosphoinositide kinase; (2) incubation with the regulatory subunit inhibited the enzymatic activity of the catalytic subunit. The inhibition was proportional with the amounts of fraction A that became bound to fraction B. Fraction A had a greater affinity for cAMP than for the catalytic subunit, because: (1) Exposure of the near-inactive form of diphosphoinositide kinase to cAMP significantly facilitated the separation of the enzyme into its subunits: the catalytic subunit, and the CAMP-bound regulatory subunit. (2) Excessive amounts of fraction B were required to displace cAMP from a fraction A-CAMP complex. (a) The significantly greater affinity of cAMP to the regulatory subunit of diphosphoinositide kinase than to any of the other enzymes observed suggests that this regulatory subunit may be one of the postsynaptic specific receptors of cyclic AMP. (b) Theoretical considerations substantiate this assumption : formation of the CAMP-receptor (regulatory subunit) generates processes that are essential for the initiation and execution of hyperpolarization, the observed postsynaptic effect of cAMP (reviewed by Breckenridge and Bray, 1970; Robison et al., 1971; Torda, 1971). On combining with CAMP, the regulatory subunit ceases to inhibit the enzymatic activity of the catalytic subunit, and DPI is phosphorylated to TPI. T P I is a more potent chelating agent than DPI (Dawson, 1965);Hendrickson and Reinertsen, 1971). Therefore, formation of T P I concurs with a quantitatively predictable increase of the membrane-bound Ca2+.This increase is sufficient to channel the local field potentials toward hyperpolarization, either directly (Diamond and Wright, 1969; Keynes, 1972; Lecar and Nossal, 1971; Magleby and Stevens, 1972), or indirectly through ionophoresis (termination of the passive transport of Na+ and K + and promotion of transport of C1- with K + ) . (c) Diphosphoinositide kinase without DPI has been isolated from the postsynaptic neuron (Torda, 1972g, 1973f).

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

41

e. Conclurions Diphosphoinositide kinase has been fractionated into a regulatory and catalytic subunit through chromatography on Sepharose 4B column. When united, the regulatory subunit inhibits the enzymatic activity of the catalytic subunit. The regulatory subunit may serve as one of the postsynaptic receptors of CAMP. On combining CAMP, the regulatory subunit ceases to inhibit the catalytic subunit, leading to phosphorylation of DPI to TPI. The concurrent events seem to initiate the necessary processes for the execution of hyperpolarization. Therefore, activation of DPI kinase by cAMP may be one of the molecular mechanisms that is able to couple in time and space the formation of the CAMP-receptor complex and membrane hyperpolarization.

2. Bioelectric (in Vivo) Observations In order to ascertain whether hyperpolarization of postsynaptic neurons following an intrapostsynaptic accumulation of cAMP is generated through activation of diphosphoinositide kinase, the effects of changes in postsynaptic diphosphoinositide kinase activity on the postsynaptic bioelectric processes has been studied in vivo in the superior cervical ganglion of rabbit, the paravertebral sympathetic chain of the frog, and the Renshaw ceIl of the cat. The results seem to validate the assumption that cAMP may generate hyperpolarization of the postsynaptic neuron through increasing the enzymatic activity of intrapostsynaptic diphosphoinositide kinase. a. Materials

cAMP and theophylline were commercially obtained. cAMP was used in concentrations of 1 x lo-' to 1 x M. Theophylline was usually given in 2-5 times higher concentrations in order to protect cAMP from hydrolysis by the intrapostsynaptic specific phosphodiesterase (Florendo et al., 1971). Diphosphoinositide kinase and its catalytic and regulatory subunits were prepared following the method of Torda (1972e) (see Section 111, C , 1, b). DPI was prepared following the method of Kai et al. (1966a).

6. Methods i. Test objects. The superior cervical ganglion of the rabbit, the paravertebral sympathetic chain of the frog, and the Renshaw cell of the cat were prepared by the method described in Section 11, E, 2 on bioelectric observations on depolarization. Similarly, the methods of stimulation, recording, and microinjections were also described in Section 11, E, 2. When required, hyperpolarizing currents were delivered through the intracellular recording electrode. The microinjections were given into the postsynaptic neuron, near the synaptic surface of the membrane. The injected substances differed. In order to protect cAMP from hydrolysis, the animals received

42

CLARA TORDA

beforehand theophylline in 2-5 times higher concentrations. Even though theophylline had only a negligible effect on the bioelectric properties of the postsynaptic neuron, in order to minimize the potential sources of error, the effects obtained by administration of cyclic AMP (with theophylline) were compared with control values obtained after administration of theophylline in comparable concentrations. In order to protect DPI from being phosphorylated before reaching its site of action, it was injected simultaneously with either cAMP or the catalytic subunit, but through separate micropipettes

.

ii. Experimental procedure. Controls (see description in Section 11). Exferimental. ( 1) Measurements of some bioelectric characteristics of the resting membrane; (2) effects of administration of diphosphoinositidekinase, the catalytic and regulatory subunits in subthreshold concentrations on some properties of the resting membrane ; (3) administration of hyperpolarizing DPI, and the regulatory current, the catalytic subunit +_ DPI, cAMP subunit & DPI during presynaptic stimulation (depolarization) with rectangular pulses of supramaximal intensity; (4) measurements of the equilibrium potential for cAMP DPI, catalytic subunit DPI, and postsynaptic hyperpolarizing pulses ; (5) membrane potential measurements in resting neurons after administration of CAMP and catalytic subunit in suprathreshold concentrations;(6) administration of two of the three hyperpolarizing agents in subthreshold amounts; (7) administration of the regulatory subunit during presynaptic stimulation with or without the added administration of hyperpolarizing pulses, cAMP DPI, or catalytic subunit .DPI. Microinjections of blanks did not affect the bioelectric processes in a measurable amount.

+

+

+

+

c. Results The basic properties of the resting membrane, the postsynaptic potential and spiking has been described together with the observations on depolarization. Administration of the catalytic and regulatory subunits of diphosphoinositide kinase in subthreshold concentrations did not seem to affect the bioelectric characteristics of the resting membrane, except for a slight increase of resistance during administration of the catalytic subunit in nearthreshold concentrations. In higher concentrations, membrane potential measurements revealed the generation of hyperpolarization of a comparable amount by hyperpolarizing pulses, CAMP,and the catalytic subunit. Within the limits of the experimental procedure, the increase appeared to be proportional to the amounts of cAMP or catalytic subunit. The hyperpolarizing effects of the catalytic subunit and cAMP were better maintained during simultaneous administration of DPI (Tables IV and X).

43

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

TABLE X EFFECTS OF INTRAPOSTSYNAPTIC MICROINJECTIONS OF DIPHOSPHOINOSITIDE KINASEAND ITS FRACTIONS ON THE RESTING MEMBRANE POTENTIALa Number of experiments Experimental procedure

Amounts of Prepaadded agents ration Cell

Presynaptic pulses Supramaximal A Hyperpolarizing pulses 1x 1x A Hyperpolarizing pulses Catalytic subunit (noDPIc) 2 Units Catalytic subunit DPI 2 Units 4 Units Catalytic subunit + DPI Cyclic AMP (no DPI) 1x M 1 x 10-6M Cyclic AMP ( + DPI) Cyclic AMP ( + DPI) 2 x 10-6M Regulatory subunit( DPI) 5 Units

+

+

40 10 10

10 10 10 10 10 10 10

200 40 42 38 40 40 40 40 40 40

Membrane potential Frog

-65 -76 -85 -75 -80 -92 -77 -79 -89 -63

k 1.7b f 3.3

k 3.5 & 2.8' & 4.3 f 5.5 f 1.9' & 2.4 f 3.1 & 4.1

Cat

-66 f 1.9 -74 f 2.8 -86 f 2.7 -77 f 3.3' -81 f 3.0 -94 f 3.3 -74 & 2.7d -80 f 3.5 -91 f 4.2 -61 2.8 ~ ~ _ _ _ _

All preparations received presynaptic supramaximal rectangular pulses (frequency l/minute) during the entire course of the experiment. All agents were added during presynaptic stimulation. Average, followed by the standard error of the mean. DPI = diphosphoinositide. The catalytic subunit of diphosphoinositide kinase and cyclic AMP usually initiated traces of hyperpolarization, but this hyperpolarization was maintained only when the catalytic subunit and cyclic AMP were given simultaneously. This suggests that cyclic AMP and the catalytic subunit generate hyperpolarization through the enzymatic activity of diphosphoinositide kinase (see text).

'

Administration of hyperpolarizing current, cAMP + DPI, and cAMP and DPI during presynaptic stimulation with rectangular pulses of supramaximal intensity generated comparable inhibition of the postsynaptic response (Table XII, Fig. 6 ) . At first the rising slope of the EPSP became less steep. The latency of spike formation increased. As long as spikes were generated they retained the usual characteristics. With a further increase of the hyperpolarizing agents the critical voltage for spike generation was not reached, and later all response may have vanished. The effects of cAMP and the catalytic subunit were better maintained during administration of DPI. The hyperpolarization generated by subthreshold amounts of any two of the three hyperpolarizing agents were additive (Table XII). Comparable equilibrium potentials were observed during test with any of the three hyperpolarizing agents (Table XI). Intrapostsynaptic microinjections of the regulatory subunit of diphosphoinositide kinase & DPI, even at relatively high concentrations (15 units)

44

CLARA TORDA

FIG.6r Effects of changes of the activity of diphosphoinositide kinase (DPIK) on hyperpolarization (Intracellular recordings). (A) Superior cervical ganglion of rabbit Column I : Effects of hyperpolarizing current inhibition of DPIK on postsynaptic depolarization. Row 1 : Postsynaptic depolarization generated by presynaptic supramaximal rectangular pulses. Arrows: Added hyperpolarizing current of increasing intensity. Rows 2 and 3: Decreased postsynaptic depolarization (first the slope of EPSP decreased, then the latency of spike generation increased, then the EPSP remained below the mV threshold of spike generation and spiking disappeared, eventually the mV of the EPSP practically vanished. Double arrow: Injections of the regulatory subunit. Row 4: Inhibition ofhyperpolarization: Postsynaptic depolarization reappeared. Column 11: Effects of catalytic and regulatory subunits of postsynaptic depolarization. Row 1: Postsynaptic depolarization generated by presynaptic supramaximal pulses. Arrows: Increasing amounts of injected catalytic subunit. Rows 2 and 3 : Increasing hyperpolarization ( = decreased postsynaptic depolarization) . Double arrow: Injection of the regulatory subunit. Row 4: Reversal of hyperpolarization.

(B) Renshaw cell of cat. the regulatory subunit of DPIK Column I: Effects of hyperpolarizing current on postsynaptic depolarization generated by presynaptic pulses. Row 1: Postsynaptic depolarization to presynaptic pulses. Arrows: Hyperpolarizing current of increasing intensity.

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

45

did not generate membrane hyperpolarization. Microinjections of the regulatory subunit during administration of any of the hyperpolarizing agents (hyperpolarizing current, CAMP, and DPI, or catalytic subunit and DPI) inhibited to a comparable degree the hyperpolarization. DPI did not modify in a measurable amount the effects of the regulatory subunit (Table XII, Fig. 6 ) .Near-complete inhibition was, usually, observed already with 5 units. The effects of the experimental procedures affected the sympathetic ganglia of the rabbit and the frog and the Renshaw cell of the cat to a comparable degree. The effective dose of either the catalytic or the regulatory subunit was usually less than 5 units.

d. Discussion Changes in the enzymatic activity of diphosphoinositide kinase seemed to affect the bioelectric processes of the postsynaptic neuron. An increase of the enzymatic activity generated hyperpolarization ; a decrease of the raised enzymatic activity (e.g., by injections of the regulatory subunit) inhibited the established hyperpolarization, regardless of the nature of the hyperpolarizing agent tried. Participation of changes in the activity of diphosphoinositide kinase in the regulation of hyperpolarization may continuously occur, since ( 1) comparable hyperpolarization was generated by hyperpolarizing pulses and the catalytic subunit, (2) the effects of subthreshold amounts of hyperpolarization, CAMP, and the catalytic subunit in generation of hyperpolarization were additive, (3) the equilibrium potentials were comparable, and (4) decreases of the enzymatic activity of diphosphoinositide kinase (e.g., by the regulatory subunit, so far the only known inhibitor) inhibited hyperpolarization by all the three hyperpolarizing agents to a comparable degree. Cyclic AMP seemed to generate hyperpolarization through activation of DPI : (1) inhibition of the enzymatic activity of diphosphoinositide kinase (e.g., by the regulatory subunit) decreased the hyperpolarizing effect of CAMP; (2) the hyperpolarizing effect of CAMP was far better sustained in presence of added DPI, the specific substrate for diphosphoinositide kinase. Rows 2 and 3 : Inhibition of depolarization. Double arrow: Regulatory subunit injected during hyperpolarizing current. Row 4: Reversal of inhibition of depolarization. Column I1 : Effects of catalytic and regulatory subunits of DPIK on depolarization. Row 1 : Postsynaptic depolarization by presynaptic pulses. Arrows: Injection of increasing amounts of the catalytic subunit. Rows 2 and 3: Increasing inhibition of depolarization. Double arrow: Injection of the regulatory subunit. Row 4: Reversal of inhibition by hyperpolarization.

TABLE XI THEEQUILIBRIUM POTENTIAL OBTAINED FOR HYPERPOLARIZING CURRENT,CYCLIC AMP ( + DPI), CATALYTIC SUBWMT OF DIPWOSPHOINOSITIDE KINASE(DPIK) ( + DPI)

Resting membrane potentialb (mV)

-65 -44 -30 -13 +10 $40 -66 -30

f 1.7’ f 1.8 f 1.5 f 2.1 f 1.2 f 1.9 f 1.9 f 2.0

4 k 0.7 +I0 +30

* 1.0 * 2.1

Hyperpolarizing current of constant intensity

Frog

Cat

Preparation

Cells

20 15 15 15 15 15 12 12 12 12 12

80 72 70 74 75 77 80 70 72 68 67

+ DPI)

No. of experiments

No. of experiments Animal

Cyclic AMP (

PSP (summit) (ml’)

-15 -10 -5 0 +4 +11 -8 -4 0 +1

f 0.5 f 0.9 1.0 f 0.3 f 1.1 f 1.7 f 1.2 f 1.0

*

k 0.2 f 0.5 + 3 2.0

*

Preparation

Cells

20 12 12 12 12 12 10 10 10 10 10

60 62 58 55 60 60 60 50 40 42 44

AND THE

Catalytic subunit of DPIK ( DPI)

+

No. of experiments PSP (summit) (mv)

-15.5 -10.2 -5.5 0 +4.5 +10.5 -7.5 -4.5 0 +1.5

f 1.0 f 1.1

2 0.7 f 0.5 1.1 f 1.5 f 1.1 f 2.0 & 0.5 f 1.0 +4.0 f 2.0

*

Preparation

Cells

20 12 12 12 12 12 10 10 10 10 10

80 52 56 76 70 62 50 50 50 47 45

PSP (summit) (mV)

-16 -12 -6 0 +3 +I2 +8.5 -4.7 0 +2.0 +4.0

f 1.4 f 1.7 f 1.3 f 0.9 f 1.9 f 1.8 f 1.2 f 0.9 f 1.0 f 0.9 f 1.4

DPI = diphosphoinositide. The method was described in the text. The membrane potential was changed from the resting level (by steady current) to the values indicated in the table and were held fixed during administration of any of the three hyperpolarizing agents. The equilibrium potential averaged 13 mV for the frog’s sympathetic ganglia, and - 1 for the Renshaw cell. Average, followed by the standard error of the mean. a

-

n

4 +

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

47

The observations seem to substantiate the assumption that one of the postsynaptic receptors of cAMP is the regulatory subunit of diphosphoinositide kinase: (1) cAMP has a far greater activating effect on diphosphoinositide kinase than on any other enzyme tested so far. (2) The regulatory subunit did have a greater affinity for cAMP than for the catalytic subunit; e.g., exposure of the purified diphosphoinositide kinase to cAMP significantly facilitated its fractionation into a catalytic and regulatory subunit, with complex formation between the regulatory subunit and cAMP (Fig. 5). (3) Biochemical in vitro experiments offered near conclusive evidence (Torda, 1972e) (see Section 111, C, 1). (4) Diphosphoinositide kinase has been isolated from the postsynaptic neuron (Torda, 1972g). This neuron lacks free DPI. During depolarization of the postsynaptic neuron, DPI is formed through dephosphorylation of TPI by triphosphoinositide phosphomonoesterase and is delivered into the postsynaptic neuron (Torda, 1972e). One may expect that this DPI cannot remain in free form inside the postsynaptic neuron, and may be phosphorylated to TPI by the diphosphoinositide kinase. Since DPI does not seem to activate the enzyme [see in vitro experiments (Torda, 1972e)l an enzyme activator is also required. Therefore, the activity of diphosphoinositide kinase depends on the presence both of an activator (CAMP)and its specific substrate, DPI. Membrane re(hyper)polarization seems to involve CAMP. Cyclic AMP may, however, use different receptors to generate the fast repolarization that follows nicotinic depolarization, and the repolarization with long latency that follows muscarinic depolarization. During the fast repolarization the receptor is the regulatory subunit of diphosphoinositide kinase (see Tables 8-12, Figs. 1, 6 ) .During the slow repolarization a postsynaptic protein kinase serves as receptor and also involves a protein phosphorylase (Greengard et al., 1972). e. Conclusions

The results suggest that fast hyperpolarization may result from activation of diphosphoinositide kinase. This process may be one of the molecular mechanisms that is able to couple the formation of CAMP-postsynaptic receptor complex and hyperpolarization of the postsynaptic membrane. In vivo hyperpolarization may be generated by increased activity of diphosphoinositide kinase. Cyclic AMP seems to be able to use the regulatory subunit as one of its postsynaptic receptors. On combining with CAMP,this regulatory subunit ceases to inhibit the catalytic subunit. This enzymatic process elicits concurrent molecular mechanisms that channel the local electric fields toward hyperpolarization (Torda, 1972g, 1973c,e; Magleby and Stevens, 1972).

TABLE XI1 HYPERPOLARIZATION-EFFECTS OF VARIOUS AGENTS ON THE POSTSYNAPTIC BIOELECTRIC PROCESSES~

n

F

~~

Frog (paravertebral sympathetic ganglia)

psp

Spike

Cell

(summit) (mV)

(peak) (mV)

25 20 25 15 15 15 16 16 14

100 78 100 70 80 55 80 88 56

14

55

No. of experiments

No. of experiments

psp

~

Experimental procedure Procedure (agent) Amount Presynaptic pulses a Hyperpolarizing pulse Catalytic subunit ( + DPI) Catalytic subunit ( + DPI) Catalytic subunit ( DPI) Catalytic subunit (no DPI) Cyclic AMP ( DPI) Cyclic AMP (no DPI) Hyperpolarizing pulse Hyperpolarizing pulse and catalytic subunit (+ DPI)

+

+

Supramaximal Suprathreshold 1 Unit 2 Unit 3 Unit 3 Unit 1 x 10-6M 1x 1 0 - 5 ~ Subthreshold Subthreshold and 1 Unit

Preparation

9 4

Cat (Renshaw cell)

0

Spike (peak) (mV)

Preparation

Cell

(summit) (mV)

26 f O.gb 75-115 12 f 2.6 Not reachedC 20 2.0 Notreached 14 f 1.0 Notreached 9 f 1.1 Notreached 12 1.6"Notreached 3 f 1.8 Notreached 8 f 2.0" Not reached 75-110 26 f 2.2

25 20 16 14 14 12 15 15 15

102 90 64 60 62 60 60 60 60

15 f 0.5 82 f 3.6 8 f 0.9 Not reached 13 It: 0.8 Not reached 10 f 1.1 Notreached 6 f 0.7 Not reached 8 f 1.ZC Not reached 3 f 0.7 Notreached 4 1.2c Not reached 14 f 1.3 80 2.5

11 f 2.4 Not reached

15

60

8 f 1.4 Not reached

g lD

Hyperpolarizing pulse and cyclic AMP ( DPI) Regulatory subunit (DPIK) Catalytic subunit ( DPI) Catalytic subunit and regulatory subunit Hyperpolarizing pulses Hyperpolarizing pulses and regulatory subunit Cyclic AMP Cyclic AMP and regulatory subunit

+

+

Subthreshold and I x 1 0 - 7 ~ 1-10 Units 5 Units 5 Units each

Strong Strong and 5 Units 1x 1 0 - 5 ~ 1x Mand 5 Units

12 12 12

54 60 62

10 f 1.6 Not reached 25 f 2.1 75-110 2 f 0.4 Not reached

15 19 15

62 80 58

7 f 1.6 Notreached 15 f 0.8 81 f 2.5 2 f 0.3 Not reached

12 12

64 60

25 2 3.3 75-110 3 f 2.4 Not reached

k5 15

58 60

14 f 2.0 82 f 2.1 1 f 1.0 Notreached

12 12

60 64

26 f 2.8 75-110 3 f 1.0 Not reached

15 12

62 60

15 f 2.0 81 f 2.2 2 f 0.8 Not reached

12

64

25 & 2.2

12

50

15 f 0.7

75-110

80 f 1.8

The presynaptic neuron was stimulated during the entire experimental procedures with supramaximal pulses at a frequency of 1/minute. The average, followed by the standard error of the mean. c Cyclic AMP or the catalytic subunit of diphosphoinositide kinase (DPIK) may have generated effects when given with or without DPI (diphosphoinositide), but the effect was maintained only when DPI was also given. This suggests that cyclic AMP acted through diphosphoinositide kinase activation, since the only function of DPI seems to be to serve as the specific substrate for DPIK. a

9

s

0” F

k9

El

0

731

2

5

r

tl9

: 0 2.

50

CLARA TORDA

IV. Current Concepts of Depolarization and Hyperpolarization

Our recent concepts of the molecular processes resulting in conduction of stimuli and generation of end-plate and postsynaptic potentials were most extensively reviewed by Katz (1966, 1969).Depolarization of the presynaptic neuron promotes influx of Ca2 into the presynaptic terminal. This leads to release of ACh. After transsynaptic diffusion (with a measured minimum of 0.1 msec duration), the ACh forms a complex with one of its postsynaptic receptors, and through a Ca2+-dependent mechanism, the postsynaptic neuron is depolarized. The model presented in the study described here is the only available insight into the molecular mechanisms between formation of the ACh-nicotinic receptor complex and depolarization of the postsynaptic neuron (see also Torda, 1972a-g, 1973 a-f). As stated before, this model is based on the experimental evidence (Torda, 1973a,b), that one of the nicotinic receptors of ACh is the regulatory subunit of triphosphoinositide phosphomonoesterase. When complexing with ACh, the regulatory subunit ceases to inhibit the enzymatic activity of the catalytic subunit of triphosphoinositide phosphomonoesterase, and TPI is dephosphorylated to DPI. The concurrent quantitatively predetermined release of Ca2 alone or in cooperation with other local processes (see p. 5) channels the local electric charges toward depolarization (e.g., opening of the Na+ channels). The random firing of nerve cells and neurons, and the distribution of the latency times as a function of stimulus strength have been studied from many points of view, with the conclusion that depolarization depends on conductance across the Na+ channels (Bennet, 1967; Blair and Erlanger, 1932; Cole, 1968; Cole et aE., 1970; Ehrenstein et al., 1970; Frankenhaeuser and Hodgkin, 1957; Frankenhaeuser and Huxley, 1964; FritzHugh, 1961; Gastwirth, 1967;Goldman, 1943; Hille, 1968, 1970a,b; Hodgkin and Huxley, 1952a,b; Hodgkin and Katz, 1949; Landahl, 1941; Lax, 1966; Lecar and Nossal, 1971; Monnier and Jasper, 1932; Pecher, 1939; Poussart, 1969; Stein, 1967; Ten Hoopen and Verveen, 1963; Verveen and Derksen, 1968). Depolarization (EPSP) consists of about a 15-25 mV decrease of the charge difference between the two membrane surfaces (inside positive), the critical membrane potential for generation of a spike in the postsynaptic neuron (Eccles, 1964; Grundfest, 1967; Hubbard et al., 1969). Depolarization may be entirely voltage dependent, having a rate constant without a memory for preceding events (Gage and Armstrong, 1968; Kordas, 1969; Magleby and Stevens, 1972).These local voltage changes may result from Ca2 movements (away) (Diamond and Wright, 1969). Most researchers assume, however, that the decrease of charge differences across the membrane results from passage of charged particles (passive transport of Na +,other cations, organic dipoles, etc.) (Frankenhaeuser, 1968;Hodgkin and Huxley, 1952a,b; Hodgkin et al., 1952a,b) . Based mainly +

+

+

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

51

on voltage-clamp experiments, the Na+ channels are believed to have a radius approximately of 3 A, a resistivity of 100 ohms (in frog membranes in Ringer’s solution), a convergence resistance of 5 x lo8 ohms, a conductance of 1 nmho. The effective fixed charge density per pore was calculated as 200 m M (Hille, 1971). The maximum carried current was measured in The Na+ flux was calculated in frog as 1.3 x lo8 ions sec-l (Hille, 1970~). squid axon as 400 ions nsec-l (Hille, 1970~).Observations on the ion selectivity of the Na+ channel and the passage of certain ions with a diameter larger than 3 A, lead Hille (1971) to the conclusion that the channel wall is lined with O,, and the ions (including Na+) are transported by forming H bonds with 0,. In the presence of tetrodotoxin the charges may be carried by Ca2 or organic ions (Bezanillaand Armstrong, 1973; Cuervo and Adelman, 1970; Geduldig and Jung, 1968; Hille, 1972; Katz and Miledi, 1967; Koppenhofer, 1967; Narahashi, 1964, 1966). The ion-sensitive membrane channels are opened and closed not by the transported ions (Latorre et al., 1972), or amounts of the Caa+bound to the orifice of the Na+ channels (Keynes, 1972), but by cooperativity (Hill and Cheng, 1971) of the conformational changes of protein subunits that form the channel walls (Hille, 1972). These conformational changes result from physicochemical reasons (e.g., formation of the ACh-receptor complex) or by minimal changes of local electric charge [e.g., membrane potential changes due to depolarizing currents, Ca2 movement (Diamond and Wright, 1969; Keynes, 1972; Magleby and Stevens, 1972)]. The model of a depolarization-hyperpolarization cycle described in the present study describes a molecular chain reaction through which ACh may generate a quantitatively predetermined movement of Ca2 and changes of ligands (Torda, 1973e). Depolarization seems to depend on, and concurs with, the release of membrane-bound Ca2 (Frankenhaeuser, 1957; Frankenhaeuser and Hodgkin, 1957; Hodgkin and Keynes, 1957; Katz, 1966, 1969). In the membranes Ca2+ seems to be bound on negatively charged groups of phospholipids (Skou, 1951; Feinstein, 1964). Changes in the concentration of Caa (of the membrane) shifts some of the parameters of the Na+ and K + conductance along the voltage axis (Brisna and Frankenhaeuser, 1972; Diamond and Wright, 1969; Eisenman, 1962 ; Fishman and Volkenstein, 1971; Fishman et al., 1971; Frankenhaeuser and Moore, 1963; Hille, 1968; Keynes, 1972; Le’car and Nossal, 1971 ; Taylor and Rojas, 1971;Yoshikami and Hagins, 1971). Possible interrelation between the action OJ ACh and phosphoinositides has been reviewed by Durell and Garland (1969). A possible role of phosphoinositides in conduction of nerve membranes has been postulated’on the following grounds: (1) Phosphoinositides occur in the myelin sheet of neurons +

+

+

+

+

52

CLARA TORDA

(Eichberg and Dawson, 1965; Hawthorne and Kai, 1970). (2) TPI and DPI have a rapid turnover rate (Dawson, 1960). (3) 32P is rapidly incorporated into TPI in the resting neuron (Yagiharaet al., 1969). (4) Membrane permeability may be under the control of TPI and DPI (Hendrickson and Reinertsen, 1969).The water-solubleTPI and DPI readily complex with protein, and both TPI and DPI (with or without protein) readily complex with Ca2+. Complexing with Ca2 renders the complexes water insoluble (Fullington and Hendrickson, 1966; Hendrickson, 1969; Hendrickson and Fullington, 1965; Hendrickson and Reinertsen, 1969). (5) Dephosphorylation of TPI to DPI (Kai and Hawthorne, 1969), and dephosphorylation of DPI (Durell and Garland, 1969) concur with release of Ca2 (6) During impulse conductance in the squid axon, Ca2+is discharged into the axoplasm from the interior surface of the axonal membrane (Hodgkin and Keynes, 1957), in amounts that are consistent with the hydrolysis of TPI during neuronal conductance (Hendrickson and Reinertsen, 1969). (7) In artificial membranes, distribution of TPI and triphosphoinositide phosphomonoesterase on one side of the membrane results in asymmetrical distribution of Ca2 and ligand charge on the two sides of the membrane (Papahadjopoulos and Ohki, 1969). A possible relationship between ACh activity and phospholipids during synaptic activity has been assumed (Durell et al., 1969) for the following reasons : (1) The speed and relative rate of synthesis to hydrolysis of ACh and TPI are similar. The hydrolysis is over 100 times faster than the synthesis (Kai et al., 1968). (2) In the presence of ACh the synapses accumulate 32Plabeled TPI faster in vitro, and labeled TPI accumulates in vitro during prolonged administration of various transmitter substances (ACh, norepinephrine, 5-hydroxytryptamine, y-aminobutyric acid, amino acids (Hokin and Hokin, 1955a,b, 1958a,b, 1960,1965,1966, 1969, 1970a, b). (3) Phospholipid catabolism has been temporarily observed to exceed the synthesis during convulsions (Torda, 1954a,b,c). (4) During prolonged presynaptic stimulation, the postsynaptic labeled phospholipids significantly increase (Larrabee, 1968; Larrabee et al., 1963; Larrabee and Leicht, 1965; Larrabee and Brinley, 1968). Unfortunately, Hokin and Hokin and Larrabee and collaborators interpreted their observations as proof that the process of neuronal activation depends on kinase-dependent synthetic processes. The technical difficulties that gave rise to such erroneous interpretation has already been pointed out by Durell et al., (1969). Experimental evidence has already been collected (Torda, 1972f,g, 1973a-d) to document that the synthetic, kinasedependent processes occur during the recovery processes, and the fast activity-dependent processes result from the hydrolysis of TPI. Relationship between TPIphosphorylation and release (mobilization)of membranebound Ca2+. TPI is a more potent chelating agent than EDTA or EGTA (Dawson, 1965; Hawthorne and Kai, 1964). Hendrickson and Reinertsen +

+.

+

53

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

(1969) have shown that at pH 7 the conversion of TPI to DPI releases 70% of bound Ca2+ and 25y0 of the ligand charge becomes altered. One may estimate that the complex formation of 1 x M ACh with its nicotinic receptor may release about K x 1.75 x M Caa+ with a concurrent decrease of K x 0.6 x ligand charge, where K is the turnover number of TPI by triphosphoinositide phosphomonoesterase. Phosphorylation of DPI to TPI results, on the other hand, in similar increase of membranebound Caa+ and changes of the ligands in the direction that results in hyperpolarization (Torda, 1973e,h, 1974a,b). Hyperpolarization (repolarization) has been calculated by Keynes and Lewis (1951) to be grossly the bioelectric equivalent of the changing sums of the equilibrium potentials of the migrating K + and C1- ions (calculated by the Nernst equation). Therefore, this hyperpolarization may be effected by selective ion transport through K channels, after these channels were opened (or the necessary conformational changes were initiated) by the voltage changes delivered through the approaching Caa ions. (Dawson, 1965; Hawthorne and Kemp, 1964; Hendrickson and Reinertsen, 1969). According to the model of depolarization-hyperpolarization described in the present study, this movement of Caa+ results from the phosphorylation of DPI to TPI by diphosphoinositidekinase, and the concurrent quantitative increase of the local chelations (Torda, 1973g, 1974c). In light of recent experiments, the concept of single ion-sensitivechannels serving to transport Na , K , and C1- in the postsynaptic neuron is being abandoned in favour of the dualistic concept (Dodge, 1961, 1963; Edelman, 1961;Koppenhofer, 1967; Narahashi et ul., 1964, 1966). This view is further supported by the experimental evidence that the conductance of a single channel at the postsynaptic membrane of the frog is 0.2 to 0.3 nmholchannel (Anderson and Stevens, 1973). Voltage-clamp experiments in presence of tetraethylammonium suggest that the K channel has unequal openings (Armstrong and Hille, 1969). Based on the rate of conductance of the K + and Na+ channels in the squid axon, the relative number of the K + and Na+ channels was calculated as 50:l (Armstrong, 1966, 1971; Armstrong and Hille, 1969; Hille, 1970). Phosphorylation of DPI to TPI concurs with binding of Caa (Dawson, 1965; Hawthorne and Kai, 1970; Torda, 19738, 1974~).The TPI-Ca2+ complex is hydrophobic and reorganizes the membrane to its original state. This binding of Caa+ (with the concurrent ligand movements) channels the local electric charges toward hyperpolarization (Diamond and Wright, 1969; Keynes, 1972; Torda, 19738, 1974a-c). +

+

+

+

+

+

V. General Discussion

Instead of conclusive proof, the collected observations offer only a probabilistic evidence about a molecular mechanism that is capable of

54

CLARA TORDA

generating one of the depolarization-hyperpolarization cycles. The coexistence of other equally valid molecular mechanisms, auxiliary and emergency measures cannot be excluded. This proposed molecular model consists of the following chain reaction. Acetylcholine appears at the postsynaptic membrane-complex formation with specific receptor (e.g., a regulatory subunit of postsynaptic triphosphoinositide phosphomonoesterase) -release (activation) of the catalytic subunit-dephosphorylation of TPI to DPI during transmembrane passage of the complex: (a) delivery of DPI into the postsynaptic neuron; (b) concurrent quantitative release of membranebound Ca2+-channeling of the local electric fields toward depolarization (probably through cooperation of various mechanisms). The arriving DPI is phosphorylated by diphosphoinositide kinase (activated by cyclic AMP) to TPI-concurrent increase of membrane-bound Ca2 (probably together with other processes) channels the local electric fields towards hyperpolarization. This model may be substantiated by already existing experimental evidence: Acetylcholine is encoded with several messages. On forming a complex with one of its postsynaptic nicotinic receptors, the message “depolarize” is read out. The selection results from the nature of the receptor molecule. On combining with ACh this receptor acquires the ability to initiate only those processes that are necessary for the execution of depolarization; e.g., on combining with ACh, the regulatory subunit of triphosphoinositide phosphomonoesterase ceases to inhibit the enzymatic activity of the catalytic subunit, and TPI is dephosphorylated to DPI. This chain reaction supplies the necessary processes to execute depolarization: (1) direct control of the local electric fields (e.g., by loss of membrane-bound Caa+); (2) indirect control by the cooperation of many factors (e.g., conformational changes, ionophoresis). i. Direct control of the local electric fields may be effected through the release of a quantitatively predetermined amount of membrane-bound Ca2+.Since DPI is a weaker chelating agent than TPI (Dawson, 1965; Hendrickson and Reinertsen, 197l), concurrently with the enzymatic dephosphorylation of TPI, membrane-bound Ca2 is released in amounts sufficient (Torda, 1973e, 1974a) to channel the local electric processes toward depolarization (Magleby and Stevens, 1972). ii. Zndirect control of the local electricfields. (a) On combining with ACh, the receptor undergoes conformational changes. These changes may directly channel the local electric fields toward depolarization and may affect it indirectly through specific changes in ionophoresis. The temporary changes in the receptor molecule act as widening of the membrane pores and may facilitate passive transmembrane transport of specific small ions, e.g., Na +,K . (b) The loss of membrane-bound Ca2 may similarly affect passive Na and K + transport. According to Hodgkin and Huxley (1952a,b), depolarization +

+

+

+

+

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

55

is the bioelectric equivalent of the changing sums of the migrating ions (calculated by the Nernst equation). (c) Several other processes may also participate. Triphosphoinositide phosphomonoesterase has been isolated from many tissues (Salway, et. al., 1967, 1968), including the postsynaptic membrane (Torda, 1972g). I t has been isolated from the myelin at high concentration, and from the microsomes (Salway et a&., 1967) and the postsynaptic membrane at low concentrations of a similar order of magnitude. Seemingly, the low enzyme concentrations are better equipped to execute processes that depend on quanta of ACh, and larger enzyme concentrations are better suited to participate in trophic processes. Enzyme-function studies revealed that other enzymes may also resort to a similar dualistic mechanism of action (Koelle, 1969). The time required between the formation of the ACh-receptor complex and the molecular chain reaction resulting in depolarization may meet the time requirements of fast synaptic processes. In chemically transmitting synapses the shortest measured synaptic delay was 0.2 msec. The formation of the ACh-receptor complex seems to be diffusion limited and may occur in a fraction of 0.1 msec. Formation of the ACh-receptor complex concurs with activation of the catalytic subunit without added time involvement. Since the catalytic subunit seems to constantly hold in place its specific substrate, the TPI (Chang and Ballou, 1967; Prottey et al., 1968), time is not required for the search for substrate. The turnover rate of TPI may be short: the maximum rate observed was 14/msec. The end products of this proposed model are: (a) membrane depolarization, and (b) delivery of DPI into the postsynaptic neuron. Hyflerpolarizationseems to result from a molecular chain reaction devised to handle the phosphorylation (and transport) of DPI by diphosphoinositide kinase, activated ,by cyclic AMP. Sufficientexperimentalevidence is already available to clarify the details of this process, e.g. :(1) mechanism generating an accumulation of CAMP during activity of the nervous system (central and peripheral) ; (2) relationship between intraneuronal accumulation of cAMP and membrane hyperpolarization: (3) mechanism of action of CAMP (e.g., a postsynaptic specific receptor, and its activation). Mechanism of readout and execution of the message “hyperpolarize” that is encoded in the cAMP molecule by the receptor. I. Mechanism of accumulation of cyclic AMP in various neurons. During stimulation of several central and peripheral presynaptic neurons dopaminergic (or catecholaminergic) interneurons are concurrently activated (Anden et d.,1966; Anton and Sayre, 1964; Biscoe and Curtis, 1966; Biscoe et al., 1966; Bjorklund et al., 1970; Carlsson et al., 1964; Engberg and Ryall, 1966; Kandel et al., 1967; Kobayashi and Libet, 1970; Libet, 1970; MacAfee

56

CLARA TORDA

et al., 1971; McLennan, 1965, 1970; McLennan and York, 1967; Matthews and Raisman, 1969; Nishi, 1970; Reinert, 1963; Riker, 1970; Ryall et al., 1963; Siegrist et al., 1968; Thoennen et al., 1967; Tosaka et al., 1968; Tosaka and Libet, 1970; Volle and Hancock, 1970; Weight and Salmoiraghi, 1966a,b; Williams and Palay, 1969; York, 1967). When activated, these dopaminergic neurons release dopamine at their postsynaptic terminals. The released dopamine activates the postsynaptic adenyl cyclase to synthesize and release cAMP into the postsynaptic neuron (Brown and Makman, 1972; Robison et al., 1971, Zanella and Rall, 1970). The released cAMP either forms a complex with one of its specific receptors, e.g., the regulatory subunit of diphosphoinositidekinase (Torda, 1972a,b,h), or perhaps the protein kinase isolated from the postsynaptic membrane by Greengard et al. (1972) ;or it is hydrolyzed by its specific phosphodiesterase located intrapostsynaptically near the synaptic gap (Florendo et al., 1971). 2. Hyperpolarization generated by CAMP. In neurons that accumulate cAMP during activity, a causal relationship seems to exist between cAMP and membrane hyperpolarization. Hyperpolarization has been also generated by an increase of intraneuronal cAMP through electrophoretic introduction of endogenous and exogenous CAMP,an accumulation by increased synthesis or release of cAMP (e.g., by administration of dopamine, norepinephrine). Decreased hyperpolarization occurs in presence of inhibitors of adenyl cyclase, and by increased activity of the specific phosphodiesterase that hydrolyzes cAMP (Bloom and Goldman, 1966; Bloom et al., 1965; Breckenridge and Bray, 1970; Breckenridge and Jonhson, 1969; Hoffer et al., 1970; Kakiuchi and Rall, 1970; Krishna et al., 1970; Libet and Tosaka, 1968; McLennan, 1970; Rall and Gilman, 1970; Rall and Sattin, 1970; Robison et al., 1971; Shimizu et al., 1970; Siggins et al., 1971; Singer and Goldberg, 1970; Steiner and Meyer, 1966; Torda, 1971; Weiss and Costa, 1967; Zanella and Rall, 1970). 3. Mechanism of generation of hyperpolarization by CAMP. cAMP is encoded with several messages. Only one of these messages is read out during complex formation with one of its specific receptors. The selected message depends for execution on the processes that are activated by the specific receptor during complex formation with the CAMP. Biochemical in vitro experiments led to the conclusion that one of the specific postsynaptic receptors is the regulatory subunit of diphosphoinositide kinase (Torda, 1972a,b,h). In order to qualify for the role of being instrumental to generate hyperpolarization, this regulatory subunit must be able to generate processes that can execute membrane hyperpolarization : e.g., channel the local electric field toward hyperpolarization by direct or indirect means. The best known quantitatively described processes that qualify are: increase of membrane-bound Caa , ionophoresis, perhaps also conformational changes. +

57

A DEPOLARIZATION-HYPERPOLAREATION CYCLE

Experimental evidence supports the assumption that on combining with CAMP, this receptor is able to generate the necessary processes to execute membrane hyperpolarization. On combining with CAMP, the regulatory subunit of diphosphoinositide kinase ceases to inhibit the enzymatic activity of the catalytic subunit and DPI is phosphorylated to TPI. Since TPI is a more potent chelating agent than DPI, the enzymatic phosphorylation concurs with the increase of the membrane-bound Caa with a quantitatively predictable amount (Torda, 1973g, 1974~).This increase may: (a) channel directly the local electric fields toward hyperpolarization (Magleby and Stevens, 1972),or (b) cause hyperpolarization by indirect means, e.g., special ionophoresis. According to Keynes and Lewis (1951) hyperpolarizationis the bioelectric equivalent of the changing sums of the equilibrium potentials of the migrating C1- and K + ions (calculated by the Nernst equation). If ionophoresis partakes in the generation of hyperpolarization, it is probably generated by the cooperation of various processes: e.g., conformational and other changes that result from the complex formation between CAMP and the receptor molecule, increase of membrane-bound Caa The changes of the special ionophoresis may result from special carrier, and from tightening the membrane pores. The closing pores terminate the passive Na+ transport, but seem to permit the transport of C1- (Durell et al., 1969; Edelman, 1961; Fishman et al., 1971; Glynn et al., 1965; Mule, 1969; Papahadjopoulos, 1970, 1971; Sheltaway and Dawson, 1966). These observations suggest that the regulatory subunit of diphosphoinositide kinase qualifies to be one of the specific receptors of CAMP, since on combining with CAMP, it is able to generate the processes required to execute fast membrane hyperpolarization. Supporting in vivo observations are: (a) cAMP is a very potent activator of diphosphoinositidekinase; (b) the regulatory subunit of diphosphoinositidekinase has a greater affinity to CAMP, than to the catalytic subunit; (c) the hyperpolarizing effect of cAMP seems to parallel the activity of diphosphoinositidekinase; (d) the hyperpolarizing effects of cAMP seems to require the presence of diphosphoinositide. Even though the dopaminergic interneurons are equally stimulated in both intact and curarized synapses, cAMP accumulates in a chemically detectable amount only in noncurarized preparations (MacAfee et al., 1971). Since diphosphoinositide is delivered into the postsynaptic neuron during depolarization (Torda, 1972e), the CAMP-activated diphosphoinositide kinase can find its specific substrate only in the active, not in the curarized, postsynaptic neuron. This suggests the possibility of a quantitative relationship between the activity of diphosphoinositide kinase and the amount of bound CAMP. The in vitro and in vizlo experiments validate the assumption that the regulatory subunit of diphosphoinositide kinase is one of the specific postsynaptic receptors of CAMP. Activation of diphosphoinositide kinase by CAMP seems to be one of the molecular mechanisms that is able to couple +

+.

58

CLARA TORDA

in time and space the formation of CAMP-receptor complex and hyperpolarization of the postynaptic neuron. VI. General Conclusions

Experimental evidence has been collected to validate the proposed model of a molecular mechanism capable to generate one of the depolarizationhyperpolarization cycles. In cholinergic synapses ACh seems to use the regulatory subunit of triphosphoinositide phosphomonoesterase as its specific postsynaptic nicotinic receptor. On combining with ACh, the regulatory subunit ceases to inhibit the catalytic subunit, and TPI is dephosphorylated to DPI. The concurrent changes channel the local electric fields toward depolarization. The entire process may be completed in 0.1 msec. Therefore, activation of triphosphoinositide phosphomonoesterase by ACh may be the molecular mechanism that couples in time and space the formation of the ACh-receptor complex and the depolarization of the postsynaptic neuron. During postsynaptic depolarization DPI is delivered into the postsynaptic neuron. Hyperpolarization seems to be the bioelectric equivalent of the biochemical and other processes generated to handle this DPI: In neurons that accumulate cAMP during activity, cAMP may use the regulatory subunit of diphosphoinositide kinase as its specific postsynaptic receptor. On combining with CAMP, the regulatory subunit ceases to inhibit the enzymatic activity of the catalytic subunit and DPI is phosphorylated to TPI. The concurrent molecular processes channel the local electric fields toward hyperpolarization. Therefore, activation of diphosphoinositide kinase by cAMP may be the molecular mechanism that couples in time and space the formation of a CAMP-specific receptor complex and hyperpolarization of the postsynaptic neuron. Should other depolarizing agents be able to activate triphosphoinositide phosphomonoesterase, and other hyperpolarizing agents activate diphosphoinositide kinase, the proposed model may assume a more extensive significance. VII. Summary

Based on biochemical and bioelectric experiments, and the available literature, a model of a molecular chain reaction able to generate a depolarization-hyperpolarization cycle has been formulated. I n cholinergic synapses, acetylcholine (ACh) may use the regulatory subunit of triphosphoinositide phosphomonoesterase as one of its nicotinic receptors. When united with ACh, this regulatory subunit concurrently ceases to inhibit the enzymatic activity of the catalytic subunit, and triphosphoinositide (TPI) is dephosphorylated to diphosphoinositide (DPI). This

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

59

dephosphorylation concurs with a quantitatively predetermined release of the membrane-bound Ca2 and ligands. The released Ca2 carries sufficient voltage changes to initiate depolarization either alone, or in cooperation with conformational changes and passive Na+ (or other means of charge) transport. The bioelectric equivalent of these processes is the depolarization of the postsynaptic neuron. Repolarization (hyperpolarization) seems to be the bioelectrie expression of the processes that concur with phosphorylation of the DPI that reached the postsynaptic neuron during depolarization (dephosphorylation of TPI). Diphosphoinositide is phosphorylated by its specific enzyme, diphosphoinositide kinase, an enzyme found in the postsynaptic neuron in an inactive form. During gresynaptic dopaminergic interneurons are activated in several synapses. When activated, these interneurons release cAMP into the postsynaptic neuron. This cAMP may use as one of its postsynaptic receptors the regulatory subunit of diphosphoinositide kinase. When united with CAMP, the regulatory subunit ceases to inhibit the enzymatic activity of the catalytic subunit, and DPI is phosphorylated to TPI. This phosphorylation concurs with a quantitatively predetermined increase of the membranebound Ca2+ and changes in ligands. Binding Ca2+ ions channels the local voltage toward hyperpolarization, either by voltage changes alone, or by cooperativity of various processes, including changes of electric charges toward hyperpolarization, K + and C1- passage through K +-sensitive channels, closure of Na channels, etc. Both the molecular chain reactions that lead to depolarization arid hyperpolarization occur with short latency. The ACh-dependent Ca2 release may be initiated in less than 0.1 msec, the cyclic AMP-dependent repolarization probably requires somewhat longer latency, but probably the minimum latency is of the order of magnitude of one or a few milliseconds. One may assume that whatever the energy requirements, they may be supplied by ATP. +

+

+

+

REFERENCES Anden, N. E., Fuxe, K.,and Larsson, K. (1966). Exfierientia 22,842. Anderson, C. R., and Stevens, C. F. (1973). Biophys. J . 13, 71a. Anton, H. E., and Sayre, D. F. (1964). J . Pharmacol. Exp. Thcr. 145, 326. Araki, T., and Otani, T. (1959). J . Neurophysiol. 18, 472. Armstrong, C. M. (1966). J . Gen. Physiol. 50, 541. Armstrong, C. M. (1971). J . Gen. Physiol. 58, 413. Armstrong, C. M., and Hille, B. (1969). J . Gen. Physiol. 59, 388. Azcurra, J. M., and De Robertis, E. (1967). Int. J . Neurofiharmacol. 6, 15. Baker, P. F. (1972). Progr. Biofihys. Mol. Biol. 24, 1. Bangham, A. D. (1968) Progr. Biophys. 18, 29. Bangham, A. D., and Dawson, R. M. C. (1962). Biochim. Biophys. Acta 59, 103. Barlow, R. B. (1969). “Introduction to Chemical Pharmacology.” Methuen, London.

60

CLARA TORDA

Belleau, B. (1967). Ann. N. Y. Acad. Sci. 144, 705. Belleau, B., and Lavoie, J . (1968). Can.J. Biochcm. 46, 1397. Bennet, M. R. (1967). Bwphys. J. 7, 151. Beychock, C. (1965). Biochem. Phmacol. 14, 1249. Bezanilla, F., and Armstrong, C. M. (1973). Biophys. J. 13, 243a Biscoe, T. J., and Curtis, D. R. (1966). Science 151, 1230. Biscoe, T. J., Curtis, D. R., and Ryall, R. W. (1966). Znt. J. Neuropharmacol. 5, 429. Bjorklund, A,, Cegrell, L., Falck, B., Ritzen, M., and Rosengren, E. (1970). Acta. Physiol. Scand. 78, 334. Blackman, J. G., Ginsborg, B. L., and Ray, C. (1963). J . Physiol. (London) 167,355 and 389. Blair, E. A., and Erlanger, J. (1932). Proc. Soc. Exp. Biol. Med. 29, 926. Bloom, B. J., and Goldman, J. M. (1966). Advan. Drug Res. 3, 121. Bloom, F. E., Costa, E., and Salmoiraghi, G. C. (1965). J. Pharmacol. Exp. Ther. 150, 244. Bray, G. A. (1960). Anal. Biochem. 1, 279. Breckenridge, B. McL., and Bray, J. J. (1970). Advan. Biochem. Pharmacol. 3, 325. Breckenridge, B. McL., and Johnston, R. E. (1969). J. Histochem. Cytochem. 17, 505. Brisna, R. T., and Frankenhaeuser, B. (1972). Acta. Physiol. Scand. 85, 237. Brown, J. H., and Makman, M. H. (1972). Proc. Znt. Congr. Pharmacol. 5th. 1972 p. 113. Carlsson, A., Falck, B., Fuxe, K., and Hillarp, N. A. (1964). Acta Physiol. Scand. 60, 112. Chang, M., and Ballou, C. E. (1967). Biochem. Bwphys. Res. Commun. 26, 199. Changeux, J.-P., and Podleski, T. R. (1968). Proc. Nut. Acad. Sci. US.59, 944. Changeux, J.-P., Podleski, T. R., and Meunier, J. C. (1965). J . Gen. Physiol. 54, 225. Chamock, J. S. (1963). J. Neurochem. 10, 219. Chen, P. B., Jr., Toribara, T. V., and Warner, H. (1956). Anal. Chem. 28, 1756. Chothia, C. (1969). Nature (London) 225, 36. Chothia, C., and Pauling, P. (1969). Proc. Nut. Acad. Sci. US.63, 1063. Cole, K. S. (1968). “Membranes, Ions, and Impulses,” University of California, Berkeley. Cole, K. S., Guttman, R., and Bezanilla, F. (1970). Proc. Nut. Acad. Sci. U.S. 65, 884. Coombs, J. S., Eccles, J. C., and Fatt, P. (1955). J. Physiol. (London) 130, 326. Cuatrecasas,J. (1970). J. Bwl. C h . 245, 3059. Cuervo, L. A., and Adelman, W. J., Jr. (1970). J. Gen. Physiol. 55, 309. Curtis, D. R. (1964). “Phgs. Tech. Biol. Res.” 6, 144. Curtis, D. R., and Ryall, R. W. (1966). Exp. Brain Res. 2, 49 and 66. Dawson, R. M. C., (1960). Ann. N.Y. Acad. Sci. 165, 744. Dawson, R. M. C. (1965). Biochem. J . 97, 234. Dawson, R. M. C., and Thompson, W. (1964). Biochem. J. 91, 244. Del Castilio, J., and Katz, B. (1957). Proc. Roy. Soc., Ser. ( B ) 146, 339. De Robertis, E. (1971). Science 171, 963. De Robertis, E., Fiszer, S., Pasquini, J. M., and Soto, E. F. (1969). J . Neurobiol. 1, 41. De Robertis, E., Lunt, G. S., and La Torre, J. L. (1971). Mol. Pharmacol. 7, 97. Diamond, J. M., and Wright, E. M. (1969). Ann. Rev. Physiol. 31, 581. Dodge, F. A. (1961). “In Biophysics of Physiological and Pharmacological Actions,” Publ. No. 69, p- 112. Amer. Ass. Advan. Sci., Washington, D.C. Dodge, F. A. (1963). Ph.D. Thesis, Rockefeller University, New York. Downie, D. E. (1970). J. Them. Bwl. 28, 297. Durell, J., and Garland, J. T. (1969). Ann. N. Y. Acad. Sci. 165, 743. Durell, J., Garland, J. T., and Friedel, R. 0. (1969). Science 165, 862. Eccles, J. C. (1961). Erg. Physiol. Bwl. Chem. Exp. Pharmacol. 51, 299. Eccles, J. C. (1964). “The Physiology of Synapses.” Springer-Verlag,Berlin and New York. Eccles, J. C. (1969). Fed. Prc:. Fed. Amn. Soc. Exp. BWl. 28, 90. Eccles, J. C., Fatt, P., and Koketsu, K. (1954). J. Physiol. (London) 126, 524

A DEPOLARIZATION-HYPERPOLARIZATION CYCLE

61

Eccles, J. C., Eccles, R. M., and Fatt, P. (1956). J . Physiol. (London) 131, 154. Eccles, J. C., Eccles, R. M., Iggo, A., and Lundberg, A. (1961). J. Physiol. (London) 159, 461. Eccles, R. M. (1952). J . Physiol. (London) 117, 181. Eccles, R. M. (1955). J. Physiol. (London) 130, 572. Eccles, R. M., and Libet, B. (1961). J. Physiol. (London) 157, 484. Edelman, I. S. (1961). Annu. Rev. Physiol. 23, 37. Edwards, C., Bunch, W., Mafray, P., Marois, R., and Meter, D. (1970).J. Membrane Biol. 2, 119. Ehrenpreis, S., Fleisch, J. H., and Mittag, T. W. (1969). Pharmacol. Rev. 21, 131. Ehrenstein, G., Lecar, H., and Nossal, R. (1970). J. Gen. Physiol. 55, 119. Eisenman, G. (1962). Biophys. J. 2, 259. Eldefrawi, M. E., Britten, A. G., and Eldefrawi, A. T. (1971). Science 173, 338. Ellis, R. B., Galliard, T., and Hawthorne, J. N. (1963). Biochem. J. 88, 125. Engberg, I., and Ryall, R. W. (1966). J . Physiol. (London) 185, 298. Erlichman, J., Hirsch A. H., and Rosen, 0. M. (1971). Proc. Nut. Acad. Sci. US.68, 731. Feinstein, M. B. (1964). J. Gen. Physiol. 48, 357. Ferrendelli, J. A., Steiner, A. L., McDougal, D. B. and Kipnis, D. M. (1970). Biochem. Biophys. Res. Commun. 41, 1061. Fishman, S. N., and Volkenstein, M. V. (1971). Biochem. Biophys. Acta 241, 697. Fishman, S. N., Choporov,,B. I., and Volkenstein, M. V. (1971). Biochim. Biophys. Acta 225, 1. FitzHugh, R. (1961). Biophys. J . 1, 445. Florendo, N. T., Barnett J. J., and Greengard, P. (1971). Science 173, 745. Folch, J. (1949). J. Biol. Chem. 177, 497 and 505. Frank, K. (1961). Annu. Rev. Physwl. 23, 357. Frank, K., and Fourtes, M. G. F. (1955). J . Physiol. (London) 130,625. Frank, K., and Fourtes, M. G. F. (1956). J. Physiol. (London) 134, 451. Frankenhaeuser, B. (1957). J. Physiol. (London) 137, 245. Frankenhaeuser, B. (1968). Prog. Biophys. Mol. Biol. 18, 97. Frankenhaeuser, B., and Hodgkin, A. L. (1957). J. PhysioE. (London) 137,218. Frankenhaeuser, B., and Huxley, A. F. (1964), J. Physiol. (London) 171, 302. Frankenhaeuser, B., and Moore, L. E. (1963). J. Physiol. (London) 169, 431. Fullington, J. G., and Hendrickson, H. S. (1966). J. Biol. Chem. 241, 4098. Fulpius, B. W., Klett, R. P., Casper, D., and Reich E. (1973). “Pharmacology and the Future of Man,” Vol. 5, p. 68. Karger, Basel. Furshpan, E. J., and Potter, D. D. (1959). J. Physiol. (London) 145, 326. Gage, P. W., and Armstrong, C. M. (1968). Nature (London) 218, 363. Galliard, T., Mickell, R. H., Hawthorne, J. N. (1965). Biochim. Biophys. Acta 106, 551. Gastwirth,J. L. (1967). “A Renewal Theoretic Approach to a First Passage Time Problem Occurring in a Neuron Firing Model,” Tech. Rep. No. 77, Dept. of Statistics, Johns Hopkins University, Baltimore, Maryland. Geduldig, D., and Jung, D. (1968). J. Physiol. (London) 189, 347. George, W. J., Polson, J. B., O’Toole, A. G., and Goldberg, N. D. (1970). Proc. Nat. Acad. Sci. U.S.66, 398. Gill, G. N. (1965). Prog. Med. Chem. 4, 39. Gill, G. N., and Garren, J. D. (1970). Biochem. Biophys. Res. Commun. 39, 335. Ginsborg, 8. L., and Guerrero, S. (1964). J. Physiol. (London) 172, 189. Glynn, I. M., and Chappell, J. B. (1964). Biochem. J. 90, 147. Glynn, I. M., Slayman, C. W., Eichberg, J., and Dawson, R. M. C. (1965). Biochem. J. 94, 692.

62

CLARA TORDA

Goldman, D. E. (1943). J. Gen. Physiol. 27, 37. Greengard, P., McAfee, D. A., and Kebabian, J. W. (1972)..Advan. Cyclic Nucleotide Res. 1, 337. Grundfest, €1. (1967). In “The Neurosciences” (G. C. Quarton, T. Melnechuk, and F. 0. Schmitt, eds.), Vol. 1, p. 353. Rockefeller Univ. Press, New York. Haase, J., and Meulen, V. D. (1961). J. Neurophysiol. 24, 510. Hawthorne,J. N., and Kai, M. (1970).In “Handbook of Neurochemistry” (A. Lajtha ed.), Vol. 3, p. 791. Plenum, New York. Hawthorne, J. N., and Kemp, P. (1964). Adu. Lipid Res. 2, 127. Hendrickson, H. S. (1969). Ann. N . Y . Acad. Sci. 165, 668. Hendrickson, H. S., and Ballou, C. E. (1964). J. Biol. Chem. 239, 1369. Hendrickson, H. S., and Fullington, J. G. (1965). Biochemistry 4, 1959. Hendrickson, H. S., and Reinertsen, J. L. (1969). Biochemistry 8, 4855. Hendrickson, H. S., and Reinertsen, J. L. (1971). Biochem. Biophys Res. Commun. 44, 1258. Hess, H., and Lewin, F. (1965). J. Neurochern. 12, 205. Hill, T. L., and Chen, Y-D. (1971). Biophys. J. 11, 685. Hille, B. (1968). Nature (London) 210, 1220. Hille, B. (1970a). Progr. Bwphys. Mol. Biol. 21, 1. Hille, B. (1970b). Biophys. J. 10, 182a. Hille, B. (1970~).J. Gen. Physiol. 58, 599. Hille, B. (1971). Proc. Nut. Acad. Sci. U.S. 68, 280. Hille, B. (1972). Arch. Intern. Med. 129, 71 1. Hille, B., and Cole, K. S. (1971). Proc. Nut. Acad. Sci. U.S. 68, 711. Hodgkin, A. L., and Huxley, A. F. (1952a). J . Physiol. (London) 116, 449, 473, and 497. Hodgkin, A. L., and Huxley, A. F. (1952b). J. Physiol. (London) 117, 500. Hodgkin, A. L., and Katz, B. (1949). J. Physiol. (London) 108, 37. Hodgkin, A. L., and Keynes, R. D. (1957). J. Physiol. (London) 138, 253. Hodgkin, A. L., Huxley, A. L., and Katz, B. (1952). J. Physiol. (London) 116, 424. Hoffer, B. J., Siggins, G. R., and Bloom, F. E. (1970). Aduan. Biochem. Psychopharmacol. 3, 349. Hokin, L. E., and Hokin, M. R. (1955a). Biochim. Biophys. Acta 16, 229. Hokin, L. E., and Hokin, M. R. (1955b). Biochim. Biophys. Acta. 18, 102. Hokin, L. E., and Hokin, M. R. (1958a). J. Biol. Chem. 233, 800, 805, 815, and 822. Hokin, L. E., and Hokin, M. R. (1959). J. Biol. Chem. 234, 1387. Hokin, L. E., and Hokin, M. R. (1960). Znt. Rev. Neurobiol. 2, 100. Hokin, L. E., and Hokin, M. R. (1965). J. Gen. Physiol. 44, 217. Hokin, L. E., and Hokin, M. R. (1966). Proc. Nut. Acad. Sci. U.S. 55, 1369. Hokin, L. E., and Hokin, M. R. (1969). J. Neurochem. 13, 179. Hokin, L. E., and Hokin, M. R. (1970a). J. Neurochem. 16, 127. Hokin, L. E., and Hokin, M. R. (1970b). J. Neurochem. 17, 357. Horhammer, L., Wagner, H., and Richter, C. (1959). Biochem. Z . 331, 155. Hubbard, J. I., Llinas, R., and Quastel, D. M. J. (1969). “Electrophysiological Analysis of Signal Transmission.” Williams & Wilkins, Baltimore, Maryland. Ito, M., Kosthuk, P. G., and Oshima, T. (1962). J . Physiol. (London) 164, 150. Kabachnik, M. I., Brestkin, A. P., Godovikov, N, Y., Michelson, M. J., Rozengart, E. V., and Rozengart, V. I. (1971). Phannacol. Rev. 22, 355. Kai, M., and Hawthorne, J. M. (1969). Ann. N.Y. Acad. Sci. 165, 761. Kai, M., Salway, J. C., Michell, R. H., and Hawthorne, J. N. (1966a). B i o c h . Biophys. Res. Cornmun. 22, 370. Kai, hl., White, G. L., and Hawthorne, J. N. (1966b). Biochem. J. 101, 328. Kai, M., Salway, J. C., and Hawthorne, J. N. (1968). Biochem. J. 106, 791.

A DEPOLARIZATION-HYPERPOLARIZATION

CYCLE

63

Kakiuchi, S., and Rall, T. W. (1968). Mol. Pharmacol. 4, 367. Kandel, E. R., Frazier, W. T., Waziri, R., and Coggeshall, R. E. (1967). J. Neurophysiol. 30, 1352. Karlin, A. (1967a). J. Theor. B i d . 16, 306. Karlin, A. (1967b). Proc. Nut. Acad. Sci. US.58, 1163. Karlin, A. (1969). J. Gen. Physiol. 54, 425. Karlin, A. (1973). “Pharmacology and the Future of Man,” Vol. 5, p. 86. Karger, Basel. Karlin, A., and Winnik, M. (1968). Proc. Nut. Acad. Sci. U.S. 60, 668. Katz, B. (1966). “Nerve, Muscle, and Synapse.” McGraw-Hill, New York. Katz, B. (1969). “The Release of Neural Transmitter Substances.” Thomas, Springfield, Illinois. Katz, B., and Miledi, R. (1967). J. Physiol. (London) 192, 407. Keynes, R. D. (1972). Nature (London) 239, 29. Keynes, R. D., and Lewis, P. R. (1951). J. Physiol. (London) 114, 119. Khomorov-Borisov, N. Y., and Michelson, M. J. (1966). Pharmucol. Rev. 18, 1051. Kier, L. B. (1967). Mol. Pharmacol. 3, 487. Kier, L. B. (1968). Mol. Pharmacol. 4, 70. Kobayashi, H., and Libet, B. (1970). J. Physiol. (London) 208, 353. Koelle, G. B. (1969). Fed. Proc., Fed. Amer. Sac. Exp. B i d . 28, 101. Koketsu, K., Cerf, J. A., and Nishi, S. (1959). J. Neurophysiol. 22, 177. Koketsu, K., Nishi, S., and Soeda, H. (1968). LiJe Sci. 7, 741. Koppenhofer, E. (1967). Ppuegers Arch. Gesamte Physiol. Menschen Tiere 293, 34. Kordas, M. (1969). J. Physiol. (London) 204, 493. Krishna, G., Forn, J., Voigt, K., Paul, M., and Gessa, G. L. (1970). Advan. Biochem. Psychopharmacol. 3, 155. Krnjevic, K., and Lisievicz, A. (1972). J. Physiol. (London) 225, 363. Krnjevic, K., Mitchell, J. F., and Szerb, J. C. (1963). J. Physiol. (London) 165, 421. Landahl, H. D. (1941). Bull. Math. Biophys. 3, 141. Langley, J. N. (1918). J. Physiol. (London) 52, 247. Larrabee, M. G. (1968). J. Neurochem. 15, 803. Larrabee, M. G., and Brinley, F. J., Jr. (1968). J . Neurochem. 15, 533. Larrabee, M. G., and Leicht, W. S. (1965). J. Neurochem. 12, 1 Larrabee, M. G., Klingman, J. D., and Leicht, W. S. (1963). J. Neurochem. 10, 549. La Torre, L. C., Lunt, G. S., and De Robertis, E. (1970). Proc. Nut. Acad. Sci. U.S. 65, 716. Latorre, R., Ehrenpreis, G., and Lecar, H. (1972). J. Gen. Physiol. 60, 72. Lax, M. (1966). Rev. Mod. Phys. 38, 541. Lecar, H., and Nossal, R. (1971). Biophys. J. 11, 1068. Lee, T. C., and Huggins, C. G. (1968). Arch. Biochem. Biophys. 126, 206 and 214. Libet, B. (1970). Fed. Proc., Fed. Amer. Sac. Ex#. B i d . 29, 1943. Libet, B., and Tosaka, T. (1968). J. Neurophysiol. 32, 43. Longo, V. G., Martin, W. R., and Unna, K. R. (1960). J. Pharmacol. Exp. Ther. 129, 61. Lowry, 0. H., Rosenbrough, N. J., Farr, A. G., and Randell, R. J. (1951). J. B i d . Chem. 193, 265. MacAfee, D. A., Schroderet, N., and Greengard, P. (1971). Science 171, 1156. MacLennan, H. (1970). “Synaptic Transmission,” 2nd ed. Saunders, Philadelphia, Pennsylvania. McLennan, H., and York, D. H. (1967). J. Physiol. (London) 189, 399. Magleby, K. L., and Stevens, C. F. (1972). J. Physiol. (London) 223, 151. Matthews, M. R., and Raisman, G. (1969). J. Anat. 105, 255. Miyamoto, E., Kuo, J. F., and Greengard, P. (1969). J. B i d . Chem. 245, 6395. Mokrash, L. G. (1967). LiJe Sci. 6, 1905.

64

CLARA TORDA

Monnier, A. M., and Jasper, H. H. (1932). C . R. Sac. Biol. 110, 547. Monod, J., Wyman, J., and Changeux, J.-P. (1965). J . Mol. Biol. 12, 88. Mule, S. U. (1969). Biochem. Pharmacol. 118, 339. Narahashi, T., Moore, J. W., and Scott, W. R. (1964). J . Gen. Physiol. 47, 965. Narahashi, T., Andersen, N. C . , and Moore, J. W. (1966). Science 155, 705. Nastuk, W. L. (1953). J . Cell. Camp. Physiol. 42, 249. Nelson, P. G., and Frank, K . (1967). J. .Veurophysiol. 30, 1097. Nishi, S. (1970). Fed. Proc., Fed. A m . Sac. Exp. Biol. 29, 1957. Nishi, S., and Koketsu, K. (1960). J . Cell. Comp. Physiol. 55, 15. Nishi, S., and Koketsu, K. (1967). Life. Sci. 76, 2049. Nishi, S., and Koketsu, K. (1968). J . Neurophysiol. 31, 109 and 718. Nishi, S., Soeda, H., and Koketsu, K. (1967). J . Neurophysiol. 30, 114. Nyman, M., and Whittaker, V. P. (1963). Biochem. J . 87, 248. O’Brien, R. D., and Giimour, L. P. (1969). PYOC.Nut. Acad. Sci. U S . 63, 496. O’Brien, R. D., Gilmour, L. P., and Eldefrawi, M. E. (1969). Proc. Nut. Acad. Sci. US.65, 438. Olsen, R., Meunier, J. C., M‘eber, hi., and Changeux, J.-P. (1973). “Pharmacology and the Future of Man,” Vol. 5, p. 118. Karger, Basel. Papahadjopoulos, D. (1970). Biochem. Biophys. Acta. 211, 467. Papahadjopoulos, D. (1971). Biochim. Biophys. Acta 241, 254. Papahadjopoulos, D., and Ohki, S . (1969). Science 164, 1075. Pecher, C. (1939). Arch. Inter. Physiol. 49, 129. Potter, L. T. (1973) “Pharmacology and the Future of Man,” Vol. 5, p. 81. Karger, Basel. Poussart, D. (1969). Proc. Nut. Acad. Sci. US.64, 95. Prottey, C., Salway, J. G., and Hawthorne, J. N. (1968). Biochim. Biophys. Acta 164, 238. Rall, T. W., and Gilman, A. G. (1970). Neurosci. Res. Program, Bull. 8, 221. Rall, T. W., and Sattin, A. (1970). Aduan. Biochem. Psychopharmacol. 3, 113. Reimann, E. M., Brostrom, C . O., Corbin, J. D., King, C. A., and Krebs, €3. G. (1971a). Biochem. Biophys. Res. Commun. 42, 187. Reimann, E. M., Walsh, D. A., and Krebs, E. G. (1971b). J . Biol. Chenz. 246, 1986. Reinert, H. L. (1963). J . Physiol. (London) 167, 18. Renshaw, B. (1941). J . Neurophysiol. 4, 167. Renshaw, B. (1946). J . Neurophysiol. 9, 191. Rikcr, W. K. (1964). J . Pharmacol. Exp. Ther. 145, 317. Riker, W. K. (1970). Fed, PYOL.,Fed. Amer. Sac. Exp. Biol. 29, 1966. Robison, G. A., Butcher, R. W., and Sutherland, E. W. (1971). “Cyclic AMP,” Academic Press, New York. Ryall, R. \\’. (1964). J . Neurochem. 11, 131. Ryall, R. W. (1970). J . Neurophysiol. 30, 251. Ryall, R. W., Stone, N. W., Curtis, 13. R., and Watkins, J. C. (1964). Nature (London) 201, 1034. Salmoiraghi, G. C . , and Stefanis, C. N. (1967). In(. Rev. Neurobiol. 10, 1 . Saiway, J. G., Kai, M., and Hawthorne, J. N. (1967). J . Neurochem. 14, 1013. Salway, J. G., Harwood, J. L., White, G. L., and Hawthorne, J. N. (1968). J . Neurochem. 15, 221. Scheibel, hl. E., and Scheibel, A. B. (1966). Arch. Ital. Biol. 104, 328. Sheltaway, A,, and Dawson, R. M. C . (1966). Biochem. J . 101, 12. Shimizu, H., Creveling, C. R., and Daly, E. (1970). Aduan. Biochem. Psychopharmacol. 3 , 135. Siegrist, G., Doiivo, N., Durant, Y . , Foroglou-Kerameus, C., de Ribaupierre, F., and Rouiller, C. (1968). J . Ultrastruct. Res. 25, 381.

A DEPOLARIZATION-HYPERPOLARIZATION

CYCLE

65

Siggins, G. R., Oliver, A. P., Hoffer, B. L., and Bloom, F. E. (1971). Science 171, 192. Silman, J., and Karlin, A. (1969). SGience 164, 1420. Singer, J. J., and Goldberg, A. L. (1970). Advan. Biocfrem. Psychopharmacol. 3, 335. Skou, J. C. (1954). Arch. Phannacol. Toxicol. 10, 325. Smythier, J. R. (1971). Int. Rev. Neurobiol. 14, 233. Soto, E. F., Pasquini, J. M., Placide, R., La Torre, J. L. (1969). J. Chromatogr. 41, 400. Stein, R. (1967). Biophys. J. 7 , 37. Steiner, F. A., and Meyer, M. (1966) Experientia 22, 58. Svensson, H. (1960). In “Analytical Methods of Protein Chemistry.” (P. Alexander, and R. J. Block, eds.), Vol. 1, p. 193. Pergamon, Oxford. Tao, M. Salas, M. L., and Lipmann, F. (1970). Proc. Nut. Acad. Sci. U.S. 67, 408. Tasaki, I. (1968). “Nerve Excitation.” Thomas, Springfield, Illinois. Taylor, R. E., and Rojas, E. (1971). Biophys. J. 11, 56a. Ten Hoopen, M., and Verveen, A. A. (1963). In “Cybernetics in Medicine.” (N. Wiener, and J. P. Schade, eds.), p. 115. Elsevier, Amsterdam. Thoennen, H., Haefely, W., Grey, K. F., and Huerlimann, A. (1967). J. Pharmacol. E x j . Ther. 156, 246. Thomas, R. C., and Wilson, V. J. (1966). Science 151, 1538. Thompson, W., and Dawson, R. M. C. (1964). Biochem. J. 91, 233 and 239. Torda, C. (1954a). Amer. J. Physiol. 177, 179. Torda, C. (1954b). Amer. J . Physiol. 178, 123. Torda, C. (1954~).Endocrinology 54, 649. Torda, C. (1971). Res. Commun. Chem. Pathol. Pharmacol. 2, 483. Torda, C. (1972a). Biophys. J. 12, 121A. Torda, C. (197213). Fed. Proc., Fed. Amer. Sac. Ex@. B i d . 31, 661. Torda, C. (1972~).Pmc. Int. Congr. Pharmacol., 5th, 1972, p. 121. Torda, C. (1972d). Proc. 2nd Annu. Meet. Sac. Neurosci. Vol. 2, p. 210. Torda, C. (1972e). Biochim. Biophys. Acta 286, 389. Torda, C. (1972f). Experientia 28, 1438. Torda, C. (1972g). “A Depolarization-Hyperpolarization Cycle. A Molecular Model.” Chicago Aligraphy, Chicago. Torda, C. (1973a). Neurobiology 3, 19. Torda, C. (1973b). Experientia 29, 536. Torda, C. (1973~).IRCS [73-31 [3-10-11. Torda, C. (1973d). Naturwissenschaften 60, 436. Torda, C. (1973e). IRCS [73-101 [3-10-221. Torda, C. (1973f). J. Histachem. Cytochem. (in press). Torda, C. (1973g). Biophys. J. 13, 239a. Torda, C. (1973h). Physiologist 16, 741. Torda, C. (1974a). Naturwissenschaften 61, 200. Torda, C. (1974b). IRCS2, 1111. Torda, C. (1974~).Lge Sci. 13. Tosaka, T., and Libet, B. (1970). Fed. Proc. Fed. Amer. Soc. Exp. B i d . 29, 716. Tosaka, T., Chichibu, S., and Libet, B. (1968). J. Neurophysiol. 31, 396. Unna, K., Kniazuk, M., and Greslin, J. G. (1944). J. Pharmacol. 80, 39. Verveen, A. A., and Derksen, H. E. (1968). Proc. IEEE 56, 906. Volle, R. L., and Hancock, J. C. (1970). Fed. Proc. Fed. Amer. Sac. Exp. B i d . 29, 1913. Walsh, D. A., Perkins, J. P., and Krebs, E. G. (1968). J. B i d . Chem. 243, 3763. Walsh, D. A., Ashby, C. D., Gonzales, D. C., Calkins, D., Fischer, E. H., and Krebs, E. G. (1971). J. Biol. Chem. 246, 1977.

66

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Waser, P. G., Hofmann, A., Schaub, M. C., Hopff, W., Karis, J. H., Rosen, G., and Chang, A. (1973). “Pharmacology and the Future of Man,” Vol. 5, p. 98. Karger, Basel. Watkins, J. C. (1965).J . Theor. Biol. 9, 37. Weight, F. F. (1972).Proc. Annu. Meet. Sod. Gen. Physiol. p. 125. Weight, F. F., and Salmoiraghi, G. C. (1966a). J . Pharmacol. Exp. Ther. 153, 420. Weight, F. F., and Salmoiraghi, G. C. (1966b).J . Pharmacol. Exp. Ther. 154,391. Weiss. B., and Costa, E. (1967).Science 156, 1750. Weller, M., and Rodneight, R. (1971). Nature 225, 187. Williams. T.H..and Palay, S. L. (1969). Brain Res. 15, 17. Wilson, V.J. (1959).J . Gen. Physiol. 42, 703. Wilson, V.J., and Burgess, P. R. (1962). J . Neurophysiol. 25, 392 and 636. WiIson, V. J., Talbot, W. H., and Kato, M. (1964).J. Neurophysiol. 27, 1063. Wolbarsht, M.L.,MacNichol, E. F., and Wagner, H. G. (1960). Science 132, 1309. Yagihara, Y.,Salway, J. G., and Hawthorne, J. N. (1969).J. Neurochem. 16, 1133. York. D. R. (1967).Bruin Izes. 5,263. York, D.R., and McLennan, H. (1967). Aurt. Exp. Biol. Sci. 45, 10. Yoshikami, S., and Hagins, W. (1971).Biophys. J. 11, 17a. Zanella, J., and Rall, T. W. (1970).Fed. Proc., Fed. Amer. Soc. Ex#. Biol. 29, 480.

ANTIAC ETYLCHOLlNE DRUGS: CHEMISTRY, STEREOCHEMISTRY, AND PHARMACOLOGY By T. D. Inch and R. W. Brirnblecornbel Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire, England

I. Introduction . 11. Chemical Evolution of Antiacetylcholine Drugs . 111. Absolute Stereochemistry and Antiacetylcholine Activity . A. Dependence of Activity on Configuration. . B. Use of Configurational Differences to Study Mechanisms of Drug Action . C. Conformation Features and Antiacetylcholine Activity . IV. Quantitative Correlation of Chemical Structure and Antiacetylcholine Activity . V. Factors That Influence the Time-Activity Profile of Antiacetylcholine Drugs . VI. Relative Potencies of Antiacetylcholine Drugs in the Central Nervous . System and Peripheral Nervous System VII. Behavioral Studies . VIII. Antiacetylcholine Drugs in the Treatment of Poisoning by Anticholinesterase Agents . IX. Metabolism . X. Pharmacological Methods . References .

67 71 77

77 86 97

101 105

111 116 125

133 137 139

1. Introduction

Dale in 1914 showed that acetylcholine and certain other esters and ethers of choline had marked effects on the cardiovascular system with low doses causing reductions in blood pressure and higher doses reductions in heart rate. These effects were abolished by atropine but not influenced by section of the vagus and so were assumed to be peripheral in origin. Larger doses of acetylcholine given to atropinized animals caused increases in blood pressure, also peripheral in origin since destruction of the spinal cord had no effect on the response, which could, however, be abolished by a dose of nicotine sufficient to paralyze all the autonomic ganglia. The depressor, cardioinhibitor action of acetylcholine was described by Dale as being of the 1 Present address: The Research Institute, Smith, Kline, and French Laboratories, Welwyn Garden City, Hertfordshire , England.

67

68

T. D. INCH A N D R. W. BRIMBLECOMBE

muscarine type since the action could be mimicked closely by that alkaloid. The pressor action was said to be of the nicotine type. The two actions are now customarily referred to as muscarinic and nicotinic, respectively. Thus the actions of acetylcholine and related agonist drugs at the postganglionic parasympathetic endings are said to be muscarinic whereas agonist actions at autonomic ganglia and the skeletal neuromuscular junction are said to be nicotinic. Similarly, there are antagonist drugs which more or less specifically block the actions of acetylcholine at these different sites. These are ganglion-blocking and neuromuscular-blocking drugs which antagonize nicotinic actions, and drugs known variously as anticholinergic, antimuscarinic, parasympatholytic, cholinolytic, atropinic, atropinelike, or antiacetylcholine, which antagonize competitively the muscarinic actions of acetylcholine and other agonists at postganglionic parasympathetic sites. This article is concerned principally with the latter type of drug. Most of the main peripheral effects of these drugs are due to their aritagoiiism of acetylcholine at muscarinic receptors : additionally they antagonize the actions of acetylcholine on certain smooth muscles which do not have cholinergic innervations (Innes and Nickerson, 1970), and they also produce effects that are central in origin. The peripheral effects are described by Innes and Nickerson (1970). I n summary, with increasing dose there is depression of the secretions of the salivary, bronchial, and sweat glands, dilatation of the pupil, inhibition of accommodation of the eye, and increase in heart rate due to vagal blockade. Higher doses cause increased micturition and decrease in the tone and motility of the gut. Inhibition of gastric secretion requires very large doses. Since the parasympathetic and sympathetic divisions of the autonomic nervous system are, in general, mutually antagonistic, the result of parasympathetic blockade by antiacetylcholine drugs is often sympathetic dominance, the effects observed simulating those resulting from overactivity of the sympathetic nervous system. Some central actions as well as peripheral actions of antiacetylcholine drugs are discussed in this article. It is well known that such central actions occur, the toxic psychosis resulting from belladonna poisoning hating been long recognized. This is characterized by disturbance of memory and of orientation, hallucinations, mania, and delirium. It is still a matter for debate whether these and other central actions of antiacetylcholine drugs result only from competitive antagonism of acetylcholine at central synapses, although the role of acetylcholine as a central transmitter substance now seems to he established beyond all reasonable doubt. T h e evidepce was summarized by Bradley (1968), who also showed that some cholinoceptive neurons in the central nervous system (CNS) respond specifically to iontophoretically applied muscarinic agonists and that these responses are

ANTIACETYLCHOLINE DRUGS

69

antagonized specifically by antiacetylcholine drugs. There is other evidence, based mainly on structure-activity relationships of tremorogenic drugs (Bebbington and Brimblecombe, 1965), for the presence of central muscarinic receptors, and there seems to be little doubt that antiacetylcholine drugs which gain access to the CNS antagonize acetylcholine actions at these receptors. Evidence is presented in this article to support such a view. Medicinally, antiacetylcholine drugs have a variety of uses. I n ophthalmology they are used as mydriatics to dilate the pupil for retinal examinations and in the treatment of acute iritis and keratitis. Larger doses cause cycloplegia (paralysis of accommodation) which may be necessary in the therapy of iridocyclitis and choroiditis or for measurement of refractive errors. I n anesthesiology antiacetylcholine drugs have an important role as premedicants. They inhibit salivary and bronchial secretions, which may otherwise become excessive, particularly when volatile anesthetics are used. Additionally they block the vagal endings, thus preventing cardiac slowing or arrest which may result from overactivity of the vagus during surgery. Because of their bronchodilator actions, the drugs have been used for the relief of bronchial spasm in asthma. The actions of drug on the gastrointestinal tract have led to their use as antispasmodics for relief of spasms of the bowels for example, in irritable colon and spastic colitis, and as antiulcer agents, because of their action in reducing the volume and the acidity of gastric secretions. The drugs are also useful in various conditions, for example colic, where contractions of smooth muscle cause severe pain. Central actions of the drugs are beneficial in the treatment of Parkinson’s disease and in the prevention of motion sickness. Antiacetylcholine drugs are also used in the treatment of poisoning with anticholinesterase agents. They are usually considered to antagonize the effects of accumulated acetylcholine at central and peripheral muscarinic sites, but not to be effective at nicotinic sites, and so are usually used in conjunction with a reactivator of the inhibited cholinesterase, for example an aldoxime. Recent evidence, discussed below, indicates that at least some antiacetylcholine drugs have other actions, for example on skeletal muscle, which may also be beneficial in anticholinesterase poisoning. Throughout this review, it is emphasized that unless accurate and reproducible pharmacological testing procedures are chosen and evaluated thoroughly, attempts to interpret pharmacological results in terms of mechanism of drug action are of little value. Numerous methods, both in uitro and in uiuo have been described for measuring the potency of antiacetylcholine drugs. The most commonly used in uitro procedure makes use of the ability of the drugs to block contractions of the isolated guinea pig ileum induced by acetylcholine or other muscarinic agonists. Schild (1947) introduced a measure, which he called PA, to express activity of antagonist drugs and the

70

T. D. INCH AND R. W. BRIMBLECOMBE

value PA, (the logarithm of the reciprocal of the concentration of antagonist which necessitates doubling the concentration of the agonist to obtain a constant response) has been widely used since. The value of pA, is theoretically equal to the logarithm of X,the affinity constant, but in practice often is not as precise a measure as the latter, especially when equilibrium conditions take relatively long times to become established. A method for measuring the affinity constant, based on that described by Barlow et al. (1963), is described in Section X. In vivo methods for assaying antiacetylcholine potency depend either on antagonizing some effect of a n exogenously administered muscarinic agonist or on measuring an effect produced by the antiacetylcholine drug in blocking the action of endogenous acetylcholine. An example of the former type of test procedure is the blockade of oxotremorine-induced salivation, usually in mice. This gives a measure of peripheral antiacetylcholine potency, and, in the same animal, a n estimate can be obtained of central antiacetylcholine potency by measuring the ability of the drug to block oxotremorine-induced tremors. The rationale of this test and practical details of its use are given later in this article. A commonly used method not involving exogenous administration of a n agonist is to use the action of the antiacetylcholine drug to produce mydriasis, also usually in mice, as a measure of its effectiveness. The mydriatic effect of antiacetylcholine drugs depends on their ability to block the postganglionic endings of the ciliary nerve which innervates the circular muscle on the iris, and thus appears to be a specific antiacetylcholine effect. This latter statement cannot be made with confidence for many other in uiuo methods in the literature, where the measured effects can only be presumed to be due to specific antiacetylcholine action of the drugs being tested. Rather than attempting to discuss ail the various aspects of the chemistry and pharmacology of antiacetylcholine drugs a selective approach has been adopted in this review. Following a brief summary of the chemical evolution of antiacetylcholine drugs (Section 11), an attempt is made in Section 111 to explore ways in which correlations of drug geometry with drug activity can enhance our knowledge of the mechanism of action of antiacetylcholine drugs, provided that the limitations of the approach are recognized and that suitable control of experimental procedures is exercised. I n Sections IV-VI, factors that affect distribution and time-activity profiles are discussed, and an attempt is made to show whether or not it is feasible to prepare drugs that retain some of the medicinal properties summarized in this Introduction, but lack others. There are also sections on the role of antiacetylcholine drugs in the treatment of anticholinesterase poisoning (VIII), on the effects of the drugs on behavior (VII), and on their metabolism (IX).

ANTIACETYLCHOLINE DRUGS

71

II. Chemical Evolution of Antiacetylcholine Drugs

Atropine and related drugs produce a wide range of pharmacological effects, and, although many of these effects probably result directly from the action of these drugs in blocking the effects of acetylchoIine at physicaIly distinct but possibly chemically similar biological receptors, there have been a large number of attempts to produce synthetic drugs in which the pharmacological effects are “separated.” The drugs produced in attempts to retain desirable pharmacological properties at the expense of undesirable ones have been prepared on the basis of broad principles of molecular modification, such as the following (Rama Sastry, 1970) (1) scission of the atropine molecule into simpler molecules containing the essential pharmacodynamic groups ; (2) molecular modification by introducing blocking groups into cholinergic drugs ; (3) changes in other antiacetylcholine drugs based on principles of bioisosterism. Historically the reasonably logical development of acetylcholine antagonists may be appreciated by reference to Table I. The aminoalkyl moieties used were in many instances “derived” from the tropane skeleton. Thus scission of its piperidine ring (Fig. 1) at point Xgives derivatives of hydroxyalkylpyrrolidines, and scission of its pyrrolidine ring at point Y makes it possible to proceed to derivatives of piperidin-4-01. The scission of both rings at point 2 leads to dialkylaminoalkanol derivatives. The acidic moieties in the

H,C-CH-CH,

HOCHRCH, \ H,C -CH, H,C-N \ HC-CH,

J

-I--.

HOCHRCH~CHRN:

R FIG. 1

72

T. D. INCH AND R. W. BRIMBLECOMBE

TABLE I EVOLUTION OF ANTIACETYLCHOLINE DRUGS FROM ATROPINE ~

Drug

~~

Reference

CHaOH Ph-C-COO

I

H Atropine (1) OH Pyman (1917)

Ph-C-COO

I

H Mandelyltropine (2) CHSOH

I I H

R

(CH2) N

Ph-G-COO

/: \:

I

von Braun et al. (1922)

R

(3)

CHSOH

I

CH3

I I

Ph-C-COOCHzCCHzNEtz

I

H

Fromherz (1933)

CH3 (4)

Ph

I

Ph-C-COOCHpCH2NEt,

I

Meier and Hoffmann (1941)

OH

I I

t

Ph-C-COOCH2CHzNMe2Et

Ing

et al.

(1945)

Randall et al. (1952)

73

ANTIACETYLCHOLINE DRUGS

esters shown in Table I are a logical progression from the tropic acid moiety of atropine. Antiacetylcholine drugs have also been designed on the basis that they can be derived from cholinergic drugs when methyl or other small substituents are replaced by bulky substituents. For example, (Table 11) replacement of the acetoxymethyl substituent in acetylcholine (8) with Ph&(OH)TABLE I1 ANTIACETYLCHOLINE DRUGS DERIVED FROM ACETYLCHOLINE-LIKE DRUGS OH

I

+ CH3COOCHzCHzNMe3

Ph-C-COO

I

+ CHaCHzNMe3

Ph

(8)

(9)

+

c

OH

3 1

NCHZCECCH~N

I

Ph-C-CH,C=CCHz Ph

3

N

afforded the potent antiacetylcholine drug benzilylcholine (9).Many other drugs based on the choline moiety are known. It should be noted that antiacetylcholine drugs of this type, unlike the cholinergic drugs from which they are derived, need not necessarily contain a quaternary nitrogen, as analogs containing tertiary nitrogen atoms may also be highly potent (Ariens, 1966). However, with only a few exceptions the antiacetylcholine quaternary nitrogen salts are more potent than their tertiary analogs in the peripheral nervous system although only the tertiary compounds have central antiacetylcholine potency. Other antiacetylcholine drugs such as 4-dimethylamino-

74

T. D. INCH AND R. W. BRIMBLECOMBE

rnethyl-:![l-aryl (or cycloalkyl)I-~ydroxy-l-phenyl]methyl-l,3-dioxolans (1 1) (in both tertiary and quaternary form) (Rrimblecombe et al., 1971a; Brimblecombe and Inch, 1970) and 2,2’-disubstituted-4-dimethylaminomethyl- 1,3-dioxolans (12) may be considered as being derived from the highly potent cholinergic drug 4-dimethylamino-2-methyl-I ,3-dioxolan methiodide (10). A third type of antiacetylcholine drug (14) contains a carbon-carbon triple bond and may be considered to be based on the tremorine structure (13) (Cho, et al., 1962). Similar acetylenic antiacetylcholine drugs have been described (Dahlbom et al., 1964; Inch and Brimblecombe, 1971). The principles used in the design of antimetabolites have been applied to the synthesis of atropinelike compounds (Table 111). For example, the ester 0

It

group (C-0) has been replaced by thioester, amide, or ether or with a chain of methylene carbon atoms. Usually the effect of these substitutions has been to reduce antiacetylcholine potency. The references for the various antiacetylcholine compounds in Tables 1-111 and elsewhere in this section are in no way comprehensive, but are given merely to illustrate the types of compounds that have been prepared. It is highly significant that despite 100 years of fairly intensive study and despite claims of superiority for some of the newly introduced synthetic agents, none possesses outstanding efficiency and freedom from adverse side effects when compared clinically with atropine and hyoscine (Rama Sastry, 1970). I n fact, it was only with the introduction of benzilates and related compounds that drugs comparable in activity with atropine began to emerge. Possible reasons for the lack of success in this field of medicinal chemistry are detailed in Sections I11 and VI. One long-acting and potent central and peripheral antiacetylcholine drug, which does not at first sight fit structurally into the general classifications listed above, is l-benzyl-4-(2,6-dioxo-3-phenyl-3-piperidyl)piperidine, or benzetimide (20) (Janssen et al., 1971). However certain structural similarities with other antiacetylcholine drugs have been emphasized (van Wijngaarden et al., 1970).

20

75

ANTIACETYLCHOLINE DRUGS

TABLE I11 EXAMPLES OF TYPES OF STRUCTURE FOUND IN ANTIACETYLCHOLINE DRUGS ~~

Structure

Name

Reference

Thioester OH

I I

Ph-C-COSCH2CH2NEtZ

Thiphenamil

Leonard and Simet (1955)

Tricyclamol

Adamson et al. (1951)

Ph (15)

Alkane

OH

I

Ph-C-CH2CHSN

I

Ph

'3

(16) Ether

OH

I

Ph-C-OCH2CH2NMe2

I

Ph (17)

Amides

Ph

I

HaNCOCCHzCH(CHdNMez

I Ph

Aminopentamide Moffett and Aspergren (1957)

(18)

CH2OH Et

I I

'0

CH-CONCH, Ph

Mydriacyl

Roche Products (1955)

(19)

Another class of compounds which have been demonstrated to have antiacetylcholine activity comprises the pyrrolizidine alkaloids (Pomeroy and Roper, 1971). Among the more potent of these alkaloids is platyphylline, which has a PA, value of 7.9 (cf. atropine = 8.7). I t has been suggested that the antiacetylcholine pyrrolizidine alkaloids may adopt conformations that allow attachment of all the key functional groups to all the binding sites usually proposed for antiacetylcholine drugs. Although many attempts have been made to define the basic structural requirements for high antiacetylcholine activity for aminoalkyl esters, the

76

T. D. INCH AND R. W. BRIMBLECOMBE

results of such attempts have usually only provided a convenient summary of antiacetylcholine compounds and have not been of any great value in the design of new and more potent compounds (Rama Sastry, 1970; Canon and Long, 1967;Lands, 1951 ; Barlow, 1964). Consequently, only a brief summary of primary structural requirements will be given here.

For convenience of discussion, the aminoalkyl esters may be divided into an aminoalkyl moiety (1) and an acyl moiety (2). The structural requirements for moiety (1) are as follows: The cationic head (either quaternary nitrogen or tertiary nitrogen probably in a protonated form, or less frequently a sulfonium or a phosphonium group) is an essential group in most antiacetylcholine compounds. The precise nature of this group may vary considerably; for example, it may be a simple trimethylammonium group or a part of a cyclic amino alcohol system (e.g., pyrollidinol, piperidinol, tropanol, quinuclidinol, granatanol) . Various claims have been made as to the optimum point of attachment of the acyl group to the cyclic amino alcohol for antiacetylcholine potency. For example, it has been suggested that the CNS potency of N-methylpiperidinols diminishes as the substituent position changes from 4 -+ 3 -+2. [With respect to substitution in the 2 position, the original paper (Abood et. al., 1959) correctly stated that the ester group is formed from a 2-hydroxymethyl substituent rather than from a 2-hydroxy substituent as indicated in a number of subsequent reviews (Abood, 1968, 1970)l. Secondary structural modifications to the structure of the aminoalkyl moiety have been detailed elsewhere (Rama Sastry, 1’970). Since small structural changes in the aminoalkyl moiety can affect the basicity of the group in addition to changing the stereochemical features of the molecule, it is not easy to assess the real significance of these changes, and indeed the pattern of structural features necessary for high activity for the aminoalkyl moiety is much less well defined than the pattern for high actions in the acidic moiety. The structural requirements for the acidic moiety (2) are as follows: When X is OH and R’ is phenyl with R” as phenyl or cycloalkyl, compounds with very high potencies are obtained. Replacement of the hydroxyl group with hydrogen or other groups usually reduces potency, as does substitution of the aromatic ring. Thienyl groups are isosteric with phenyl groups. The

ANTIACETYLCHOLINE DRUGS

77

presence of one aromatic system is desirable but not essential; e.g., when R‘ = R“ = cyclohexyl, potent antiacetylcholine compounds may be obtained. Tropic acid derivatives R’ = Ph, R” = CH,OH, X = H although potent are less effective than benzilic acid derivatives. Detailed descriptions of the many variations in the structure of the acidic moiety have appeared elsewhere (Rama Sastry, 1970). It has been observed that both the antiacetylcholine and psychotomimetic potency of the aminoalkly esters of a variety of acids seem to parallel the acid strength of the carboxylic acid involved in their structure and also their stability to acid hydrolysis (Abood and Biel, 1962;Abood, 1968). Among evidence quoted was the fact that in a series of piperidin-3-01 esters the order of potency and acid strength is benzilate > diphenylacetate > phenylacetate > acetate. It was further observed that the duration of mydriasis could be correlated with the times of half acidic hydrolysis for many esters. Although the observed correlation between antiacetylcholine potency and acid strength are interesting, there does not appear to be any theoretical justification, and indeed there are a number of anomalies. For example, aminoalkyl esters of 2-cyclohexyl-2-hydroxy-2-phenylacetic acid (a weaker acid than benzilic acid) are more potent and longer-lasting antiacetylcholine drugs than aminoalkyl esters of benzilic acid (Brimblecombe et al., 1971b). Further, in other series of drugs (e.g., dioxolans and acetylenes), antiacetylcholine potency increases, up to a certain level, as the bulk of the substituents increases. Since many of the reported correlations in the ester series between potency and acid strength might be equally as convincing as correlations between “substituent bulk” and potency, it seems more acceptable to assume that “bulk” rather than acid strength is the key factor. This interpretation of the observed correlations is consistent with a concept of a highly lipophilic area for drug binding and with theories concerned with the duration of action of these drugs, and it provides a consistent rationale for the similarities between the ester and non-ester series of antiacetylcholine drugs. Indeed it has been demonstrated that there is in some series of drugs a good correlation between antiacetylcholine activity and partition properties (n values) (Section IV) . 111. Absolute Stereochemistry and Antiacetylcholine Activity

A. DEPENDENCE OF ACTIVITY ON CONFIGURATION The differences between the biological activity of optical isomers have been of continuing interest to the pharmacologist and the medicinal chemist, and many varying claims have been made concerning the value of studies of such differences (Cushny, 1926; Casy, 1970). Although undoubtedly the fact that nonenzymatic biological systems can display considerable stereoselectivity toward optical isomers is one of the most important reasons for

78

T. D. INCH AND R. W. BRIMBLECOMBE

the concept of “biological receptors,” the extent of the contribution of pharmacological comparisons of isomers to our present knowledge of the receptor is not easy to assess. Since the dextro- and levorotatory isomers of atropine were among the first compounds examined that exhibited differing potencies, it is particularly relevant in this section to consider in detail the results from studies of a range of optically active antiacetjdcholine drugs in a n attempt to provide a critical assessment of the contribution such studies make toward our knowledge of “biological receptors.” Additionally, and probably of more importance, the relevance of isomeric comparisons as an approach for obtaining more general information about the mode of action ofantiacetylcholine drugs will also be discussed. It must be emphasized at the outset that most of the principles involved and conclusions reached are equally relevant to other types of drugs. Most of the antiacetylcholine drugs listed in Tables IV-VI contain an asymmetric benzylic carbon atom, and in the majority of examples where such a n asymmetric center is present, significant differences in the potency of the enantiomers or isomers are observed. In all the examples that contain one asymmetric center (Table IV) and in which the absolute configuration of the benzylic carbon atom has been established unequivocally, there is no doubt that the relative disposition of groups about the benzylic carbon atom is the same in all the active enantiomers. [It should be noted that although and (-)-hyoscyamine (1) has a configuration that is designated ( - ) -dimethylaminoethyl-2-cyclohexyl-2-hydroxy-2-phenylacetate (22) has a configuration that is designated ( R ) , the spatial disposition of groups about the asymmetric carbon atom may be considered to be analogous as shown in the formulas.] Furthermore, there is no reason to suppose that the active isomers of other drugs which contain asymmetric benzylic centers for which no unequivocal configurational proof has yet been provided, hiffer in their absolute configuration. Usually there is some evidence, such as the rotational properties of tricyclamol (35) and relirted drugs, which suggests that the active enantiomers are configurationally related to the active enantiomers of esters such as (-)-hyoscyamine. 0 For the examples quoted (Table IV) of drugs which contain one asymmetric center that is not a benzylic carbon atom, it is clear that the potency of these drugs is not so critically dependent on the absolute configuration, and for this class of antiacetylcholine drugs enantiomeric potency ratios are small. The results obtained for studies of antiaeetylcholine drugs which contain two or more asymmetric cexters (Tables V and VI) emphasize the importance of the configuration of the asymmetric benzylic carbon centers and the considerably lesser importance of the other asymmetric center or centers. a The R and S notation for describing absolute configurations is that proposed by Cahn ct al. (1956)

TABLE I V AND ANTIACETYLCHOLINE ACTIVITY OF DRUGS WITH ONEASYMMETRIC CENTER OPTICALISOMERISM Reference" (a)

Compound

Test system

Isomeric ratio'

References (b)

Esters with one asymmetric center

16

CHIOH

0 S-( - )-Hyoscyamine (1)

16

0 S-(- )-Hyoscine (21)

Guinea pig ileum (PA,) Rabbit ileum Mydriasis in mice (c.s.) Mydriasis in mice (topical) Mydriasis in mice EEG (rats, dogs, monkeys) Vasodepression (dog) Salivation (dog) Mouse chimney test Antitremorine (mice, s.c.) Antiarecoline (mice, s.c.) Cat blood pressure Salivation (dog) Rabbit ileum Guinea pig ileum (PA,) Mouse chimney test Mouse fighting test Spontaneous motility (rats) Antitremorine (mice, s.c.) Antiarecoline (mice, s.c.) Mydriasis in mice (c.s.) Mydriasis in mice (topical)

32 (5) 110 29 41 200 8-50 200 170 20 31 21 47 23 17 15 54 46 5 20 63 76 65 28

3 1 9 9 1 10 1 1 8 9 9 9 9 11 12 9 9 9 9 9 9 9 9 (Continued)

CD -4

co

TABLE IV (Continued OPTICAL ISOMERISM AND ANTIACETYCHOLINE ACTIVITY OF DRUGS WITH ONEASYMMETRIC CENTER Reference“ (a)

Compound

Test system

Isomeric ratioc

15

Guinea pig ileum (log K) Mouse mydriasis (i.v.) Antioxotremorine salivation Antioxotremorine tremors

100 (2.14) 123 (2.23) > 100 (1.84) > 22

15

Guinea pig ileum (log K) Mouse mydriasis (i.v.) Antioxotremorine salivation Rat jejunum (PA,)

200 (2) 38 (1.68) 147 (3.33) 100

Guinea pig ileum (log K) Mouse mydriasis (i.v.) Antioxotremorine salivation Antioxotremorine tremors

272 (2.45) 20 (2.01) 43 (1.72) 16 (5.86)

Guinea pig ileum Mouse mydriasis Antioxotremorine salivation

100 (5.0) 2.3 (1.31) 17.6 (0.88)

15

15

0

References (b)

7 7 7

ro

ro 0 0

m

s

ANTIACETYLCHOLINE DRUGS

I

g

. +

m

4

81

TABLE IV (Continued) OPTICAL ISOMERISM AND ANTIACETYLCHOLINE ACTNITYOF DRUGS WITH ONEASYMMETRIC CENTER Reference" (a)

Compoundb

Test system

Nonesters with one asymmetric center

In tests for log K, mydriasis and antagonism of oxotremorine-induced salivation and tremors, there was no really significant difference between the R and S enantioqers, which were at least 65y0 optically pure. The general trend of the results showed that the S-( +)-isomer was probably more potent.

13

+ NMez

Guinea pig ileum (PA,) Guinea pig ileum (log K ) Mydriasis (i.p.)

Isomeric References ratioC (b) 13

c3

U

49 (2)

375

> 18 (1.7)

2 6 2

Procyclidine (34) Me1 Tricyclamol

(35) EtI

(36)

Benzhexol (37)

Guinea pig ileum (PA,) Guinea pig ileum (log K) Mydriasis (i.p.)

160 (2.4)

Guinea pig ileum (pA2) Guinea pig ileum (log K ) Mydriasis (i.p.)

290 (2.5) 226 200 (1.9)

Guinea pig ileum (pA2) Guinea pig ileum (log K) Mydriasis (i.p.) Rabbit ileum

9.8 (2.5) 5.5 4.8 (1.9) 160

a7 62 (1.8)

2 6 2

Me1 (38)

3

6

Dog salivation Dog vasodepression Guinea pig ileum (PA,) Guinea pig ileum (log K ) Mydriasis (i.p.) Rabbit ileum Dog salivation Dog vasodepression Mydriasis Guinea pig ileum (PA,)

> 500 > 500 48 86 33 (2.4)

30

1 1 2 6 2

30

1 1 1 1

> l o o 0 (2)

19

> 30 > 30

18

S-(+) Benzetimide (20) a Reference column (a) : determination of absolute configuration. Reference column (b) : determination of enantiomeric potency ratio. Key: 1. Long et al., 1956; 2. Duffin and Green, 1955; 3. Marshall, 1955; 4. Randall et al., 1952; 5. Ellenbroek et al., 1965; 6. Barlow, 1971; 7. Brimblecombeet al., 1971b; 8. Cushny, 1926; 9. Buckett and Haining, 1965; 10. Domino and Hudson, 1959; 11. Cushny and Peebles, 1905; 12. Cushny, 1921; 13. Inch and Brimblecombe, 1971; 14. Belleau and Pauling, 1970; 15. Inch et al., 1968; 16. Fodor and Csepreghy, 1961; 17. Beckett ef al., 1963; 18. Spek et al., 1971; 19. Janssen et al., 1971. Configuration and sign of rotation of active isomers. Ratio of active isomer to racemate.

co

w

TABLE V ANTIACETYLCHOLINE ACTIVITY OF DRUGS WITH Two ASYMMETRIC CENTERS Reference

1

Compound OH

I

+ Ph. 7.COOCH(CH3)CHPNMe3 I

2

Isomer

RR SR RS

ss RR

OH

I

P h . C . COOCH2CH(CH3)NMe2

Affinity constant

Salivation

Mydriasis

8.9 6.9 8.3 6.6

Rs

9.88 10.0

0.55 0.55

2.78 1.63

RR RS

10.08 10.04

0.20 0.27

4.25 3.16

I

2

OH

I

+ Ph. C . COOCH2CH(CH3)NMe3

I

3

+

(43) a

trans-Racemate D-traRS (2S,4S) L-franc (2R,4R) &-Racemate D-cis (2R,4S) L - C ~ S(2S,4R)

8.41 8.56 8.24 8.34 8.34 8.73

15.0 4.2 20.2 6.2 16.6 2.9

Key to references: 1 . Ellenbroek et al., 1965; 2. Brimblecombe el al., 1971b; 3. Brimblecombe and Inch, 1970.

0.61 1.21 0.29 0.98 0.74 2.29

ANTIACETYLCHOLINE DRUGS

85

TABLE VI ANTIACETYLCHOLINE ACTIVITY OF THE EIGHTISOMERIC HYDROCHLORIDES AND EIGHTISOMERIC METHIODIDES OF 4-DIMETHYLAMINOMETHYL-2-(I-CYCLOHEXYL1-HYDROXY-1-PHENYL)METHYL-1,3-DIOXOLANn(44)

Configuration

Guinea pig ileum affinity constants (log K)

Antagonism of oxotremorineinduced salivation in mice (ED,,, pmoles/kg)

Hydrochlorides D-C~S,4S,2R(R)

D-trans, 4S,2S(R) D-cis, 4S,2R(S) D-trans, 4S, 2S(S) L-cis, 4R,2S(R) L-trans, 4R2R(R) L-cis, 4R,2S(S) L-trans, 4R,2R(S) Methiodides D-cis, 4S,2R(R) D-trans, 4S,2S(R) D-cis, 4S,2R(S) D-trans, 4S,2S(S) L-cis, 4R,2S(R) L-tram, 4R,2R(R) L-cis, 4R,2S(S) L-trans, 4R,2R(S) a

7.07, 7.06 8.36, 8.41 6.56 < 7 8.79, 8.89 7.35, 7.40, 7.25 < 6.5 6.24, 6.28 7.28, 7.41 9.37, 9.33 6.56, 6.52 11.09, 11.08 7.60, 7.53 6.77, 6.79

40.6 1.5 Inactive at 100 pmoles/kg Inactive at 100 pmoleslkg 1.7 28.3 Inactive at 100 pmoleslkg Inactive at 100 pmoles/kg 46.6 0.22 Inactive at 75 pmoleslkg 16.2 0.07 17.7 Blocks 2/5 at 100 pmoles/kg Blocks 2/5 at 100 pmoleslkg

Brimblecombe et al. (1971a).

For example, in the /3-methylcholine derivatives (40) there was little dependence of activity on the configuration of the amino alcohol moiety, but considerable dependence of activity on the configuration of the benzylic center in the acidic moiety. In the dioxolan derivatives (43) in Table V. there was only slight stereoselectivity, but again the predominant feature was the configuration of the benzylic center. From Table VI it can be seen that there was considerable variation in the potency of the isomers of (44) and that only compounds with the R configuration at the benzylic center were of high activity. The configuration at the benzylic center of the active isomers shown in Tables IV-VI is the same. There appears to be an interesting dependence of activity in the quaternary derivatives upon the configuration at the nitrogen atom, where unlike substituents can be in axial or equatorial position, e.g., tropane derivatives

86

T. D. INCH AND R. W. BRIMBLECOMBE

where R, = ethyl and Re = methyl were more active than when R, = meth,yl and Re = ethyl (D6da et al., 1963; Fodor, 1967). It is possible, however, that these configurational assignments may need revision (Brown et al., 1967; Thut and Bottini, 1968).

DIFFERENCES TO STUDYMECHANISMS OF B. USEOF CONFIGURATIONAL DRUGACTION The full significance of the results listed in Table IV is difficult to assess for a number of reasons. First, many of the results may be considerably in error because they were obtained by methods that were designed primarily as screening procedures and were not developed sufficiently to produce reasonably precise quantitative results. Second, in many of the reported studies the optical purity of the isomer was uncertain and in some cases the absolute configuration was unknown. I n most, if not all of the examples, this latter information could now be provided by chemical correlation or by use of modern physicochemical methods. Indeed for many substances configurational assignments were made long after isomers-were separated and subjected to pharmacological evaluation. The difficulties that exist in any attempts to establish absolute optical purity unequivocally are rhuch more serious. For compounds that contain one asymmetric center, such as atropine and hyoscine, the usual procedure for obtaining the enantiomers of these drugs is one of classical resolution. For the antiacetylcholine drugs which are usually amines, optically active acids are the resolving agents usually used. The efficiency of a resolution procedure must be monitored by some physical chemical method, and classically the specific rotation of resolved enantiomers is taken as a measure of optical purity. When enantiomers are obtained with equal rotations of opposite sign, the resolution is considered to be complete. More recently gas chromatogaphic and nuclear magnetic resonance spectroscopy and other refined techniques have been developed to measure optical purity or to separate enantiomers. Unfortunately no physical method is capable of detecting less than 1-2% of one enantiomer as a contaminant of the other, and so, unless great care is exercised in resolution procedures, doubts about the optical purity of isomers must remajn (Wilen, 1971; Eliel, 1962). Usually a small amount of optical impurity will not seriously affect the conclusions obtained from pharmacological results, but

ANTIACETYLCHOLINE DRUGS

87

it should be noted that where enantiomeric differences are large (e.g., 100) as little as 1% of the active isomer present as a contaminant in the less active isomer could account totally for the apparent activity of the latter. Errors caused by such impurities could have a marked effect on enantiomeric potency ratios (Barlow et al., 1972b). This source of error has long been recognized, and many attempts have been made to minimize it. For example, one approach has been to resolve enantiomers by classical procedures and to use constant biological activity as a measure of optical purity (Long et al., 1956). Another approach has been to prepare optically pure enantiomers by stereospecific synthesis starting from naturally occurring, presumably optically pure, compounds (e.g., carbohydrates) (Brimblecombe et al., 1971b). Recently the situation has been analyzed mathematically, and the effect of various proportions of optical impurities on enantiomeric potency ratios has been determined. I t has been shown that within a closely related series of compounds, it is possible to assess the importance of possible optical impurities by careful consideration of measured specific rotations and potency ratios (Barlow, 1971). I n the context of the above discussion, it is clear that the results in Table I V should not be taken at face value and that the true significance of each result be assessed after reference to the original papers to find the details of the pharmacological procedures and details of the chemical analysis of the isomers. Usually the experimental procedures that have been employed necessarily limit the amount of information obtained. However, despite many deficiencies the results of studies collated in Table I V provide a basis for developing a clearer insight into the type and extent of information comparisons of isomers should be able to provide. On the assumption, which will be shown later to be fully justified, that the observed potency differences of enantiomers reflect mainly the efficiency of interaction of drug with receptor, it is pertinent first of all to consider what the gross stereochemical differences and the listed enantiomeric potency ratios tell us about the nature of the receptor (or receptors) a t which antiacetylcholine drugs interact. It has been suggested (Pfeiffer, 1956) that the large difference between the potencies of enantiomers for highly active drugs compared with the much smaller differences in the potency of enantiomers of less active drugs reflects the better geometrical “fit” of the former for the receptor. If this suggestion is true, it would be reasonable to consider highly potent drugs as template models for the receptor, and thus as good models €or measuring distances between key areas or functional groups on the receptor. (This argument presupposes that the conformations as well as the configurations of the drugs are known.) Unfortunately, the results obtained from studies with antiacetylcholine drugs do not substantiate the above suggestion and implications. For example, it has been shown by

88

T. D. INCH AND R. W. BRIMBLECOMBE

Barlow (1971) that for the series of drugs procyclidine (34), tricyclamol ethiodide (36), tricyclamol methiodide (35), benzhexol methiodide (38), and benzhexol (37) the enantiomeric potency ratio decreased in the order 375, 226, 87, 86.3, and 5.5, respectively, whereas the affinity constants (log K ) were in the order 8.2, 8.6, 8.7, 9.1, and 8.7. Thus, in this series the enantiomeric potency ratio was highest for the least active compound. Also in some optically active acetylenic drugs it has been shown that there is little difference in the potency of enantiomers whereas for closely related drugs of similar potency the enantiomeric differences were large (Inch and Brimblecombe, 1971). The above results, which demonstrate that the differences in the potency of enantiomers can vary considerably within closely related series of compounds that contain essentially similar asymmetric centers, invalidate to a large extent some conclusions about the importance of certain areas of drugs for the drug-receptor interaction that have been drawn from studies of drugs that contain more than one asymmetric center (Ariens, 1966). For example, one suggestion was that the 8-methyl group in 2-cyclohexyl-2-hydroxy-2phenylacetyl-p-methylcholine (40) (Table V) was not essential to drug action because, whereas the potency of this compound depended critically on the absolute configuration of the benzylic center, there was essentially no dependence of potency on the configuration of the 8-methylcholine moiety. Evidence cited in support of this suggestion was that (40) was considerably less potent than the corresponding choline derivative (22) (Table 4). The value of this evidence is now considerably reduced because it has been shown that the potency of 2-cyclohexyl-2-hydroxy-2-phenylacetyl-cl-methylcholine (42) is independent of the stereochemistry of the a-methylcholine moiety although this drug is more potent than the corresponding choline derivative. The above results collectively demonstrate that it is not justified to consider, as has often been done previously, drug-receptor interactions in terms of three point (or multipoint) models, but that the stereospecificity observed must be considered to be a function of the entire molecule, not of parts of the molecule. At present, therefore, it seems reasonable to conclude that comparisons of enantiomers and related stereochemical studies of compounds with more than one asymmetric center reveals little about the arrangement of groups on the receptor but instead demonstrate many limitations of the approach of using small molecules to “map” the surface of large molecules which presumably are conformationally mobile. On the basis of the above argument, it is questionable whether it is justified to use a stereochemical argument to provide information about whether or not acetylcholine and related drugs and antiacetylcholine drugs share common points of attachment with a biological receptor. However, since stereochemical studies have been invoked in discussions of whether

ANTIACETYLCHOLINE DRUGS

89

atropine and related drugs are true competitive antagonists of acetylcholine (see Section V), and because the arguments used certainly demonstrate a possible application of stereochemical studies in providing information about similarities or differences in receptors, the arguments will be outlined below. Comparison of the stereochemical requirements for high cholinergic activity and for high antiacetylcholine activity may be made most conveniently in the following manner (Brimblecombe et al., 1970b). Acetylcholine may be converted into an antiacetylcholine drug by replacement of the acetyl substituent by a more bulky substituent such as 2-cyclohexyl-2hydroxy-2-phenylacetyl, and the stereochemical comparison between the two types of drug may be made by reference to the following formulas: + CH,CO. OCHR’ CHRNMe3

Ph\ ,OH

P\GO-OCHR’. CHR&Me3

CCeHii a R = R = H c R = Me, R = H e R = H, R = Me

bR=R‘=H d R = Me, R’ = H f R = H, R’ = Me

1. Replacement of any of the N-methyl substituents in a by other alkyl groups reduces cholinergic activity whereas in b the nature of the N substituents may vary over wide limits without appreciably reducing potency, and in some instances there may be an increase in potency. Also in antiacetylcholine drugs the nitrogen may be tertiary or quaternary whereas only quaternary compounds are potent agonists. 2. Replacement of one of the a-protons in a with methyl to give acetyl a-methylcholine c causes a considerable reduction in muscarinic potency (although the nicotinic potency is little affected). Also the muscarinic potency is dependent on the absolute configuration of the methyl substituted carbon, the R-enantiomer of c being 8 times more active than the S-enantiomer. On the other hand, replacement of an a-proton with methyl in b to give d enhances antiacetylcholine potency, and potency no longer depends on the configuration of the methyl-substituted carbon atom. 3. The S-enantiomer of acetyl /I-methylcholine e is equiactive with acetylcholine whereas the R-enantiomer is much less active. Substitution of the /I-carbon of b with methyl to give f affords a product which is less active than b and in which the absolute configuration of the /3-substituted carbon atom is of little importance. 4. Replacement of the alcoholic oxygen atom in acetylcholine by sulfur considerably reduces muscarinic potency, but replacement of alcoholic oxygen by sulfur in antiacetylcholine compounds has little effect on antiacetylcholine potency.

90

T. D. INCH AND R. W. BRIMBLECOMBE

5. The antiacetylcholine activity of b, d, and f depends on the absolute configuration of the benzylic center. Thus apart from the observation that the antiacetylcholine drugs may be derived formally from acetylcholine by the replacement of acetyl by a bulky substituent, the stereochemical requirements for high acetylcholine-like and high antiacetylcholine activity bear no resemblance and make it unlikely that the two types of drug share points of attachment with a common receptor. Whether antagonists and agonists share completely or in part, or not at all, common points of attachment with a receptor has been a topic of constant debate (Ariens, 1966; Ariens and Simonis, 1967; Stubbins et al., 1972; Moran and Triggle, 1969). It may be relevant, however, that following a nuclear magnetic resonance study of the association of atropine and several of its analogs with acetylcholinesterase, as indicated by the line widths of the N-methyl and phenyl group resonances of the smaller molecule, it was concluded that atropine and its analogs bind to a site distinct from the active center of the enzyme (Kato et al., 1970; Kato and Yung, 1971). The above discussion about the information that studies of isomers can provide was concerned mainly with the interpretation of observed differences from in uitro tests, which may be considered to arise mainly from differences in the interaction between drug and receptor. Studies of enantiomeric antiacetylcholine drugs can in principle provide information of the similarity or otherwise of antiacetylcholine receptors in daxerent species and in the central and peripheral nervous systems of these species. For such studies, however, in uiuo as well as in uitro tests have to be used, so before discussing this statement it is necessary to consider which factors other than differences in the efficiency of the drug receptor interaction can affect the measured enantiomeric potency ratio (sometimes referred to as the stereospecific index). It has been suggested that the following factors can contribute to observed differences in the potency of enantiomers (Pfeiffer, 1956): (a) differences in the rates of absorption, destruction, and excretion of isomers ; (b) competitive inhibition between isomers; (c) the possible racemization in uiuo of one or both isomers; (d) differential penetration of enantiomers to the site of drug action; (e) differences in the interaction of drug with receptor. I n principle it is possible to establish which of these factors contribute to the observed difference in potency in any particular case. For example, if the active enantiomer is appreciably more active than the less active isomer and twice as active as the racemate, there is good reason for discounting factors (b) and (c) as making any significant contribution to the observed potency difference. Unfortunately, not all the reports referenced in Table I V describe measurements of the racemates as well as the isomers. I t is also possible to carry out pharmacological experiments taking the time-activity

ANTIACETYLCHOLINE DRUGS

91

profile into consideration. If drug potency is assessed at the optimum time, the contribution of factors (a) and (d) to observed potency differences may be minimized. Again, unfortunately, only in a limited number of experiments were observations made at optimum times. Finally, some indication of the contribution of the factors listed in (a) may be obtained by carefully controlled comparisons using different routes of drug administration and by the use of different species. Such studies, backed up where necessary by autoradiographic and other radiochemical experiments, would provide considerable information about the stereoselectivity of metabolic enzymes as well as about the effects of absorption, destruction, and excretion of isomers on the enantiomeric potency ratio. Against this background, it is justified to suppose that if the factors listed as (a), (b), (c), and (d) make only minor contributions to observed enantiomeric potency differences or can be eliminated or corrected for in the test procedures, then constant enantiomeric potency ratios in diverse pharmacological tests would be an indication of receptor similarity whereas widely varying potency ratios would be an indication of receptor differences. The fact that the absolute stereochemistry at the asymmetric benzylic center of most of the active enantiomers in Table I V is the same provides some indication of receptor similarity in different species and in different organs, but the varying potency ratios reported for a number of enantiomeric pairs clearly make it necessary to demonstrate that these variations are likely to result from one or more of factors (a) to (d) before any conclusion can be reached. I n practice it is easy to discount factors (b) and (c), since for many of the compounds and tests in Table I V the results show that the active isomer is twice as active as the racemate. Additionally one study has been reported which analyzes the results in a way which shows that the receptor for antiacetylcholine drugs in the central and peripheral nervous systems of different species are likely to be essentially of similar composition and that adequate reasons for the observed variations in enantiomeric potency ratios are available (Brimblecombe et al., 1971b; Inch et al., 1973). It was observed that the enantiomeric potency ratio of R- and S-dimethylaminoethyl 2-cyclohexyl-2-hydroxy-2-phenylacetate(22) was remarkably constant [as measured from affinity constants (guinea pig ileum), mydriasis (mice), antioxotremorine salivation and tremors (mice), amd EEG data (cats)] whereas with closely related but slightly more potent drugs (e.g., 24 and 25) there were marked variations in the enantiomeric potency ratios. Closer inspection of the results showed that, because the R- and S-enantiomers of (22) were both quick-acting drugs, differences in their time-activity profiles [factors (a) and (d)] were not important, but that such differences could affect the potency ratios of the other pairs. However, such differences could not account for the differences in ratios observed with the affinity

92

T. D. INCH AND R. W. BRIMBLECOMBE

constants and mydriasis results, which were both obtained at optimum times. For example, for (25) the affinity constant ratio was 100 and the mydriasis ratio was only 2.3. The explanation (Brimblecombe et ul., 1971b) of this variation is that with antiacetylcholine drugs a certain minimum dose is necessary in vivo to produce a maximum effect. Subsequently (Inch et al., 1973) it was shown that drugs with affinity constants of about log K 2 9.49 on the guinea pig ileum are active at this minimum dose (cu. 0.03-0.05 pmole/kg) in vivo to elicit a maximum response. For compounds such as (25), the less active isomer has a log K of 9.08 and is thus active almost at the minimum effective dose level. Thus although on the ileum the enantiomeric potency ratio of the R and S isomers of (25)is 100, measurements in vivo indicate little stereoselectivity. These observations obviously invalidate the generalization that high potency ratios necessarily parallel high drug potency for in vivo experiment^.^ The concept that drugs cannot elicit maximum effects in vivo when administered below a certain minimum dose provides an explanation of why drugs significantly more potent than hyoscine and atropine have not been prepared. The cause of this phenomenon is at present only a matter for speculation. From the results with the isomers of atropine and hyoscine and from the results with derivatives of the isomers of 2-cyclohexyl-2-hydroxy-2-phenylacetic acid, there seems to be little doubt that the observed variations in potency ratios arise from deficiencies in the experimental method or because of the intrinsic necessity of a minimum dose requirement for maximum antiacetylcholine effect, not because of variations in the drug-receptor interaction. This is a n extremely important conclusion because it implies that “separation” of antiacetylcholine effects can be achieved only by modifications to properties that affect drug distribution, not by modification of properties that affect the interaction of drug and receptor. Two recent studies of the effect of stereochemical differences on drug potency have been reported. I n one of these studies (Biggs et al., 1972), the conclusion was reached that the receptors on the guinea pig ileum differed from those in the circular muscles of the eye because, whereas on the ileum diastereoisomers had different potencies, in the eye no differences were observed. I t is probable that this conclusion is in error because of deficiencies in the procedure used for measuring mydriasis, where measurements were made a t fixed times without establishing the time-activity profiles of the drug examined. The second paper (Barlow et al., 1972a) compared the affinity constants of a series of antiacetylcholine drugs in the ileum, bronchial muscle, and iris of the guinea pig. I t was shown that, although the compounds 3 In this connection, it is of interest to speculate whether or not the fact that within a series of compounds no peak of the enantiomeric potency ratio is reached is indicative of the possibility that more potent compounds with that particular type of action exist.

93

ANTIACETYLCHOLINE DRUGS

examined differed up to a millionfold in affinity, most of the estimates of log affinity constant for the bronchial muscle and iris differed only slightly from those on the ileum. It was also shown, however, that there was a small variation in enantiomeric potency ratios on the three receptors for the enantiomers diethylaminoethyl 2-cyclohexyl-2-hydroxy-2-phenylacetate athiodide and related compounds. Although it was suggested that the differences in potency ratios could result from small changes in the receptor protein, e.g., replacement of just one amino acid by another, perhaps the true explanation for the differences results from differences in the procedures used for measuring the affinity constants on the three preparations (Barlow et al., 1972a). It was shown that for highly potent drugs the procedure required to establish equilibrium conditions is more demanding than for less active drugs. Thus when the affinity constants for diethylaminoethyl 2-R-2cyclohexyl-2-hydroxy-2-phenylacetatehydrochloride was measured isometrically the log K value rose from 9.791 to 9.979 as the contact time between carbachol and ileum was increased from 40 to 300 seconds. Under isoteric conditions, a log K value of 9.600 was obtained. Although these differences are small, they have a pronounced effect on enantiomeric potency ratios, and the conclusion to be drawn from the work of Barlow et al., (1972a) on measurements of affinity constants in vitro appears to be that the receptors in different tissues of the guinea pig are essentially similar and in good agreement with the results of the in vivo studies. The discussion previously in this section has been concerned primarily with evaluating the information which studies of isomers provide about drug receptors. It is now pertinent to consider the role of such studies in evaluating certain more general aspects of the mode of action of these drugs. First, it is worth repeating that the concept that a minimum dose was necessary to produce maximum antiacetylcholine effects was developed to explain certain variations in potency ratios. The finding is of obvious importance in any theory of the mode of action of antiacetylcholine drugs. Other findings of potential importance include those that relate to the times to onset and duration of action of antiacetylcholine drugs. For example, it has been shown that for any enantiomeric pair, the active isomer has a longer duration of action than the less active isomer. This result precludes direct acylation of the receptor by, as has been suggested (Abood, 1968) a carbonium ion mechanism, e.g.,

I

R

I R

R’

I n such a mechanism the asymmetry of the tetrahedral carbon atom is

94

T. D. INCH AND R. W. BRIMBLECOMBE

destroyed when the trigonal carbonium ion is formed, so both R- and Sisomers should have equal durations of action. Of additional interest are metabolic and pharmacological results which show that although in some species the R- and S-enantiomers of alkyland of benzeaminoalkyl 2-alkyl (or cycloalkyl)-2-hydroxy-2-phenylacetates timide (Janssen et al., 1971) are metabolized by different pathways and at different rates whereas in other species the metabolic pathways for the enantiomers are similar, the metabolic variations do not appear to affect the difference in the potency of the enantiomers (D. G . Upshall, personal communication, 1972). For a drug-receptor situation represented by Drug

+ receptor

h

[DR]

4

this result is acceptable only if k2 is very much smaller than the rate of metabolism. That is, the results appear to show that, once antiacetylcholine receptors are occupied, metabolic processes do not affect the situation if the rate of dissociation is much slower than the rate of metabolism. It is not so easy to analyze observed differences in the times to onset of action of enantiomeric pairs in terms of mechanism of drug action. Typical of observed differences is that when administered a t equal concentrations on the guinea pig ileum S-(+ ) -N-methylpiperidin-4-yl 2-cyclohexyl-2hydroxy-2-phenylacetate reaches equilibrium with the ileum more rapidly than the corresponding R-enantiomer (24). Also, in mydriasis experiments, a t the ED,,, level, the S-enantiomer produced maximal effects more quickly than the R-enantiomer. The observed differences at the guinea pig ileum, where the isomers were compared in equal concentrations, indicates that there must be a basic difference, not simply a concentration-dependent difference, in the rates of reaction of enantiomers with the receptor. (This point is discussed more fully in Section V.) Although this basic difference probably contributes to some of the observed in uiuo differences, these differences may be enhanced by non-specific absorption effects, which will be more pronounced for compounds given at low dose. It is unlikely that the cause of the difference in rates of reaction at the ileum is related only to the absolute configuration, for, as shown in Section V, the time of onset and duration of action of antiacetylcholine drugs appears to be dependent primarily on the affinity constants in such a way that drugs with different substituent groups but with similar affinity constants will have similar timeactivity profiles. I n the latter situation the factors related to properties such as partition coefficient and pK, values must be considered, so it is probable that studies of differences in the time-activity profiles of enantiomers where such properties are the same may have considerable advantages over comparisons of drugs where these properties may vary.

ANTIACETYLCHOLINE DRUGS

95

The work of two groups of workers that is summarized in Table I V merits discussion in some detail since it provides an indication of the potential value of comparisons of enantiomers in pharmacological procedures and at the same time also provides examples of the limitations of the approach if enantiomers of absolute optical purity are not available or if the pharmacological procedures are not refined beyond the standard satisfactory for simple drug screening. Domino and Hudson (1959) compared (+)- and ( -)-hyoscyamine using six measures of CNS activity and two measures of peripheral nervous system (PNS) activity. The ( -) :(+) potency ratios of the enantiomers varied from 8 to 50, but it was not always clear whether the main source of this variation lay in the test method or in the purity of the (+)-hyoscyamine. This is particularly unfortunate in view of the fact that the test methods used by Domino and Hudson are of considerable interest. The methods used for assessing CNS activity were concerned with measuring the effects of the antiacetylcholine drugs on the spontaneous EEG, on the EEG arousal resulting from sciatic nerve stimulation in dogs immobilized with decamethonium, on the spontaneous EEG in monkeys with chronically implanted electrodes, on EEG arousal to various afferent stimuli in monkeys with chronically implanted electrodes, on gross monkey behavior and on conditioned avoidance behavior in the rat. PNS activity was estimated by the blockade of the depressor response to methacholine and the increase in heart rate in unanesthetized dogs immobilized with decamethonium. I n one series of experiments the ( - )-hyoscyamine : ( + ) -hyoscyamine potency ratios were close to 50 as measqed by effects on spontaneous EEG in dogs, EEG arousal to sciatic nerve stimulation in dogs, methacholine responses and heart rate in dogs. Since the latter two procedures can reasonably be assumed to measure fairly specific antiacetylcholine activity, it seems probable that the EEG effects result from similar actions. I n another similar series of experiments the potency ratio for (-)-: (+)-hyoscyamine fell to 8-16 because of the impurity of the enantiomers. I n experiments with monkeys, both isomers produced increases in the amount of slow wave activity in the EEG and elevations in the threshold for arousal to various stimuli. The effects were not, however, quantified very precisely and for this reason it is impossible to extract isomeric potency ratios from the reported results. Similarly, in the rat conditioned avoidance experiments, although it was established that 32 mg/kg of the (-)-isomer caused a 50% decrease of the avoidance response, the only result of the (+)-isomer was that it had no effect at this dose level. [Perhaps the (+)-isomer would be toxic at the dose necessary to cause effects in this test.] Such experiments merely establish that one isomer is more potent than the other and do not provide information about their mechanism of action. .

96

T. D. INCH AND R. W. BRIMBLECOMBE

Buckett and Haining (1965) compared the activities of (+)- and (-)hyoscine and hyoscyamine. Similar ( -) :(+) enantiomeric potency ratios (54 and 32, respectively) were obtained for hyoscine and hyoscyamine when determined as pA, values (Schild, 1947), by measurement of mydriasis in mice after subcutaneous injection, and for the antagonism of oxotremorineinduced tremors. When the drugs were applied to the cornea directly and their mydriatic effects were measured, the potency ratios obtained for hyoscine and hyoscyamine were lower than for the corresponding subcutaneous (s.c.) procedure. This variation possibly reflects the unreliability of the topical application procedure, which has obvious disadvantages. Two behavioral tests used by Buckett and Haining, which gave enantiomer potency ratios similar to those obtained by the above procedures, were the chimney test in mice and the arecoline antagonism test in rats. The chimney test (Boissier et al., 1960) is said to be a method for detecting tranquilizing activity and depends on the animal’s ability to progress along the length of a narrow copper tube. In the arecoline test, rats were conditioned to jump onto a pole in response to a buzzer. This response was then blocked with arecoline, and the antiacetylcholine drugs were tested for their ability to reverse the arecoline blockade. Since in both these tests the ( - ) :( + ) potency ratios for hyoscine and hyoscyamine were similar to those obtained from pA, values, mydriasis, and antagonism of oxotremorine-induced tremor experiments, which are clearly tests for antiacetylcholine activity, it is probable that these latter two procedures are also genuine tests for antiacetylcholine activity. For hyoscine, Buckett and Haining (1965) observed ( -) :(+) enantiomeric potency ratios of 20 and 5, respectively, in tests for spontaneous activity in rats and abolition of the fighting response in previously isolated mice. These ratios, which are considerably lower than those obtained in the other tests, casts doubt on these tests as measures of antiacetylcholine activity. Another interesting observation from studies of the enantiomers of antiacetylcholine drugs is that the more potent antiacetylcholine enantiomer is not necessarily the more toxic enantiomer. For example it was shown that R-(+)-hyoscyamine had a n LD,, of 81 mg/kg and was more toxic than S-(-)-hyoscyamine (LD,, 95 mg/kg) although less potent as an antiacetylcholine drug (Buckett and Haining, 1965). Similarly, ( -)-procyclidine was a more potent antiacetylcholine drug and was less toxic than (+)-procyclidine (Duffin and Green, 1955), and in various esters of amino alcohols the same phenomenon was shown (D. Swanston, personal communication). The mechanistic significance of these observations has not been established. From the results described in this section, it is apparent that there are considerable advantages to be gained from pharmacological comparisons of the enantiomers of a series of drugs with differing potencies, provided

97

ANTIACETYLCHOLINE DRUGS

that a number of accurate pharmacological test procedures are available. It should also be emphasized that studies of enantiomers and their racemates have the advantage that a built-in check of the assay procedure is available. For example, for drugs where one enantiomer is much more active than the other, the active enantiomer should be twice as active as the racemate. If this is not so, the assay procedure must be unreliable or there must be mechanistic differences between the enantiomers. There are usually no great problems in distinguishing between the possibilities. I t is unfortunate therefore that in many of the enantiomeric comparisons listed in Table IV the corresponding data for the racemates were not available. Equally unfortunate is the fact that, although atropine has been shown to be only half as active as (-)hyoscyamine in increasing the heart rate in man pretreated with propanolol, no data on (+)-hyoscyamine were provided (Bagshaw et al., 1970). Such information would have provided evidence for the similarity or otherwise of human and animal antiacetylcholine receptors.

C. CONFORMATION FEATURES AND ANTIACETYLCHOLINE ACTIVITY Abood and coworkers (Gabel and Abood, 1965; Abood, 1968; 1970) have suggested repeatedly that the antiacetylcholine and psychotomimetic

R

R

(III)

(11)

decreasing potencies

h

R

R

+ decreasing potencies

0 II

R = e . g . , Ph,C(OH)C-O

FIG.2

(Iv)

98

T. D. INCH AND R. W. BRIMBLECOMBE

potency of aminoalkyl esters of glycolic acids is in some way connected with the availability of the “charge and the one-electron pair” on the tertiary nitrogen atom in these compounds. Since this viewpoint has been propagated widely (Burger, 1970), it is pertinent to consider the precise meaning of this suggestion and to summarize the evidence for and against it. The initial suggestion was based on the observation that in a series of psychotomimetic benzilates the potency, as assessed by the behavioral disturbance index, decreased as indicated in Fig. 2. The known ready accessibility of the lone pair of electrons on nitrogen in the highly potent quinuclidinol derivative triggered the idea that the interaction of the lone pair of electrons with an electrophilic biological receptor could be important for high potency. Subsequently an argument was presented in terms of conformational analysis that for the compounds in Fig. 2 the decrease in the accessibility of the lone pair of electrons on nitrogen paralleled the decrease in potency. Although some of the conformational arguments may be ~ r i t i c i z e d some , ~ evidence in support of this notion has been provided recently (Nogrady and Algieri, 1968) by experiments which have shown that the ease of quaternization and ease of change complex formation show trends that may be correlated with biological activity. I t is not absolutely clear from the literature published by Abood and his coworkers whether or not correlations between electron availability and psychotomimetic activity alone (Gabel and Abood, 1965) or between electron availability and both psychotomimetic activity and antiacetylcholine activity (Abood, 1968) are to be expected. Also it is not absolutely clear whether the hypothesis about the electron lone pair relates to the ease of direct interaction of the lone pair of electrons with a biological receptor, or to For example, it is suggested that esters of piperidin-3-01 are less potent than esters of piperidin-4-01 because the piperidin-3-01 derivatives can undergo an intramolecular interaction in the chair conformation that can reduce the accessibility of the electron lone pair on nitrogen, whereas for a similar interaction the piperidin-4-01 derivatives must adopt an energetically unfavorable boat conformation. Abood and his co-workers (1958) appear to have overlooked the point that, in the chair conformation depicted, the 3substituent is axially oriented and not in the energetically favored equatorial orientation in which no interaction with the electron lone pair can occur.

Ph Ph\

I

,c-c-0

0

II

ANTIACETYLCHOLINE DRUGS

99

the ease of protonation of the tertiary nitrogen and subsequent interaction of the protonated species with an anionic receptor site. This latter interpretation, which is adopted by Burger (1970), is the more acceptable, since most of the drugs cited have pK, values of 7.5-8 and thus a t physiological p H may be expected to exist at least 50% in the protonated form. Also the concept of interaction of the nitrogen-containing region of cholinergic and antiacetylcholine drugs is widely accepted and is consistent with the observation that usually quaternary antiacetylcholine drugs are more potent peripherally than their tertiary analogs. In this context it is probable that, although it is the tertiary form of the drugs that passes through the blood-brain barrier into the CNS, it is the protonated species that interacts with the antiacetylcholine receptor in the CNS. [Evidence in support of this contention has been provided by comparisons of enantiomeric potency ratios (Brimblecombe et al., 1971b).] The suggestion has been made that hydrogen bonding between the hydroxyl proton in atropine and glycolic acid analogs and the antiacetylcholine receptor contributes to antiacetylcholine potency (Abood, 1968). I t has also been suggested that intramolecular hydrogen bonding will reduce the ability of the hydroxyl to undergo intermolecular hydrogen bonding with the receptor site. I n this connection, the availability of the lone pair of electrons for intramolecular bonding has been considered as a possible reason for the lower antiacetylcholine potency of piperidin-3-01 derivatives compared with quinuclidin-3-01 and piperidin-4-01 derivatives (Burger, 1970). This suggestion is not consistent with the concept that the protonated form of antiacetylcholine drugs that contain tertiary nitrogen is the active species, and also the same criticisms as mentioned previously must apply. I n summary, it is probably fair to say that the relevance of the availability of the lone pair of electrons on nitrogen for biological potency in the sense of the drug-receptor interaction has been overemphasized. The effect of the steric environment of the electron lone pair will be principally to influence the pK, of the drugs and hence their degree of protonation at physiological pH. The consequences of this will affect such properties as transport through biological membranes and the effective drug concentration at the receptor site. Other conformational studies of antiacetylcholine drugs have been reported. A recent observation of interest (Biggs et al., 1972) was that one ester of N-methylpiperidin-3-01 was much less active (PA, values) in quaternary salt form than as the tertiary salt. It was suggested that this difference could have resulted from the piperidine ring adopting a conformation in which the ester group was axial in the tertiary salt but equatorial in the quaternary derivative. In our opinion this observation is suspect because of possible error in the pharmacological procedures.

100

T. D. INCH AND R. W. BRIMBLECOMBE

Various suggestions have been made with regard to possible conformations of receptor-bound atropine (Long et al., 1956; Bowman et al., 1968), and some of these suggestions involve the possibility that the boat form of the tropane residue interacts with the receptor. The difficulties of arriving at a n unequivocal conclusion are considerable as has been pointed out recently (Hunt and Robinson, 1972). A number of crystallographic and nuclear magnetic resonance spectroscopic studies of the conformations of antiacetylcholine drugs in the crystal and in solution are of general interest although it remains to be shown whether such studies make any significant contribution to our knowledge of the mechanism of action of such drugs. Crystal structures have been determined for (S)-hyoscine hydrobromide (Pauling and Petcher, 1969), quinuclidinyl benzilate hydrobromide (Meyerhoffer and Carlstrom, 1969), benzetimide (Spek et al., 1971), and the active enantiomer of atropine, (S)hyoscyamine hydrobromide (Pauling and Petcher, 1970). Nuclear magnetic resonance studies have shown that the conformations in solution of benzilyl derivatives of choline and a- and /3-methylcholines differ little from the corresponding acetyl derivatives (Inch et al., 1970). On the basis of their studies which show conformational similarities between acetylcholine-like and antiacetylcholine drugs, Spek, Peerdeman, and co-workers as well as Pauling and Petcher have suggested that both types of drugs interact in similar ways with the muscarinic receptor. It remains an open question whether ground-state conformations in solutions or crystals necessarily bear any relation to the conformations of drugs when bound to receptor protein. In this connection, it is perhaps relevant that it has been demonstrated that benziloylcholine and benziloylthiocholine are equipotent as anti-acetylcholine drugs (Parlies, 1955) although they differ in ground-state conformation. In the former the +NMe, and ester substituents about the C-C bond have a gauche relation and in the latter the +NMe3 and thio ester substituents have a trans relation (Inch et al., 1970; Cushley and Mautner, 1970). From studies of the configuration and conformations of antiacetylcholine drugs, no clear picture has emerged of the nature of the receptor with which they interact. Perhaps, the recent approach adopted by Smythies (1971) provides the best hope of obtaining a unifying hypothesis for accommodating all the stereochemical data available at present. Smythies has assumed that the receptor is a complex of polypeptide (with arginine and glutamate components) together with nucleophospholipids, prostaglandin, and Ca2+ and has shown that the extensive structure-activity data on cholinergic agonists and antagonists is consistent with receptors in the muscarinic, neuromuscular, and ganglionic synapses, which differ simply in the amino acid sequences in the polypeptide. The various drugs are assumed to adopt the conformations necessary to fit the receptor.

101

ANTIACETYLCHOLINE DRUGS

IV. Quantitative Correlation of Chemical Structure and Antiacetylcholine Activity

Generally drug potency is a function of several physical properties operating simultaneously to produce the observed effect. Since these physical properties are changing more or less independently as compounds are compared one with another in a series, it is often not possible to discern the underlying patterns of change by mere inspection of the structure-activity data. Indeed, it is often a difficult task to establish which physical properties have an important influence on potency and which do not. I n an attempt to overcome some of the usual difficulties, Hansch and others (Hansch, 1967, 1968, 1969; McFarland, 1970) have developed linear free-energy relations in which drug potency (and in some cases the rate of drug action) may be related to physical parameters of the drug, such as partition coefficient, Hammett constant, dipole moment, and steric factors. Since this approach has been applied to a limited extent to antiacetylcholine drugs and because it is possible that a much deeper insight into certain aspects of the action of these drugs might be obtained by use of this approach, it is pertinent to outline the method and its potential relevance for studies of antiacetylcholine drugs. The initial approach was to relate drug potency to the Hammett constant u and a hydrophobic parameter II by an equation-such as

1 logc = k1II

+ k,o +

k3

where C is some measure of potency and ll = log P, - log Px where P, is the octanol-water partition coefficient of a derivative and PH is the partition coefficient of the parent molecule. In some forms of this and other equations, II is replaced by log P where P is the partition coefficient of the drug. It is possible to calculate u and I'I for many members of a particular drug series, providing the values for one member of the series are measured, by utilizing values of the parameters that have been calculated for most common substituents (Leo et al., 1971). The constants kl, k,, and k, are obtained by regression analysis using data from many compounds. The simple linear relation does not give adequate correlations of physical parameters and biological activity in many cases. For example, it was soon recognized that in many instances log (l/C) could be related parabolically, k3a k,. not linearly, to II by equations such as log (l/C) = klI12 + k,II Examples of the use of this type of equation are available (Hancsh and Anderson, 1967; Hancsh et al., 1967). The idea behind this equation was that molecules which are highly lipophilic will not penetrate lipophilic barriers readily and hence will have a low probability of reaching the biological sites of action in the test interval.

+

+

102

T. D. INCH AND R. W. BRIMBLECOMBE

Molecules having very high log P values will be strongly held by the first lipophilic material they encounter and thus will be hindered in their approach to the sites of action. Thus there will be a maximal log P for high potency. A theoretical analysis has justified the parabolic equations which have been used empirically to relate drug effectiveness with lipophilic character, and it has also been shown that the rate of penetration through skin and other membranes can be very dependent on partition properties (Pennington et al., 1969). It has been found possible to calculate the ideal lipophilic character (usually expressed as log Po)for drugs acting by a common mechanism. For example, optimal log Po for (a) a high rate of skin penetration by alkylphosphates was - 1.58; (b) rate of penetration of insecticides into cockroach cuticle was - 1.46 (Pennington et al., 1969); (c) antibacterial activity was 4 for drugs active against gram-negative bacteria, 6 for drugs active against gram-positive bacteria (Lien et al., 1967) ; (d) was 2 for compounds, such as barbiturates, that have activity in the central nervous system (Hansch et al., 1967). Further extensions (McFarland, 1970) of the Hansch equations have led to equations of the form log (l/C) = a n 2

+ bn + cu + dp2 + e(a - a=) + f

where p is the dipole moment, a is the polarizability of a substituent and aH is the polarizability of a nearby group on the receptor. I n other equations a steric substituent constant, usually defined as E,, is used. The general analytical approach is to include available experimental or derived data into a number of the above equations, to carry out regression analyses and to examine the correlation coefficients of the constants obtained. Equations where all the correlation constants are of high significance provide an indication of those physical parameters which are important to drug potency. Bowden and Young (1970) have examined a series of esters of type RCOOCH,CH,NEt,HCl, where R usually contained aromatic groups which were sometimes substituted. It was found (for this series of compounds where there was no aromatic substituent) that the antiacetylcholine activity as assessed by the ability of the drugs to antagonize the response of the guinea pig ileum to acetylcholine, and the antiacetylcholine activity calculated from the Eq. (1) were in excellent agreement. log (l/Cso) = 0.317 112

+ 2.596II + 6.46 u - 0.197

(1)

where C,, is the dose required to produce a 50% inhibition of spasm produced by acetylcholine. The optimum partition constant of nofor R in RC02CH2CH2NEt2HC1 was calculated from this regression to be 4.08. It was suggested that the dependence of the activity on polar effects and the independence of steric

103

ANTIACETYLCHOLINE DRUGS

effects-the absence of a steric term in Eq. (1)-appear to be directly related to the drug's ability to effectively interact with the receptor. Any interaction involving spatial demand at the carbonyl group or, for example, H-bonding from the receptor to the C=O therefore appeared unlikely. Regression analysis of the nuclear substituted compounds in the above series indicated that substituents were unfavorable for interaction because of steric rather than electronic effects. A second study of the use of correlation techniques has concerned some 3-tropanyl 2,3-diarylacrylates (Craig et al., 1970). Regression techniques were used to correlate II and u functions with antispasmodic activity. For 14 compounds, the best results were obtained with the equation log l/C = 4.744 - 1.215 'U - 0.842 u In the series of compounds examined, log P was 4.3 & 1.2, so it is not surprising that there is little apparent dependence of antispasmodic activity on partition coefficient. It is of interest that the results of this analysis showed a dependence on u but not on II. The importance of the electronic distribution around the aromatic ring is not usually considered important for antiacetylcholine effects. This result must raise the question whether the spasmolytic activity of these diarylacrylates is really connected with antiacetylcholine activity. Another approach which uses the Hansch II treatment has been made by Triggle (1971) in an attempt to interpret some data (Abramson et al., 1969) comprising the variation of affinity constant (log K) with structure for a series of drugs of general formula R-+NMe,. By assigning Ph(Ch,), +NMe, the value X I l = 0, Triggle calculated ZI'I values for the members TABLE VII II-ANALYSIS FOR CHOLINERGIC ANTAGONIST SERIES R-+NMe3

R

No.

XI 0

1 Ph(CHz),

2 Ph(CHz)zO(CHz)z 3 4 5 6 7 8 9 10 11 12

PhCHzCOO(CHz)2 CeHii(CHz), C s H i i(CHz) zO(CHz)z CeHliCHzCOO(CHz) 2 PhzCH(CHZ)* Ph,CHCHZO(CHz), PhZCHCOO(CH2)a (CeHid zCHCHzO (CHz) z (CeHli)aCHCOO(CH,)z Ph(CeH11)CHCOO(CHz)z

-0.51 - 1.28 0.62 +0.11 - 0.66 1.89 1.38 0.3 1 2.62 1.85 1.23

+

+ + + + + +

log K 5.180 4.702 4.533 5.387 5.282 5.067 7.015 6.413 7.159 7.254 7.686 8.438

104

T. D. INCH AND R. W. BRIMBLECOMBE

0.0

0"

[L o9

6.0

5.0 4.5

- 1.0

+4.0 +2.0 Ln FIG. 3. Log K - l l plot for muscarinic antagonists listed in Table VII. 0

of the series listed in Table VII. From a plot of log K against ZII (Fig. 3), Triggle showed that apart from the esters (3,6,9, 11, 12 in Table VII) there was an essentially linear dependence of activity upon II so that the antagonistic activity increases with increasing hydrophobic character of the ligand. The ester series show a similar but less well defined dependence of activity upon II. It was concluded that these results revealed the possible existence of two separate interaction mechanisms, one dependent only on the hydrophobic character of the R group in +RNMe, and the other dependent on both hydrophobic and polar character. Other attempts to use a quantitative approach for correlating antiacetylcholine activity with partition properties have been reported. Pratesi and his co-workers (1969) observed a pA,-lI correlation for the antiacetylcholine activity of a series of N-alkyl-N-phenyl-Z-aminoethyl-( N-piperidino) ethiodides deviating from linearity with large alkyl groups ( > C,H,,) that had presumably exceeded the size of the nonpolar binding areas. Chang et al. (1972) have reported a reasonable correlation of PA, and ll values for some 2,2-disubstituted derivatives of 1,3-dioxolane-4-dimethylaminomethyl methiodides. I t should be noted that although the correlation-type approach may be valid there are inherent dangers. For example, depending on the II values

ANTIACETYLCHOLINE DRUGS

105

chosen it may be shown that three separate correlations (for esters, ethers, and polymethylene chains) may be obtained instead of the two correlations found by Triggle. Also attention has been drawn to the fact (McFarland, 1970) that electrostatic attraction between a charged site on the receptor and one on the drug is of such great energy that it could easily override the importance of other factors. For dissociable drugs that in solution exist as mixtures of ionized and nonionized forms (such as antiacetylcholine drugs like atropine), equations have been used that take into account the dissociation constant of the drug (Fujita and Hansch, 1967). For example, a pKa-dependent term was used in correlations of the rate of metabolism (MR) of amines (Hansch et al., 1968). log M R = -0.231 log P2

+ 0.972 log P + 0.48 pKa - 3.973

Unfortunately there are considerable difficulties associated with accurate methods for measuring directly the pKa of most antiacetylcholine drugs (mixed solvents must be used), and partition measurements a t p H values greater than 7.5-8 are complicated because hydrolysis of the esters occurs. Despite these difficulties, the results obtained by the use of quantitative correlation methods and the results obtained from studies of antiacetylcholine drugs make it clear that useful information can be obtained about the nature of the drug-receptor interaction and, probably more important, about physical features that influence the rate and extent of drug distribution. Thus, for antiacetylcholine drugs, the possibility of calculating optimum values of partition coefficients for the penetration of tertiary bases into the central nervous system and of showing whether or not steric or electronic factors have any significant influence on distribution is clearly worthy of exploration. Attention has been drawn elsewhere (Section VI) to the importance of distribution factors on the activity of antiacetylcholine drugs.

V.

Factors That Influence the Time Activity Profile of Antiacetylcholine Drugs

Attention has been drawn (Section 111, B) to the fact that although the rates of onset of antiacetylcholine effects in uiuo (and also in uitro) are strongly influenced by the dose or concentration of the drug, other factors also have a pronounced effect (Brimblecombe et al., 1971b; Inch et al., 1973). The conclusion was reached that the time-activity profiles of antiacetylcholine drugs are related to their affinity constants so that both times to onset and duration of effects increase as the affinity constant increases for drugs that are administered at equipotent doses. A typical situation is illustrated in Fig. 4, where it is shown that although dimethylaminoethyl and N-methyl(compounds 22 piperidin-4-yl ( 2 4-2-cyclohexyl-2-hydroxy-2-phenylacetates and 24, log K = 9.06 and 10.92, respectively) both produce maximum

106

T. D. INCH AND R. W. BRIMBLECOMBE

,

COMPOUND 22 (R) Time to peak response

COMPOUND 22(SI

20

10

X

10 3

0

0

b

5 40 20 40 80 160

Time to reach 50% of peak response COMPOUND 2 4 ( S I

COMPOUND 24 ( R )

0

5 10 20 40 80l6032064Ol280

0 5 I0 2 0 4 0 80 160320

Time (minl log scale

FIG.4. Graphs showing production and duration of action of mydriasis induced by isomers of compounds 22 and 24. Compounds 22(R) 24(R): x = 0.1,O = 0.05,O = 0.025 pmole/kg i.v. Compound 24(S) : x = 2, 0 = 1, 0 = 0.5 pmole/kg i.v. : x = 4, 0 = 2, 0 = 1 pmole/kg i.v. Compound 22(S) Compound 24(R) :A = 0.005 pmole/ml topical application Compound 24(S) :A = 0.125 pmole/ml topical application

mydriasis in mice at 0.1 pmole/kg, the effects were produced more rapidly in the former and were more persistent in the latter. Also it was shown that at a concentration of 4 x 10-B M the time required for the methiodides of R-(-)- and S-(+ )-N-methylpiperidin-4-yl 2-cyclohexyl-2-hydroxy-2phenylacetate (compound 25 and its S enantiomer) (log K = 11.08 and 9.08, respectively) to equilibrate with the guinea pig ileum were 107 and 28 minutes respectively. These and other results from studies of the ability

~

ANTIACETYLCHOLINE DRUGS

107

of antiacetylcholine drugs to antagonize oxotremorine-induced salivation and tremor (Inch et al., 1973) showed that the relationship between affinity constants and time-activity profiles appears to hold in vitro and in vivo for both PNS and CNS activity. The relation between duration of action and affinity constants can be demonstrated only if great care is taken in measurements of antiacetylcholine activity using the guinea pig ileum. An example of the care required, and perhaps of the need to standardize pharmacological procedures, may be provided by reference to benzetimide and its active enantiomer dexetimide (Janssen et al., 1971). The published time-activity profiles for mydriasis produced by these relatively long-acting drugs were consistent with log K values for benzetimide and dexetimide of 10 and 10.3, respectively (affinity constant of an active enantiomer should be twice the affinity constant of the racemate, i.e., a difference of log K = 0.3). However, Janssen, Niemgeers, and their co-workers recorded PA,, values of 7.56 and 8.10 for benzetimide aild dexetimide, values which are equivalent to log K values of 8.51 and 9.05, and with such values benzetimide and dexetimide would be expected to be relatively short-acting drugs like atropine (log K = 9.00). However, when log K values were determined under conditions that ensured equilibrium between the drugs and the guinea pig ileum, values of 9.95 and 10.25 were obtained (Inch et al., 1973). This result is particularly important because benzetimide has a chemical structure quite distinct from that of most antiacetylcholine drugs. The apparent dependence of the time-activity profiles of antiacetylcholine drugs on their affinity constants, coupled with the fact that for the glycolates it was observed (see Section 111, B) that the differences in potency of enantiomers resulted mainly from differences in the drug-receptor interaction, not from general distribution or metabolic factors, make it necessary to seek an explanation for differences in time-activity profiles on the basis of the interaction of the drug with the receptor. Most of the fundamental studies concerning the times to onset and duration of action of antiacetylcholine drugs and their relation to the drugreceptor interaction have been carried out to explain one basic anomaly which is relevant to most discussions of antiacetylcholine drugs. The anomaly is that although atropine and related drugs generally behave as competitive antagonists of acetylcholine and related compounds, it is not easy to reconcile with the concept of true competitive antagonism the finding that although atropine has a slow dissociation rate, acetylcholine can produce a prompt response in atropinized preparations and that large quantities of acetylcholine do not apparently facilitate atropine washout. Three basic explanations would appear to be available to accommodate such discrepancies: (a) Atropine and related drugs do not act at the receptor site

108

T. D. INCH AND R. W. BRIMBLECOMBE

but act by a n indirect or regulatory mechanism. (Steric evidence which shows that agonist and antagonists do not share common areas of attachment with a receptor, has been described in Section 111, B. (b) Activator molecules need only occupy a small fraction of receptors to produce a maximum response and hence can produce this response even when a substantial portion of the receptors are blocked by a slowly dissociating antagonist. (c) Activator molecules need occupy only a small fraction of receptors to produce a maximum response and actually dissociate very quickly from the receptor but that access to and from the receptor from the aqueous phase is hindered by a biophase which does not limit access of the activator molecule. Measurements of the time course of action of antiacetylcholine drugs have been primarily concerned with distinguishing between explanations (b) and (c). Explanation (b) is consistent with the reaction-rate limiting theory, in which it is considered that the rates of onset and offset of drug action are dependent on the rate coefficients k, and k2 of the association and dissociation of drug with receptor, i.e., Drug

+ receptor

k,

. A Drug - receptor ks

Explanation (c) is consistent with the access-limited theory, in which it is considered that the dissociation and association reactions are very fast and that the observed reaction rates depend on the rates of diffusion into and out of (or through) a biophase which surrounds the receptor, i.e., Receptor

+ biophase + drug

ka

A ~

,[drug

- biophase] + receptor

.ti

[drug

- receptor] + biophase

I t has been shown that most kinetic results can be considered to be consistent with either model provided that suitable values of k,, k,, k3,and k4 are chosen. (It should be noted that both models involve the “spare receptor” theory, an assumption that is not necessarily valid.) It was suggested by Rang (1966) that a distinction can be made between the reaction rate-limited and access rate-limited models from studies of the antagonism produced by combinations of fast- and slow-acting antagonists. According to the reaction rate-limited model, addition of a fast-acting antagonist to muscle preequilibrated with a slowly dissociating antagonist, will result in equilibration of the former agents with that fraction of the receptors unoccupied by the slowly dissociating antagonist before any shift has occurred in the equilibrium between the receptors and the slowly dissociating antagonist. Consequently there should exist an overshoot of antagonism that will decline to the new

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equilibrium occupancy at a rate determined by the dissociation constant of the slowly dissociating drug. It was suggested by Rang (1966) but disputed by Thron and Waud (1968) that this phenomenon would not be expected on the basis of the access-limited model. I n short, the experimental evidence which has been invoked to distinguish between the two models is conflicting and readers are referred to the papers by Rang (1966) and Thron and Waud (1968) for a clear exposition of much of the available data. Perhaps the observation that is most relevant to the finding that rate of action is related to affinity constant is one by Thron and Waud (1968), who have shown that the differences in the affinities of the drug for the receptor provide the key to explaining differences in their rate of action. The explanation follows from a consideration of the “virtual space” presented by receptors. When a drug diffuses into a simple compartment, the rate of equilibration depends on the rate of diffusion and on the volume to be filled. If the compartment contains binding sites or concentrating mechanisms, these tend to increase its apparent volume so that a longer time is required for the establishment of diffusion equilibrium. If tissue containing M moles of receptor per gram is equilibrated with drug concentration x and if K is the affinity (or association) constant, then the receptors will take up a n amount of drug equal to Mx/(k, x ) moles per gram of tissue. This is the same quantity that would be taken up by a physical compartment of volume M / ( l / k , + x ) assuming a partition coefficient of unity. Thus the receptors present a “virtual space” equal to M / ( l / k , + x ) . For a weakly bound antagonist (where k, is small) this virtual space is relatively small, and accordingly the establishment of diffusion equilibrium is relatively fast. For a strongly bound agent, the virtual space will be large and, particularly at low concentrations, the rate of equilibration will be slow. At high concentration the receptor virtual space tends towards zero and the rate of equilibration approaches that of an unbound or weakly bound agent. Finally, if several agents are compared at concentrations at which they all produce the same degree of receptor occupancy the virtual space will be proportional to the affinity constant and the rate of equilibration will decrease regularly with K. This explanation is attractive because of its simplicity and because it explains most of the observed results. It is also attractive because it overcomes the difficulty presented by most access-limited models that agonists act almost immediately and appear to experience no diffusive delay whereas even weak antagonists require several minutes to equilibrate with receptors. Independent evidence suggests that affinity constants of agonists are low. Another attractive feature of this access-limited model is that it provides an explanation for the difficulty that is usually experienced in measuring accurate affinity constants of highly potent antiacetylcholine drugs. On the

+

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basis of the access-limited model, it is possible for receptor occupancy to rise faster initially than the antagonist concentration at the receptor environment, and thus it is likely that some measurements of affinity constants for highly active drugs are measured before true equilibrium is reached. The theory put forward by Thron and Waud (1968) does not require that the rate of diffusion be the rate-limiting step, but only that the volume to be filled determine the rate. Thus it provides a rationale of why drugs with different configurations and partition properties act at the same rate if they have the same affinity constant. Conversely, however, it does not provide an explanation of why rates of dissociation increase as the affinity constant decreases or why pairs of enantiomers may dissociate a t markedly different rates. I n any biophase model that predicts diffusion controlled offset of action, it is necessary to assume that appreciable differences exist in the rates of diffusion of enantiomers through the biophase. Existing evidence shows that enantiomers usually show little difference in their rates of penetration through biological membrane. Thus the fact that the enantiomers of antiacetylcholine drugs which differ in potency also differ in their durations of action may be considered as evidence that the duration of action is dependent on the rate coefficient for the dissociation of drug from receptor, providing the rate of diffusion is much slower than the rate of metabolism and essentially independent of subsequent diffusion processes. I n the foregoing discussion it has been shown that, although no unequivocal explanation of the factors controlling the rates of onset and offset of action of antiacetylcholine drugs is possible, available evidence is consistent with a model in which time to onset is dependent on the rate of diffusion into the compartment containing the receptors [it has been shown recently (Thron, 1972) that simple mathematical models are not available which are consistent with such a simple mechanism] and that duration of action is dependent on the rate coefficient for dissociation of the drug-receptor complex, particularly for long-acting drugs. However, the available data about rates of reaction in no way militate against the concept of entirely separate sites of action for acetylcholine agonists and antagonists. It has been demonstrated (Inch et al., 1973) that for any one drug the times of onset of antiacetylcholine effects are essentially the same in the CNS (as measured by antagonism of oxotremorine-induced tremors in mice) as in the PNS (as measured by antagonism of oxotremorine-induced salivation in mice) and that this time of onset increases with affinity constant. The result that the relation between affinity constants and time-activity profiles in vivo is independent of the nature of the in vivo test and the result that time-activity profiles are mainly controlled by events in the immediate vicinity of the receptor provide strong evidence that the chemical nature of the receptor with which antiacetylcholine drugs interact is essentially the same in the

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CNS and PNS of mice and probably of other species as well. Thus evidence has been obtained from studies of time-activity profiles of antiacetylcholine drugs that supports the conclusion drawn from comparisons of enantiomeric potency ratios. The general conclusions expressed previously in this section are supported by some additional results that provide information about the rates and durations of action of antiacetylcholine drugs. Atropine sulfate administered a t a dose of 1.2 mg/kg i.p. to mice afforded protection against a LD,, dose of oxotremorine only if administered less than 60 minutes before the oxotremorine. Higher doses of atropine sulfate afforded protection for longer periods (Natoff, 1970). The distribution of atropine in rats by i.p. and S.C. administration have been compared for a period of 300 seconds. I n both cases, liver and plasma levels of the drug rose rapidly (Lukas et al., 1971), although the results indicated that compounds administered i.p. are absorbed primarily through the portal circulation and must pass through the liver before reaching other organs. Gill and Rang (1966) compared benzilylcholine mustard (N-P-chloroethyl-N-methyl-2-aminobenzilate)and benzilylcholine and found that, although the drug potencies were similar and the time to onset of effects either on isolated tissue or in causing mydriasis were similar, the mustard had a far more persistent action. The complex with the receptors formed by benzilycholine mustard was a reversible one, but alkylation occurred rapidly the rate of alkylation being considerably greater than that of dissociation of the reversible complex. VI. Relative Potencies of Antiacetylcholine Drugs in the Central Nervous System and the Peripheral Nervous System

For any antiacetylcholine drug it is important, for many reasons, to know the relation between its potency in the CNS and its potency in the PNS. For example, (a) antiacetylcholine drugs with high CNS and low PNS activity are desirable for the treatment of Parkinson’s disease; (b) for the purpose of developing more efficient drugs than atropine for the treatment of anticholinesterase poisoning (Section VIII), it is important that the contribution of the CNS and PNS effects of these drugs be properly assessed; (c) since in studies of behavioral changes caused by antiacetylcholine drugs the central effects are often complicated by accompanying peripheral effects, it is essential to know the relative PNS and CNS potencies of these drugs (Section VII). I n this section, methods for comparing CNS and PNS potencies will be described and their limitations discussed. Methods for comparing the potencies of antiacetylcholine drugs in the CNS and PNS must be carried out in uiuo and should ideally be measures of the efficiency of these drugs in antagonizing the effects of endogenous

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acetylcholine. However, although measurements of mydriasis are convenient for estimating antiacetylcholine activity in the PNS (even although central effects may, for certain drugs, make a small contribution to observed mydriatic effects), there is no correspondingly simple test for CNS antiacetylcholine activity. Most procedures for measuring antagonism of CNS activity involve behavioral tests which are very time consuming and particularly difficult to quantify (Longo, 1966). Thus it is a logical choice to seek to compare antiacetylcholine drugs on the basis of their ability to antagonize the effects of exogenous drugs which act like acetylcholine in the CNS and PNS. Since quaternary drugs do not easily penetrate the blood-brain barrier, comparisons of antagonism of exogenous acetycholine and related quaternary drugs can be made only if the central effects are assessed after intracerebral administration of the cholinergic drug (Madill et al., 1968; Decsi et al., 1963). This is not an entirely satisfactory procedure for routine usage. Consequently, many workers have attempted to use tertiary muscarinic drugs that can penetrate the blood-brain barrier to produce centrally similar effects to exogenous acetylcholine. Drugs that have been used include pilocarpine, arecoline, and oxotremorine, but of these drugs probably only oxotremorine (Cho et al., 1962) fulfills all the requirements necessary for estimating antiacetylcholine activity in the CNS and PNS. Pilocarpine suffers from the disadvantage of being a partial agonist (Barlow, 1964) with only weak CNS activity, and arecoline from the disadvantage that relatively high doses are required to produce central effects that are of very short duration (Zejmal and Votava, 1961). Arecoiine also has nicotinic properties. Oxotremorine suffers none of these disadvantages; it is an extremely potent muscarinic agent in the PNS, and these effects are completely blocked by atropine and its congeners (Friedman and Everett, 1964; Bebbington and Brimblecombe, 1965). It has essentially no nicotinic action, nor is there any significant effect on cholinesterase (Cho et al., 1962; Holmstedt et al., 1965). On isolated ileum it behaves similarly to quaternary acetylcholine-like drugs (Brimblecombe et al., 1970b). The central actions of oxotremorine are also blocked by atropinelike compounds, and central and peripheral effects can be separated by means of a quaternary drug that can block only peripheral responses. The central effects of oxotremorine are duplicated by other muscarinic agents, such as carbachol, when applied directly to appropriate sites in the brain (Connor et al., 1966), although it is possible that all peripherally similar cholinergic drugs do not act in the same way in the CNS (Ankier et al., 1971) and a good correlation can be made between the central tremorogenic effects of oxotremorine and its analogs and their in vitro parasympathomimetic activities (Bebbington et dl., 1966). A good correlation has been demonstrated between potencies of antiacetylcholine drugs in antagonizing oxotremorine-induced tremors and potencies in elevating EEG

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arousal thresholds (Brimblecombe and Green, 1967, 1968; Inch et al., 1973). There has been considerable discussion as to whether oxotremorine acts directly on muscarinic receptors or whether it acts indirectly by releasing acetylcholine. Recently available evidence (Cox and Hecker, 1971; Cox and Potkonjak, 1969) favors a direct interaction. However, as has been pointed out (Brimblecombe, 1970), it is relatively unimportant from the viewpoint of measurement of antiacetylcholine potency which mechanism operates provided that muscarinic receptors are involved. The recent finding (KarlCn et al., 1971) that oxotremorine distribution is affected by antiacetylcholine drugs does not appear to invalidate the use of oxotremorine for comparing central and peripheral activities of antiacetylcholine drugs. For the above reasons PNS :CNS potency ratios for antiacetylcholine drugs have been determined in this laboratory by measuring the antagonism of salivation (a PNS effect) and tremors induced by equal doses (Brimblecombe and Green, 1967, 1968) or equipotent doses (Inch et al., 1973) of oxotremorine. It should be pointed out, however, that other CNS effects of oxotremorine, such as hypothermia (Spencer, 1965) and analgesia, are antagonized by antiacetylcholine drugs. Oxotremorine-induced hypothermia is also antagonized by sympathomimetic amines, such as amphetamine, and by quaternary antiacetylcholine drugs, and so antagonism of this effect cannot be used as a measure of central antiacetylcholine activity. It has been demonstrated, however (Leslie, 1969), that antagonism of oxotremorineinduced analgesia can be used for assessing central antiacetylcholine activity, since for a series of drugs there was a very good correlation between the results obtained in the induced analgesia test and those obtained in an oxotremorine-induced tremor test. Indeed, it is possible that for some purposes antagonism of oxotremorine-induced analgesia, which may be more specific than antagonism of oxotremorine-induced tremors, may be a better procedure for estimating central antiacetylcholine activity. Various sympathetic blocking drugs (e.g., phenoxybenzamine, propanolol, and reserpine) are effective in inhibiting oxotremorine-induced tremor in rats (Cox and Potkonjak, 1969), and it is essential therefore to show that a drug is a competitive antagonist of acetylcholine (i.e., by measuring affinity constants) before antioxotremorine tests can be used as measures of antiacetylcholine activity. Two pieces of information illustrate that comparisons of antagonism of oxotremorine-induced salivation and tremors accurately reflect the activity of antiacetylcholine drugs in the PNS and CNS. It has been demonstrated by comparing the antiacetylcholine activity of a large number of drugs in these tests (Inch et al., 1973; Brimblecombe and Green, 1968) that no antiacetylcholine drug is appreciably more potent in the CNS than PNS. This observation was consistent with the finding that the receptors with which

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antiacetylcholine drugs interact are essentially the same in the CNS as in the PNS (Sections I11 and V.) The second piece of evidence to support the use of the oxotremorine procedure is that the PNS:CNS potency ratio obtained for atropine by this procedure is close to that which may be calculated from results of experiments of antagonism of endogenous acetylcholine with atropine in man. In man the dose of atropine required to produce peripheral effects is 0.25-2 mg per man, whereas to produce central effects which relieve symptoms of Parkinson’s disease 54 mg per man per day have been given (Martindale, 1968); further, single doses of 30-50 mg per man are required to produce central (psychotomimetic) effects (Ostfeld and Aruguette, 1962). From these figures the PNS:CNS ratio in man appears to be approximately 50. Against this background, it is of interest to consider some of the reports which suggest that antiacetylcholine drugs may be obtained that have high CNS: PNS potency ratios and which at first sight are contrary to the results obtained by oxotremorine procedure and are not consistent with the concept that the antiacetylcholine receptors are essentially the same in the CNS as in the PNS. Parkes (1965) found that pyrollenoethyl benzilate had a CNS :PNS ratio fifty times greater than hyoscine. This result, however, does not indicate a compound significantly more potent in the CNS than PNS because, when compared as an antagonist of oxotremorine-induced salivation and tremors, hyoscine has a PNS :CNS ratio of 22 from which a CNS :PNS ratio of 2 for pyrollenoethyl benzilate may be calculated. Parkes (1965) determined CNS potency by four methods : potentiation of morphine-induced analgesia in mice, antagonism of morphine-induced tail erection in mice, emesis in pigeons, and a behavioral test involving spontaneous alternation in rats. Although these tests most probably reflect the potency of drugs producing a central effect, it is not known whether cholinergic systems are directly involved, and therefore the tests, cannot be considered to give a specific estimate of central antiacetylcholine activity. When pyrollenoethyl benzilate was examined by the oxotremorine procedures it was found to be a weak antiacetylcholine drug with a PNS: CNS ratio > 1. In 1970, Lingdren and others found that one N-(perhydroazepinoalkyny1)succinimidederivative was twelve times more potent in antagonizing oxotremorine-induced tremors (a central effect) than in producing mydriasis (a peripheral effect) in mice. No attempt was made to determine potencies at optimal times, and the other serious disadvantage of this procedure is that whereas the CNS potency determinations depend on the use of an exogenous agonist, the mydriasis test depends presumably on antagonism of endogenous acetylcholine. In 1969, Brown et al. described some pharmacological properties of benapyrazine (BRL 1288),which was reported as being capable ofpreventing

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oxotremorine-induced tremor in mice at doses that showed very little peripheral antiacetylcholine activity as measured by mydriasis. Subsequently it was found (Leslie and Conway, 1970) that benapyrazine in mice was only 2.5, 1.3, and 3.1 times more potent centrally in antagonizing oxotremorineinduced tremor, analgesia, and hypothermia, respectively, than peripherally, where peripheral activity was assessed by arbitrary screening of salivation, defecation, lacrimation, and sweating. Although these CNS :PNS potency ratios were similar to that obtained by direct comparison of oxotremorineinduced tremors and salivation (Inch et al., 1973), the procedures may be criticized because no attempts were made to determine time-activity profiles and because oxotremorine was given a t the high dose of 2 mg/kg. Indeed, by Leslie and Conway’s procedures, the PNS :CNS potency ratio for atropine was unity. More recently it has been claimed that KAO-264 (6,6-9-trimethyl-9azabicyclo [3.3.1] non-3-P-yl a7a-di(2-thienyl)glycolate hydrochloride is more potent in the CNS than PNS (Kojima et al., 1971). The central antiacetylcholine activity was determined by protection tests against tremorine-induced tremor and physostigmine-induced death in mice, whereas the peripheral tests were production of mydriasis and a test for protection against pilocarpine-induced salivation. In these procedures the CNS:PNS potency ratio for KAO-264 and atropine were 1.96 and 0.28, respectively. The low PNS :CNS ratio for atropine reflects the inadequacy of the experimental procedures for providing a realistic estimate of PNS: CNS potency ratios. The limitations of published experimental procedures for comparing CNS and PNS potencies of antiacetylcholine drugs are clear, and to our knowledge there have been no reported examples to show that antiacetylcholine drugs may be appreciably more active in the CNS than PNS. On the basis of the results presented in Sections I11 and V, it is unlikely that any such drugs will be prepared. Although it is not possible to have antiacetylcholine drugs with high CNS :PNS potency ratios, many antiacetylcholine drugs do have high PNS :CNS potency ratios. Undoubtedly the ability or otherwise of antiacetylcholine drugs to penetrate the blood-brain barrier accounts for the fact that some drugs are much less potent in the CNS than PNS (Brodie et al., 1960). For example, quaternary antiacetylcholine drugs, which penetrate the blood-brain barrier only slowly, are usually devoid of central antiacetylcholine activity whereas highly lipophilic tertiary antiacetylcholine drugs usually have pronounced activity in the PNS and CNS (Golikov and Pechenkin, 1963). Indeed, it has been shown that there is some relation between the CNS and PNS activity of antiacetylcholine drugs and their partition coefficients (Herz et al., 1965). It has also been demonstrated that

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the distribution of a series of acetylenic amines between the CNS and PNS could be correlated with their pK, value (Karltn and Jenden, 1970), and the relation between pK, and partition coefficients is well established (Butler, 1953). It is important to emphasize, however, that it is the rate of penetration of drugs into the CNS, not directly their degree of penetration, that is dependent on their partition properties. Thus in a hypothetical situation where metabolism and other general absorption processes are absent, it might be expected that any given antiacetylcholine drug would be equipotent in the CNS and PNS but that the rate at which CNS effects were produced would be related to the partition properties of the drug. In practice, however, the results of pharmacological (Inch et al., 1973) studies and distributional studies appear to indicate that distribution in vivo is rapid and that the percentage of any antiacetylcholine drug that penetrates into the CNS does so rapidly irrespective of the partition properties of the drug. The PNS does not act as a reservoir from which slow release of antiacetylcholine drugs into the CNS takes place. There are many difficulties apart from the pharmacological problems discussed previously in this section, which made it difficult to correlate partition properties precisely with the relation between CNS and PNS effects. Correlations between central antiacetylcholine activity and partition coefficients measured at physiological pH, such as those described by Herz and his co-workers (1965) are not necessarily valid because even it it is justified to use aqueous buffer-organic solvent systems as models for the in vivo situation, the partition coefficients obtained by such procedures are subject to thermodynamic control whereas in vivo kinetic factors will assume greater importance. Thus the rate of penetration from the PNS to CNS may possibly be correlated more realistically with the partition coefficient of the non-ionized drug than with the partition constant measured at any set pH. Unfortunately, physicochemical procedures have not yet been adopted to permit accurate measurement of the former parameter for relatively insoluble, alkali-labile, esters of amino alcohols. At present, although it is very clear that drugs like atropine and hyoscine which partition preponderantly into the aqueous phase from n-butanol have high PNS :CNS potency ratios whereas highly lipophilic drugs have PNS: CNS potency ratios that approach unity, it is not possible to establish whether optimal CNS potency is associated with particular partition characteristics. VII. Behavioral Studies

Interaction of muscarinic agents, notably oxotremorine, with central acetylcholine receptors results in tremors, often accompanied by akinesia and muscular rigidity, and so it is likely that central cholinergic systems are involved in the coordination of movement. This view is supported by the

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fact that antiacetylcholine drugs have been used in the treatment of Parkinson’s disease since the middle of the last century and it seems that, in part, hyperactivity of cholinergic systems are involved in the pathogenesis of Parkinsonism. This matter is discussed in detail by Brimblecombe and Pinder (1972), who concluded that tremors and other movement disorders are associated with decreased levels of catecholamines on the one hand and increased levels of acetylcholine and histamine on the other. As well as this involvement in the coordination of movement, central cholinergic systems also seem to be involved in other, more general, aspects of behavior. Antiacetylcholine drugs have been widely used as tools to explore this possibility. I n man the psychotic manifestations of belladonna poisoning have been recognized as being due to atropine, and more recently the psychofomimetic properties of glycolic acid esters have been fairly widely studied (see, for example, Abood, 1968), but precise information concerning their potency in man is lacking, and until this is forthcoming it is extremely difficult to draw any conclusions concerning their modes of action, in particular whether the behavioral effects result from central antiacetylcholine actions. There have been many studies of the behavioral effects of antiacetylcholine drugs in experimental animals, but although many interesting and significant results have emerged it is difficult to relate them to the largely subjective behavioral changes that the drugs produce in man. Nevertheless there is a good deal of evidence, some of which is given below, for the involvement of cholinergic systems in specific aspects of behavior. When the types of behavior being studied are complex, the evidence is perhaps less convincing; this is to be expected since noncholinergic systems are certainly also involved in complex behavioral responses, and effects on cholinergic systems may, therefore, be partially masked. Behavioral studies prior to 1966 were reviewed by Longo (1966), and no attempt will be made here to give an exhaustive review. Rather, a few studies will be considered in some detail. One characteristic behavioral effect of centrally acting antiacetylcholine drugs is to produce hyperactivity in animals. Spontaneous motor activity is easily measured in rodents, so that this effect has been much studied. Harris (1961) found that atropine, hyoscine, and several other drugs all produced an increase in the spontaneous activity of mice as measured using photocell cages. Their potency in this respect was not, however, correlated with their mydriatic potency. Since the latter effect is mainly a manifestation of a peripheral action of the drugs, the lack of correlation is probably not surprising. Harris also showed, using d- and Ghyoscyamine and d- and Gtrihexyphenidyl, that only the more active isomers with appreciable antiacetylcholine activity caused increases in spontaneous activity; i.e., that antiacetylcholine activity was necessary for the stimulatory effect. Quaternary

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salts with the exception of methylhyoscine were inactive, and, since even this drug was about one hundred times less effective in increasing spontaneous activity than the corresponding free base, it seems reasonable to conclude that the effect was central in origin. Abood (1968) studied the effects of some glycolic acid esters on spontaneous activity in rats. Within some series, e.g., piperidyl, potency in producing hyperactivity seemed to correlate with psychotomimetic potency. I n others, e.g., piperazinyl, there was only weak psychotomimetic activity despite marked ability to produce hyperactivity. Brimblecombe and Buxton ( 1972) have measured the antiacetylcholine activities of eight drugs using a variety of methods and have compared these activities with the potencies of the same drugs in producing increases in spontaneous activity of rats measured using photocell cages. The results are given in Table VIII. Although there was no statistically significant correlation between potency in producing hyperactivity and any of the measures of peripheral antiacetylcholine potency, there was a significant correlation with potency in antagonizing oxotremorine-induced tremors (# < 0.05). This constitutes very strong quantitative evidence for the involvement of central cholinergic mechanisms in this particular behavioral response. The dose levels used in experiments of this kind are of relevance, Brimblecombe and Buxton (1972) found that the doses of drugs required to produce hyperactivity in rats and to produce a 50% blockade of oxotremorineinduced tremors in mice never differ by more than a factor of 4. This seems to add more support to the view that central cholinergic mechanisms are involved in both these actions of the drugs. Another commonly studied aspect of the action of antiacetylcholine drugs is their effect on processes variously described as “learning” or “memory,” i.e., the acquisition or retention of some response, often a conditioned response. I n this context a number of workers have studied drug actions on spontaneous alteration, i.e., the behavior characteristics of rats when placed in a simple T maze, of entering opposite limbs of the T on successive trials in the maze. There is still debate over the mechanisms concerned in this behavioral pattern, but it seems reasonable to assume that memory is involved since the direction taken in any given run of the maze is influenced by the previous run, which must, therefore, be recalled. Meyers and Domino (1964) reported that both hyoscine (0.13 or 1.05 mg/kg) and l-hyoscyamine (1.0 or 8.0 mg/kg) caused a reduction in spontaneous alternation. I-Hyoscyamine at a dose of 1.0 mg/kg reduced alternations to the level which would be expected by chance, and both doses of hyoscine and the higher dose of I-hyoscyamine reduced the level to one significantly lower than

TABLE VIII PERIPHERAL AND CENTRAL ANTIACETYLCHOLINE POTENCIES OF EIGHTDRUGS AND THEIR POTENCIES IN PRODUCING INCREASES IN SPONTANEOUS ACTIVITY OF RATS

Antiacetylcholine drugs Atropine Hyoscine N-Methyl-3-piperidyl benzilate N-Ethyl-3-piperidyl benzilate N-Methyl-3-piperidylphenylcyclopentyl glycolate N-Ethyl-3-piperidyl phenylcyclopentyl glycolate N-Ethyl-2-pyrrolidylmethylphenylcyclopentyl glycolate Ditran

Approximate minima1 effective dose (pmoles/kg) producing hyperactivity in rats

Antagonism Of effects in mice. ED50 (pmoles/kg) for antagonism of Salivation Tremors

Antagonism of aceProduction of tylcholine on isolated mydriasis in mice guinea pig ileum (potency relative to atropine) (1% KB) 1.o

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would be expected by chance. These results suggest an amnesic action of the antiacetylcholine drugs, although the effect of hyoscine cannot be explained solely on this basis, and it seems possible that the drugs produced response perseveration. Similar effects have been reported in animals with lesions in the hippocampus; thus Meyers and Domino postulated a possible hippocampal site of action for the antiacetylcholine drugs. Again the doses of hyoscine employed (0.3 and 2.4 pmoles/kg) are very close to those shown by Brimblecombe and Buxton to have other central actions. Parkes (1965) also studied the effects of hyoscine on spontaneous alternation and found that it was reduced to a level that would be expected by chance. The dose causing a significant interference with spontaneous alternation in 50% of trials was 0.1 mg/kg. Atropine had a similar effect but was less active by a factor of between 20 and 50; i.e., doses of 2 to 5 mg/kg were required. This ratio of potencies correlates reasonably well with measures of the relative central antiacetylcholine potencies of these drugs. For example, Brimblecombe and Green (1968) found that atropine was about 15 times less active than hyoscine in blocking oxotremorine-induced tremors. BureH (1968) using a slightly different form of apparatus elaborated the left-right alternation by punishing noncorrect (i.e.,nonalternating) responses with electric shock. The alternation habit was clearly impaired by both atropine at 6 mg/kg (i.e., 14.5 pmole/kg) and physostigmine a t 0.5 mg/kg. The same animals were used repeatedly throughout this series of experiments and so became. progressively overtrained. Presumably as a consequence of this, the drugs became progressively less effective. The other special feature of these experiments was that the alternation habit was acquired in the presence of the drug, and this may be an example of drug-dependent learning where the response is not transferred from the drugged to the nondrugged state. Thus, as was pointed out by BureS, his results do not necessarily imply that the drugs produce a loss of recent memory. The same statement can probably be made about all the studies using spontaneous alternation; nevertheless, it seems lik%lythat learning or memory or recall are affected by antiacetylcholine drugs. There have been many experiments designed to study the effects of the drugs on conditioned behavior, and, as with the spontaneous alternation experiments, many of these have given results that suggest an amnesic effect. However, amnesia, which in this context is an inability to reproduce or recall a conditioned response, can be a result of drug action on acquisition, retention, or recall of the response, and in order to differentiate between these the experimental design needs to be carefully conceived. In fact, not all studies by any means show decrement in performance. Longo (1966) reviewed some twenty reports of the effects of atropine and hyoscine on

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discrete trial avoidance, mostly in rats. Half of these showed no effect of the drugs on performance, three showed an increment, and seven a decrement. I n analyzing these studies, Longo noted that decrement was most likely to occur when the conditioned response was recently acquired or when it involved relatively complex motor performance. I n ten further studies analyzed by Longo in which an element of discrimination was introduced into the avoidance procedure, atropine and hyoscine were without effect on performance in four investigations, but produced a decrement in six investigations. I n these studies the variations in effects on performance seemed to be independent of dose of drug used. I n six studies of the effect of atropine and hyoscine on continuous avoidance, i.e., lever pressing in a n operant conditioning situation, there was a general tendency for the rate of response to be increased but for there to be either no effect or a reduction in the efficiency of avoidance responding. Finally, in seventeen studies of the effects of the two drugs in conditioned response situations with positive rather than negative reinforcement, decrements in performance were always reported. These may be due, in part, to the anorexic action of the drugs, but on the whole they appear to represent specific actions. The picture to emerge from these studies reviewed by Longo is a complex one with examples of both increments and decrements of performance resulting from antiacetylcholine drug treatment. One particular problem is whether apparent increments are due merely to the hyperactivity produced in the animals by drugs. This matter has been investigated by Brimblecombe and Buxton (1972), who studied the eight drugs listed in Table V I I I for their effects on the acquisition of a conditioned avoidance response by rats in a shuttlebox. There were two training sessions separated by 24 hours, each session comprising 50 trials. The drugs, initially in a dose of 25 mg/kg, were given 15 minutes before the first session only. The results, summarized in Table X, show that three drugs resulted in significant increases in the numbers of avoidances during the first session, while three produced significant decreases in avoidances in the second session. All the drugs caused hyperactivity, manifested by significant increases in the number of intertrial barrier crossings, i.e., crossings unrelated to the conditioned stimulus (a tone). I t is possible that this hyperactivity is responsible for the increased avoidances noted after three of the drugs, but the results showed that while avoidances progressively increased during the first session, the number of intertrial crossings reached a peak about half-way through the session and then decreased. A check was also made of the frequency of barrier crossing throughout the trial period. All animals crossed the barrier during the 6-second shock period, so that if the frequency of crossing during the remainder of the trial period (18 seconds intertrial and 6 seconds of conditioned stimulus) were

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random, the ratio would be 3 :1 intertrial: conditioned stimulus. I n practice the ratio was nearer 2 :1, suggesting a learned response to the conditioned stimulus. Although not conclusive, this evidence suggests that increased avoidance was an effect not due entirely to the hyperactivity of the animals. Oliverio (1967) produced evidence to support this view. He studied the effects of hyoscine (2 mg/kg) on avoidance conditioning of mice in a shuttlebox and found that it enhanced the performance of naive animals. I n comparison, the drug markedly impaired the performance of animqls which had been given five training sessions prior to drug administration and so had acquired the conditioned response. However, if the nature of the stimulus was changed, the drug again enhanced performance in the sense that the response to the new stimulus was acquired more readily than in control animals, i.e., the enhancement seemed to be related to the novelty of the stimulus. I n further experiments, Oliverio showed that naive mice given the same dose of hyoscine before a session of 50 trials showed enhanced performance compared with that of controls. When re-tested 24 hours later, the control and drug-treated groups did not differ in performance. Hyoscine given to animals which had previously received a training session of 50 trials impaired performance in subsequent sessions, but only during the period within 24 hours of the drug administration. Thus, in these experiments both the enhancing and impairing actions of hyoscine seemed to persist for less than 24 hours. Other workers, including Brimblecombe and Buxton (1972) in the work referred to above, have reported effects of longer duration. The picture to emerge from these and many other similar studies is a confused one. I n general it seems that antiacetylcholine drugs enhance performance in naive animals but impair performance in animals already trained. The former effect appears to be a specific one not due entirely to the hyperactivity of the animals. There also appear to be differences between species, and it is clear that the precise circumstances of the experiment can influence the kind of result which is obtained. I t is still not clear whether the drugs primarily affect the process of acquisition or that of retention, and so amid all this confusion it is not profitable to speculate on their modes and sites of action. Similar, apparently conflicting, results are also easy to find among studies of the effects of antiacetylcholine drugs on operant conditioning. Again there are many examples of both enhancement and impairment of performance, and little purpose would be served in listing them here. One series of experiments by Carlton does warrant consideration in some detail, however, since he has made an attempt to draw together various results into a reasonable hypothesis. I n 1963 Carlton suggested, as a result of consideration of a number of

ANTIACETYLCHOLINE DRUGS

123

studies of antiacetylcholine drugs on operant conditioning, that the drugs led to an increase in the number of unrewarded responses due to a disinhibition of the system which normally suppresses such responses and which is presumably cholinergic in nature. A reciprocal activating system was also assumed to be present mediated by amines, since amphetamine produced effects on operant conditioning very similar to those seen after antiacetylcholine drugs. Later, in 1968, Carlton developed the hypothesis, and suggested that antiacetylcholine drugs were influencing the process of habituation whereby repetition of a stimulus results in the gradual loss of its effect. Experimental evidence was derived from a situation in which rats were allowed 15 minutes to explore a novel chamber. One day later thay were water-deprived for 1 day and then reintroduced into the chamber, which then contained a water bottle. These animals located the water bottle more readily than waterdeprived animals which had not received previous experience of the chamber, presumably because the bottle was the only novel feature of the chamber to the animals with previous experience; i.e., they had habituated to all aspects of the chamber except the water bottle. Rats which were given hyoscine prior to the first session in the chamber, despite the fact that they were hyperactive and hyperexploratory, behaved like naive animals on their second exposure; i.e., habituation had not taken place. Carlton proceeded to discuss the commonly expressed view that antiacetylcholine drugs interfere with “memory” and to suggest that most experimental results which have been interpreted in this way could equally well be explained on the basis of his habituation hypothesis. Partial support for these views of Carlton was provided by Wagman and Maxey (1969). They used rats in an operant conditioning situation where one lever (the work lever) had to be operated in order to allow reinforced presses of a second lever (the reward lever). Doses of greater than 0.75 mg/kg hyoscine resulted in disruption of performance on the work lever but enhancement of performance on the reward lever, which was cued by an external clicker stimulus. The latter result might suggest that the animals were responding more adequately, i.e., becoming habituated less readily, to the stimulus-onset cue. Carlton was not able to show disruption of performance by hyoscine in a situation where positive reinforcement (sweetened milk) was used. He suggested that antiacetylcholine drugs mainly interfere with the respome to an “initial stress,” i.e., a noxious stimulus to which it has not yet learned an appropriate response. Normally the initial response to shock is a general suppression of behavior; this is possibly interfered with by the drugs in such a way as to result in improvement in performance in the early stages of conditioning. The subsequent decrement in performance in animals which

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T. D. INCH AND R. W. BRIMBLECOMBE

have already learned the response can be accounted for by the intrusion of unrewarded responses into the behavioral pattern. Carlton, like Meyers and Domino (1964), noted a similarity between the effects of hyoscine and hippocampal lesions and suggested that the drug might be producing a functional lesion of the hippocampus. Bignami and Rosic (1970) considered that Carlton’s hypothesis and their own, i.e., the response disinhibition hypothesis (Bignami, 1967), could be considered as a single working hypothesis for further analyses of the effects of antiacetylcholine drugs. Essentially Bignami’s hypothesis was that the reappearance of nonreinforced responses and of those responses whose suppression is necessary to obtain positive reinforcement or to avoid punishment was due not to inhibition of drive but more on the motor side of the stimulusresponse chain. Bignami and Rosic (1970) make a distinction between “drive disinhibition” produced by LSD-25 and “response disinhibition” produced by antiacetylcholine drugs. The overall conclusion to be drawn from behavioral studies with antiacetylcholine drugs seems to be that relatively simple behavioral responses like increase in spontaneous motor activity are clearly controlled by acetylcholine-mediated systems and thus are produced by antiacetylcholine drugs acting as central antagonists of acetylcholine. With responses of greater complexity the relationship is less clear, presumably because noncholinergic systems are also involved. Additionally, in order to disrupt these more complex responses it has often been necessary to use large doses of the drugs, which are thus likely to produce effects other than antagonism of acetylcholine. The significance of these effects on animal behavior in terms of the behavioral changes produced by the drugs in man is still by no means clear since insufficient quantitative data from human experiments are available. Equally unclear is the relationship between drug-induced changes in the electrical activity of the brain and behavioral effects. A consistent effect of antiacetylcholine drugs was first reported by Wikler in 1952. He showed that dogs given atropine had an EEG pattern normally associated with drowsiness or sleep (high amplitude, low frequency waves) irrespective of whether the animals were alert or drowsy. This he called an EEG-behavioral dissociation, and he later suggested (Wikler, 1954) that the systems subserving synchronization of the EEG were independent of the systems involved in ideation, mood, level of awareness, motility, and sensation. The former systems were presumably cholinergic in nature since they were modified by antiacetylcholine drugs, but not the latter. Quantification of this drug-induced dissociation is possible using the cat enct@hale isole’ preparation (Bradley and Key, 1958) in which behavioral and EEG arousal can be induced by electrical stimulation of the brain stem

ANTIACETYLCHOLINE DRUGS

125

reticular formation. After antiacetylcholine drugs, the threshold for behavioral arousal remains unchanged while that for EEG arousal is elevated. Brimblecombe and Green (1967, 1968) measured the potencies of a series of antiacetylcholine drugs in producing elevation of the EEG arousal threshold in this preparation and showed that they correlated well with their potencies in blocking oxotremorine-induced tremors in mice. This provided quantitative evidence to support Wikler’s postulate that the systems subserving synchronization and desynchronization of the EEG were cholinergic in nature. Whether it is possible to accept fully the remainder of Wikler’s view is perhaps open to question. Certain aspects of behavior, especially those concerned with alerting, appear not to be subserved by cholinergic systems, but others, especially those concerned with motility, are, as has already been shown, fairly clearly mediated through acetylcholine. VIII. Antiacetylcholine Drugs in the Treatment of Poisoning by Anticholinesterase Agents

All the important acute manifestations of poisoning with anticholinesterase agents are due to the accumulation of acetylcholine as a result of the inhibition of acetylcholinesterase, the enzyme normally responsible for its hydrolysis. The anticholinesterase agents may also have direct effects, but these are less obvious and probably of much less practical importance. The symptomatology of anticholinesterase poisoning is thus explainable in terms of the actions of accumulated acetylcholine at peripheral muscarinic (postganglionic parasympathetic endings), nicotinic (autonomic ganglia and skeletal neuromuscular junction) and central sites. The therapeutic value of atropine against anticholinesterases, which was first recognized in 1864 by Kleinwachter is usually considered to depend, at least in part, on the ability of antiacetylcholine drugs to act as competitive antagonists to acetylcholine at peripheral muscarinic sites and in the CNS. However, equally clearly the limited effectiveness of antiacetylcholine drugs in treatment of anticholinesterase poisoning is due to their apparent inability to antagonize the action of acetylcholine at nicotinic sites (although some antiacetylcholine drugs may have some therapeutic value depending on activities other than the antimuscarinic one, as will be discussed later). In order to find more efficient antiacetylcholine drugs as replacements for atropine for the treatment of anticholinesterase poisoning, mgny attempts have been made to establish, for example, whether the central or peripheral antiacetylcholine properties are of preponderant therapeutic importance or whether less clearly defined effects of antiacetylcholine drugs play an important role. Many such studies were reviewed by Wills (1963), who considered the importance of antimuscarinic effects in the PNS and CNS and also the possible use of drugs which antagonize the actions of acetylcholine

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T. D. INCH AND R. W. BRIMBLECOMBE

at nonmuscarinic synapses. It was not possible to reach any definite conclusions about the chemical or pharmacological requirements for therapeutic efficiency since most therapeutic studies have been complicated by the need to study the treatment of anticholinesterase poisoning by mixtures of antiacetylcholine drugs and cholinesterase reactivators such as oximes. Although the situation is very confused, it seems pertinent in this review to consider some of the studies which were in part, at least, designed to provide information about the relative importance of PNS and CNS effects for treatment of poisoning by anticholinesterase agents, against the background of the results in Sections V and VI. I n one study of 35 substituted tertiary amine antiacetylcholine drugs (Karczmar and Long, 1958), the assumption was made that the therapeutic efficiency depended on the central activity of the compounds. This assumption was supported by the finding that atropine methyl nitrate, which penetrates poorly into the CNS, was much less effective than atropine sulfate. I t was then found that there was a good correlation between mydriatic potency (a peripheral antiacetylcholine effect) and antitetraethylpyrophosphate activity of the drugs. This result was considered as providing evidence for the similarity of the receptor sites for antiacetylcholine drugs in the CNS and PNS. On the basis of our present knowledge that, for example, atropine is much more active in the PNS than CNS whereas some of the other drugs examined by Karczmar and Long are equiactive in the PNS and CNS, the correlation found should have been interpreted as indicating that the PNS effects were of major importance. Two more recent studies also provide conflicting evidence for the relative importance of CNS and PNS effects of antiacetylcholine drugs in the treatment of poisoning by anticholinesterase agents. One of these studies (Madill et al., 1968) led to the conclusion that high levels of therapeutic effectiveness were directly related to high levels of central antiacetylcholine potency. I n this study nine drugs were compared with atropine both as therapeutic agents and in tests for central and peripheral antiacetylcholine potency. The therapy experiments were carried out by administering the antiacetylcholine drug (10 pmoles/kg, i.m.) and the oxime l,l-trimethylene-bis-4formylpyridinium bromide (TMB4) (10 mg/kg, i.m.) immediately after injection. of sarin. Central antiacetylcholine activity was expressed as the ability to prevent seizures after intracerebral carbachol in mice and as the lowest dose to cause significant effect in mouse screen climbing. Peripheral antiacetylcholine effects were measured by standard mydriasis and other procedures. One exception to the general pattern was provided by the drug caramiphen, which showed significantly higher therapeutic effectiveness than atropine a t equimolar dosages, yet was less active as a central or peripheral antiacetylcholine drug.

TABLE IX THEPROTECTIVE ACTIONS AGAINST THE LETHAL EFFECTS OF SARINOF SOME ANTIACETYLCHOLINE DRUGSWHENUSEDALONE OR IN COMBINATION WITH THE OXIME P2Sa IN MICE,RATS,AND GUINEAPICS~ Protection ratios: LDS0of drug-treated animals: LDSoof nontreated animals Mice Antiacetylcholine drug (50 pmoles/kg)

1.3

Guinea pigs

+

Drug + P2S (140 pmoles/kg)

Drug alone

Drug P2S (140 pmoles/kg)

Drug alone

2.6 2 .o

1.2 1.2

27.5 9.3

1.4

35.8 21.4

1.1

2.5

4.1

11.8

1.1

18.6

1.4

6.3

9.3

1.5

58.3

Drug alone 1.1

Atropine Hyoscine

Rats

Drug + P2S (140 pmoles/kg)

1.3

Caramiphen

G3063 (Conlinurd)

c.

N

U

TABLE IX (Continued) THEPROTECTIVE ACTIONS AGAINST THE LETHAL EFFECTS OF SHIN OF SOME ANTIACETYLCHOLINE DRUGS WHEN OR IN COMBINATION WITH THE OXIME P2s" IN MICE,RATS, AND GUINEA PIGSb

USED

ALONE

Protection ratios: LD,, of drug-treated animals: LDso of nontreated animals Rats

Mice Antiacetylcholine drug (50 pmoles/kg)

Drug alone

Drug + P2S (140 pmoles/kg)

Guinea pigs

Drug Drug alone

+

+ P2S

(140 pmoleslkg)

Drug alone

Drug P2S (140 pmoles/kg)

9

P c7 X

1.5

3.0

2.0

62.3

1.5

84.2

1.4

2.4

2.0

33.6

1.4

70.2

1.2

-

PMCG

Ditran P2S a

*

P2S = pyridine-2-aldryime methyl methanesulfonate. Brimblecombe et al., 1970a.

b

ANTIACETYLCHOLINE DRUGS

129

In the second study (Brimblecombe et al., 1970a) the effectiveness of a number of antiacetylcholine drugs alone and in conjunction with the oxime P2S (pyridine-2-aldoximemethyl methanesulfonate)in protecting mice, rats, and guinea pigs from sarin was investigated. The results are summarized in Table IX. None of the antiacetylcholine drugs when used alone at a dose of 50 pmolelkg gave protection ratios greater than 1.5 in mice or guinea pigs; in rats, caramiphen and its analog G3063 gave somewhat better protection. When the oxime P2S (30 mg/kg) was used in addition to the antiacetylcholine drugs, markedly improved degrees of protection were obtained. Although the number of drugs studied was small, the results indicated that there was no correlation between their protective potency and their antiacetylcholine potency either in the PNS [log K , mydriasis, antagonism of oxotremorine-induced salivation (Table X)] or in the CNS [antagonism of oxotremorine-induced tremors (Table X)]. It should be recognized that in the protection experiments single large doses of the antiacetylcholine drugs were used irrespective of their antiacetylcholine potency. For example, for atropine a dose of 50 pmoles/kg is only 3 x ED,, for antagonism of oxotremorine-induced tremors, whereas for Ditran 50 pmoles/kg represents 50 ED,o in the tremor test. Thus it is perhaps not surprising that correlations between antiacetylcholine potency and protective efficiency are difficult to observe even if they do exist. Furthermore, in the protection experiments no attempts have been made to take differences of time-activity profiles of antiacetylcholine drugs into account, and few authors record the important parameter, time to death, which could be an important guide to the relevance of true antimuscarinic effects. In experiments recently carried out in this laboratory in which animals pretreated with oxime have been given sarin immediately followed by an antiacetylcholine drug, it is clear that PMCG is effective at a dose level some fifty times lower than that of atropine in affording a significant degree of protection against the lethal effect of the anticholinesterase agent. PMCG is nowhere near being fifty times more effective than atropine as an antiacetylcholine drug either peripherally or centrally. Although presently available information about antiacetylcholine drugs clearly provides some direction for the design of experiments for establishing relations between antiacetylcholine activity and therapeutic efficiency, the work of Coleman et al., (1962a,b, 1963) has shown just how complex any relations are likely to be. From a consideration of the symptomatology of cholinesterase poisoning, Coleman, Little, and Bannard assumed that drugs which have higher CNS activity than atropine or drugs having the ability to inhibit more of the various acetylcholine effects than atropine, would be superior to atropine €or the therapy of poisoning by anticholinesteraseagents. In one paper, Coleman

c.

w

0

TABLE X ANTIACETYLCHOLINE POTENCIES OF DRUGS USEDIN PROTECTION EXPERIMENTS IX) (SEETABLE

Drug Atropine Hyoscine Caramiphen G3063 PMCG Ditran

Antagonism of carbachol on isolated guinea pig ileum (logK* SE)

*

8.95 9.49 7.34 8.75 9.2 9.4

0.04 f 0.02

* 0.02

& 0.05 f 0.05 f 0.05

4

P

Antagonism of oxotremor- Antagonism of oxotremorProduction of mydriasis ine-induced salivation ine-induced tremors in mice in mice in mice (ED,,, pmoleslkg with (ED,,, pmoles/kg with (potency relative to 95% limits) atropine with 95y0 limits) 95y0 limits) 1.o 5.0 0.02 0.33 1.0 0.63

(3.9-6.4) (0.01-0.03) (0.31-0.35) (0.82-1.2) (0.50-0.83)

0.44 (0.33-0.66) 0.05 (0.02-0.08) 23.5 (13.2-41.2) 5.45 (3.4-8.6) 1.7 (0.4-7.1) 0.77 (0.15-1.2)

16.2 (10.G26.0)

1.1 40.3 4.7 3.5 0.88

(0.6-2.5) (24.0-67.5) (1.9-11.3) (2.2-5.7) (0.50-1.4)

1 9

a

rj

?J

r

8

B

-i

M

n

0

x

ANTIACETYLCHOLINE DRUGS

131

et al. (1962a) reported studies of 34 antiacetylcholine drugs (at 50 pmoles/kg used in conjunction with P2S a t 30 mg/kg) for their effectivenessin protecting mice from the lethal effects of sarin. They found that 20 of the drugs used gave protection equal or superior to that given by atropine sulfate. In similar studies with rats, 11 out of 18 drugs were equal or superior to atropine sulfate. However, when the active compounds were compared with atropine for their ability to protect against 2 LDBO’s of sarin, none of the compounds was superior to atropine sulfate which had an ED,, of 2.5 pmoleslkg. A partial explanation for this discrepancy was provided when it was shown that with atropine sulfate and certain other compounds the degree of protection increased initially with increasing dose of antiacetylcholine drug but then reached a plateau, with no further increase of protection with dose. I n contrast, with other drugs there was a progressive increase in protection with increasing doses between 10 and 100 pmoles/kg. I t was not possible, however, to provide a reason for the differences in therapeutic behavior of the antiacetylcholine drugs examined. I t is ofparticular interest with respect to the work described in Seotion I11 that Coleman et al. (1962a) compared (-)- and ( +)-2‘-diethylaminoethyl 2-cyclohexyl-2-hydroxy-2-thienylacetateas the ( ) -bitartrate salts. Whereas the former isomer afforded good protection for mice and rats poisoned with sarin, the latter isomer was relatively ineffective. This result provides some indication that the stereochemical requirements for therapeutic ability are similar to those for antiacetylcholine activity at muscarinic receptors. I n another study (196213) Coleman and his colleagues investigated the actions of the same series of antiacetylcholine drugs as adjuncts to, rather than substituents for, atropine sulfate when used in conjunction with the oxime P2S. With most of the drugs there was a clear association between activity as adjuncts and as substitutes, but a few drugs were relatively ineffective as substituents but relatively effective as adjuncts and vice versa. In a third study Coleman and his colleagues (1963) compared tertiary and quaternary drugs and mixtures of tertiary and quaternary drugs. They had hoped that the tertiary drugs would afford CNS protection and that as a result optimal protection would be provided by the mixtures. The results were extremely confusing, the protective ability varying with the nature of the salt and in some cases quaternary drugs were even more potent than tertiary ones. This latter result calls to question all claims that emphasize the importance of the CNS effect of atropine in the treatment of anticholinesterase poisoning. I t must be pointed out that the criticisms applied to other work apply also to the work of these Canadian workers who used high doses of antiacetylcholine drugs in their studies and neglected time effects, etc. The results described above show that there is probably a need to consider actions of antiacetylcholine drugs other than their competitive antagonism

+

132

T. D. INCH AND R. W. BRIMBLECOMBE

of acetylcholine if the therapeutic efficiency of such drugs is to be explained fully. Potential additional sites of action for these drugs are the skeletal neuromuscular junction and the skeletal muscle fiber. There was an early observation by Botkin (1862) that large doses of atropine cause paralysis, apparently peripheral in origin, in experimental animals, and there followed a series of reports of neuromuscular blockade produced by the, same drug (Cushny, 1903; Haffner, 1914; Dale and Gaddum, 1930; Abdon, 1945). This became known as the “curarelike” action of atropine, but Bulbring (1946) showed an important difference between the actions of curare and atropine. Using the rat phrenic nerve-diaphragm preparation, she found that both drugs abolished the depressant action of rapid motor nerve stimulation, but atropine, in contrast to curare, did not abolish the depression caused by endogenous acetylcholine. Bulbring likened the action of atropine to that of procaine in that it seemed to have a presynaptic action as well as a postsynaptic or curarelike action. The presynaptic action of procaine seems to result in a reduction in acetylcholine output (Harvey, 1939; Jaco and Wood, 1944), so this might also be expected of atropine. As well as this, still incompletely understood, neuromuscular blocking action of atropine, there is also evidence for a facilitating action of the drug on muscle contraction, especially when lower contractions are used. Bulbring (1946), Dutta (1949), and Segawa et al. (1967) all reported potentiation by atropine of twitches of the isolated rat diaphragm elicited by stimulation of the phrenic nerve. Brimblecombe and Everett (1969a, 1970a) reported a similar dose-dependent biphasic action of the drug in vivo in anesthetized cats with doses of less than 8 mg given intraarterially causing potentiation of twitches elicited by direct or indirect stimulation in both a fast-twitch (flexor hallucis longus, FHL) and slow-twitch (soleus) muscle of the cat’s hind limbs. Higher doses depressed twitches of both muscles. Low-frequency tetani were potentiated, higher-frequency tetani depressed and nonmaintained. Other antiacetylcholine drugs have similar actions, but the most potent studied to date is PMCG (Brimblecombe and Everett, 1969a, 1970a), which is effective on the rat-phrenic nerve diaphragm preparation at a concentration of 10 pg/ml and in vivo in cats at an intraarterial dose of 0.25 mg. It is of interest to speculate whether this rnusculotropic action of PMCG and other antiacetylcholine drugs is of any significance in their effectiveness as antidotes to anticholinesterase poisoning. This matter was investigated by Brimblecombe and Everett (1969b, 1970b), who showed that in anesthetized cats PMCG was capable of preventing or reversing the effect on skeletal muscle of the organophosphorus anticholinesterase agent sarin. T h e dose of PMCG used was rather high (2 mg intraarterially), and so it is still

ANTIACETYLCHOLINE DRUGS

133

an open question whether this action of PMCG plays any role in its therapeutic value in anticholinesterase poisoning. Further experiments are required to settle the matter. I t is still not inconceivable that the conventionally recognized effects of antiacetylcholine drugs both centrally and peripherally make only relatively minor contributions to the treatment of anticholinesterase poisoning (Ramachandran, 1966, 1967). There is still no unequivocal evidence for the importance of central antiacetylcholine effects in this context and certainly available data suggest that the drugs are relatively ineffective when employed without concurrent use of a reactivator of inhibited cholinesterase or in treatment of poisoning by “oxime-resistant” agent like Soman (Heilbronn and Tolagen, 1965; Harris et al., 1969). There are conflicting data as to whether atropine prolongs the time to death in anticholinesterase-poisonedanimals (Sanderson, 1965; Coleman et al., 1961), perhaps because this depends in part on the time interval between administration of the anticholinesterase agent and atropine. Increasing the dose of atropine above a certain level does not improve its antidotal effectiveness in anticholinesterase poisoning (Kords et al., 1968). In most reported studies combinations of antiacetylcholine drugs with reactivators of the inhibited enzyme have been used, since neither type of drug is particularly effective when used alone (Fleisher et al., 1970). I t has been suggested that atropine and related drugs afford some protection in the first critical hours after poisoning, helping to restore circulation rates to normal and to maintain high concentrations of oxime in the blood, although the reason why oxime levels should be higher in animals treated with atropine than in animals not so treated has not been established (Mayer and Michalek, 1971). I t has also been suggested that atropine may affect the metabolism of brain acetylcholine (MiloSeviC, 1970). A decrease in total brain acetylcholine content by atropine was first described by Giarman and Pepeu (1962) and has since been confirmed by Holmstedt and Lundgren (1965) in animals treated with anticholinesterases and atropine. Most of the studies which have been carried out to investigate the nonconventional antiacetylcholine drugs have involved studies with atropine and different oximes. There is clearly a need to study the effects of those antiacetylcholine drugs which appear to be therapeutically more efficient than atropine on blood oxime levels and on brain acetylcholine levels, etc., and also to compare in these processes the enantiomeric potency ratios of optically pure antiacetylcholine drugs. IX. Metabolism

As a result of pharmacological observations, it is suggested in Section II1,B that rates of metabolism have little influence on the time-activity profiles of

I34

T. D. INCH AND R. W. BRIMBLECOMBE

antiacetylcholine drugs, unless perhaps abnormally high doses are administered, and that nonspecific absorption of antiacetylcholine drugs (i.e., absorption which does not necessarily lead to conventional antiacetylcholine effects) is both rapid and extensive. The implication of this view is that drug metabolism and distribution studies need not be related to observed pharmacological effects and perhaps do not provide any meaningful indication of the key sites of action of antiacetylcholine drugs. In this section an attempt is made to review some of the available resultsof metabolic and distributional studies of antiacetylcholine drugs and to show that the results from these studies are not contrary to the pharmacological conclusions. Since most of the reported studies of antiacetylcholine drugs relates specifically to atropine these studies will be discussed first. The distribution and metabolic degradation of atropine has been studied in the mouse (Evertsbusch and Geiling, 1956; Gosselin et al., 1955; Albanus et al., 196813, 1969b; Gabourel and Gosselin, 1958), the rat, kitten, and guinea pig (Kalser et al., 1957), the dog (Albanus et al., 1969a,b), rabbits (Lendle and Paul, 1964), and in man (Albanus et al., 1969b; Gosselin et al., 1960; Kalser and McLain, 1970). There are undoubted species variations in the metabolism of atropine, and in this respect it is of interest to compare reported urine contents of unmodified atropine. In mice (Gosselin et al., 1955) only about 25% of an intravenous or intraperitoneal dose could be recovered as such. Rats excreted less of the atropine label in their urine, but the proportion of this radioactivity present as intact drug was high so that a mean of 39% of an intravenous dose was so eliminated (Kalser et al., 1957). The ultimate recovery of unchanged atropine in mouse urine was the same after intravenous, intraperitoneal, and subcutaneous doses. In rats, however, Tonnesen, (1950) observed a marked difference in the urine content of atropine or atropinelike substances (33% and 10% of an intravenous and a subcutaneous dose, respectively). For other common laboratory animals, urinary recovery of pharmacologically active materials have been reported as follows: onethird of the dose in the dog (Wiechowski, 1901),almost 25 '7, in the dog after 24 hours following subcutaneous injection (Albanus et al., 1968a), about one-quarter of the administered dose in guinea pigs (Kalser et al., 1951; Oelkers et al., 1932), essentially none in the cat (Cloetta, 1911) or about the same as in the rat (Kalser et al., 1957). The quantities in rabbit urine are apparently low but variable because many rabbits possess a genetically determined plasma esterase (atropinase) which hydrolyzes this alkaloid (Cloetta, 1911 ; Tonnesen, 1950). In man, 40-50% of unchanged atropine in the urine has been reported (Gosselin et al., 1960), but more recent studies (Kalser and McLain, 1970) show that perhaps the urinary component tentatively identified as atropine was in fact a glucuronide. These recent

ANTIACETYLCHOLINE DRUGS

135

studies provide a stern warning that no metabolic study of atropine has been reported in which the metabolic products have been identified unequivocally and thus that all discussions of atropine metabolism must be treated with extreme caution. From the viewpoint of assessing the effect of metabolism on time-activity profiles of antiacetylcholine drugs, the precise nature of the metabolic products is of much less importance than their rates of excretion. However, even where only excreted radioactivity is measured, comparison of rate of excretion are difficult to interpret because preponderant routes of excretion vary considerably with species. For example, after 6 hours 50% of a 0.5 mg/kg S.C.dose in the dog was excreted in the urine and 16.7% was excreted in the bile (Albanus et al., 1968a); after 6 hours in man, 60-80x of a 2 mg per man i.m. dose was excreted in the urine with at least 3% (from a 14CH3-N< label) expired as 14C0, (Kalser and McLain, 1970); after 6 hours in mice, 75% of the S.C.administered dose was excreted in the urine (Gosselin et al., 1955). I t has been claimed (Albanus et al., 1968a) that dogs are about ten times more sensitive to atropine with regard to effects on the central nervous systems than mice or rats and that this difference can be explained by differences in the rate of metabolism. It is our opinion that reported data permit no unequivocal conclusions, but the indications are that although different species metabolize atropine to varying extents by different routes, there are no greater differences between species in overall rates of metabolism. Consequently, even if the claim that dogs are more sensitive than mice to atropine is correct, and the evidence for this is tenuous, the reason for this difference certainly cannot be ascribed to differences in metabolism. Studies of the rate of metabolism of atropine provide little evidence either for or against the suggestion that time-activity profiles of antiacetylcholine drugs depend little on rates of metabolism. Perhaps, however, comparisons of antiacetylcholine effects in rabbits which do and do not have the specific atropine esterase would be rewarding, for it has been shown (Lendle and Paul, 1964) that the rate of elimination of atropine from rabbits which have the esterase is much greater than from those without it. Many of the more recent papers mentioned previously in this section described radiochemical analyses of various tissues from animals treated with radiochemically labeled atropine. One autoradiographic study with 3H-labeled atropine in mice has been described (Albanus et al., 1968b). I t was found that 3H-labeled atropine was taken up rapidly by the iris and ciliary body, the lung, salivary glands, and certain endocrine glands, such as the pituitary, thyroid, parathyroid, pancreatic islets, and adrenal medulla. I n contrast, low concentrations were found in the heart and smooth muscles, although their function is affected by small doses of atropine. This result

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T. D. INCH AND R. W. BRIMBLECOMBE

emphasized the difficulties associated with attempts to relate drug distribution with pharmacological activity. A very detailed study of the distribution and metabolism of ( -)-hyoscine (Werner and Schmidt, 1968) in mice, rats, guinea pigs, and the common marmosets is of considerable interest. I t was shown that no racemization of (-)-hyoscine or its metabolites occurred in vivo. I t is also known (Glick and Glaubach, 1941; Ammon and Savelsberg, 1949) that the less active enantiomer (+)-hyoscine is hydrolyzed two orders more slowly than (-)-hyoscine and is probably more persistent in vivo. These results provide independent evidence that the differences in potency of (+)- and (-)-hyoscine result from differences in their interactions with the receptors. Other observations of particular interest were that the relative proportions of metabolites did not vary greatly as the administered dose was increased from 10 to 1000 pg per mouse and that tissue concentrations of (-)-hyoscine decreased rapidly as little as 10 minutes after injection of hyoscine. Unfortunately, pharmacological effects were not determined in these experiments. Perhaps the most detailed study of distribution and metabolism of an antiacetylcholine drug which may be related to its pharmacological effects concerns benzetimide (20) and its enantiomers (van Wijngaarden, 1969, 1970; van Wijngaarden and ,Soudin, 1968; Janssen et al., 1971). The excretion and metabolism of lac-and 3H-labeled benzetimide and its enantiomers was studied after S.C.injection of these compounds a t a dose of 0.5 mg/kg into male Wistar rats. In Table X I are summarized the TABLE XI RADIOACTIVITY EXCRETED DURING THE FIRST4 DAYS ADMINISTRATION OF (&)-, (+)- AND ( -)-14C-BENZETIMIDE TO RATS

PERCENTAGE OF THE AFTER

~

1 2 3 4 Total

~ _ _ _ _

37.8 0.9 0.8 0.2 39.7

INJECTED

~

~

27.7 1.5 0.3 0.0

29.5

60.0 1.0

0.3 0.3 61.6

42.0 10.4 0.7 0.5 53.6

32.5 20.2 2.9 0.0

55.6

23.8 2.7 0.7 0.1 27.3

79.8 11.3 1.5 0.7 93.3

60.2 21.7 3.2 0.0

85.1

83.8 3.7 1 .o 0.5 88.9

percentages of injected radioactivity in urine and feces. Most radioactivity was excreted within the first 24 hours; in the urine the excretion of the (-)enantiomers was more rapid. The differences in the rates of excretion in no way accounted for the difference in potency of the two enantiomers: the ( +) isomer is of similar potency to atropine in the PNS and more active in the CNS whereas the

137

ANTIACETYLCHOLINE DRUGS

( - ) isomer was inactive at a dose of 40 mg/kg S.C. I n connection with the high CNS and PNS activity of dexetimide, (+)-benzetimide, compared with the ( - )-enantiomer the possibility of selective uptake and/or retention of dexetimide in the eye, the iris, the brain, and the caudate nucleus of the rat was investigated, the results are summarized in Table XII. After 1 hour, TABLE XI1 THEABSOLUTE AMOUNT OF BENZETIMIDE (pg/mg wet wt) 1 AND 4 HOURS AFTER ADMINISTRATION IN RATSOF BOTHISOMERS (2.5 mg/kg s.c.) 1 Hour

4 Hours

Tissue

(+I

(-1

(+I

Eye Caudate nucleus Rest of the brain

234 2 74 226

214 124 142

52 141 74

(-1 85 46 54

the concentration of the (+)- and (-)-isomers in the eye were almost the same; after 4 hours, in spite of the sharp decline in the concentration of the (+)-isomer, mydriasis was still maximal. At that moment the concentration of the (-)-isomer was twice that of the (+)-isomer. I n contrast, the concentration of the (+)-isomer always exceeded that of the (-)-isomer in the caudate nucleus and the rest of the brain. These studies, in which care was taken to identify metabolites and to ensure that metabolites were inactive, clearly emphasize that studies of metabolism and distribution are particularly difficult to relate to pharmacological activity. Furthermore, it is pertinent to comment that, whereas it has been shown (Paton and Rang, 1965) that 10-lOM atropine interacts with I gm of tissue from guinea pig ileum, the amount of atropine or other antiacetylcholine drugs (or more accurately radiochemical equivalent) found in various organs from different species in some of the papers discussed in this section is many orders greater than this. X. Pharmacological Methods

A recurring theme in this article has been a criticism of methods used by many workers for measuring antiacetylcholine activity. I n particular, we feel that not enough attention has been paid to the time course of action of the drugs, measurements often being made before equilibrium conditions have been established in vitro or before the peak of the effect has been reached in vivo. Additionally, some effects have been assumed to be due to antagonism of endogenously released acetylcholine on very flimsy grounds and without any serious attempt being made to validate this assumption. I n view of these criticisms, we feel that we should describe the methods used in our laboratory, which seem to us to give accurate assessment of antiacetylcholine activity in vitro and in vivo, peripherally and centrally.

138

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The in vitro method used is essentially that described by Barlow et al., ( 1963) in which a segment of guinea pig ileum is suspended in a 2.5-ml organ bath containing Ringer-Tyrode solution maintained at 37” and gassed with a 95% 0 2 , 5% CO, mixture. The fluid in the bath is changed by upward displacement either by Tyrode solution alone or by Tyrode solution containing drugs at predetermined concentrations. The sequence of events in the assay is controlled automatically. Regular contractions of the ileum are obtained for two different concentrations of carbachol (contact time 15 seconds, interval between doses 1.5 minutes), then the Tyrode solution in the bath is replaced by Tyrode solution containing the antiacetylcholine drug, and the concentrations of carbachol is increased to obtain contractions similar to the original ones. The dose ratio corresponding to a particular concentration of antagonist can then be calculated. (Dose ratio is equal to the dose of agonist required to produce a given response in the presence of an antagonist, divided by the dose required to produce the same response in the absence of the antagonist = A / a ) . The affinity constant of the antagonist can then be calculated from the equation BK = A / a - 1 (Gaddum, 1957), where B is the concentration of the antagonist and K its affinity c o n ~ t a n t . ~ The important aspect of this procedure is that measurements are made only when a steady response to the agonist in the presence of the antagonist is obtained. Thus, for drugs with low affinity constants (log K < 9 ) , the antagonist and the ileum come into equilibrium in less than 10 minutes; for drugs with higher affinity constant, much longer times are required. Two in vivo methods are employed, one depending on antagonism of the effects of an injected muscarinic agonist and the other on antagonism of endogenous acetylcholine. Oxotremorine is used as a muscarinic substance. In our earlier studies 100 pg of oxotremorine per kilogram was injected intravenously into mice 15 minutes after the intraperitoneal injection of the antiacetylcholine drug being studied. The animals were examined at 5, 10, and 15 minutes after the oxotremorine injection for the presence of salivation or tremors. ED50’~ for block of salivation and of tremors were calculated by probit analysis and were used, respectively, as measures of peripheral and central antiacetylcholine potency. More recently (Inch et al., 1973) we have refined the method in three respects. The interval between the injections of antiacetylcholine drug and oxotremorine is adjusted to take account of the time course of action of the former drug. Both drugs are given intravenously. Different animals are used for measuring EDIO’s for salivation and for tremors, the doses of oxotremorine used being, for each, 1.5 x the ED,, for production of salivation and tremors; i.e., equipotent rather than equal doses of oxotremorine are used. 6 Precise details are given in “Pharmacological Experiments on Isolated Preparations.” Edinburgh Staff, Livingstone, 1968.

ANTIACETYLCHOLINE DRUGS

1.39

The method involving antagonism of endogenous acetylcholine depends on the measurement of mydriasis in mice. Antiacetylcholine drugs are given intravenously, the pupil diameters are measured a t intervals to cover the total period of action of the drug. Potency relative to atropine can then be calculated, the maximum mean pupil diameter reached at each of three doses being used in the calculation. Estimates can also be obtained of duration of action of the drug. These three methods together give accurate information concerning the potencies of antiacetylcholine drugs in uitro and in vivo. REFERENCES Abdon, N. 0. (1945). Acta Physiol. Scand. 1, 153. Abood, L. G. (1968). In “Drugs Affecting the Central Nervous System’’ (A. Burger, ed.), p. 127. Dekker, New York. Abood, L. G. (1970). In “Psychotomimetic Drugs” (D. N.Efron, ed.), p. 67. Ravetl Press, New York. Abood, L. G., and Biel, J. H. (1962). Znt. Rev. Neurobiol. 4, 218. Abood, L. G., Ostfeld, A., and Biel, J. H. (1958). Proc. SOC.Exp. Biol. Med. 97, 483. Abramson, F. B., Barlow, R. B., Mustafa, M. G., and Stephenson, R. P. (1969). Brit. J. Pharmacol. 37, 2 0 7. Adamson, D. W., Barett, P. A., and Wilkinson, S. (1951). J. Chem. SOC.,London p. 52. Albanus, L., Sundwall, A., Vangbo, B., and Winbladh, B. (1968a). Acta Pharmacol. Toxicol. 26, 571. Albanus, L., Hammerstrom, L., Sundwall, A., Ullberg, S., and Vangbo, B. (1968b). Acta Physiol. Scand. 73, 447. Albanus, L., Aquilonius, S. M., Sundwall, A., and Winbladh, B. (1969a). Acta Pharmacol. Toxicol. 27, 8 1. Albanus, L., Sundwall, A., and Vangbo, B. (1969b). Acta Pharmacol. Toxicol. 27, 97. Ammon, R., and Savelsberg, W. (1949). Hoppe Seyler’s 2.Physiol. Chem. 284, 135. Ankier, S. I., Brittain, R. T., and Jack, D. (1971). Brit. J. Pharmacol. 42, 127. Ariens, E. J. (1966). Advan. Drug Res. 3, 235. Ariens, E. J., and Simonis, A. M. (1967). Ann. N . Y. Acad. Sci. 144, 842. Bagshaw, H., Chamberlain, D. A., and Turner, P. (1970). Brit. J. Pharmacol. 40, 600P. Barlow, R. B. (1964). In “Introduction to Chemical Pharmacology,” p. 185. Methuen, London. Barlow, R. B. (1971). J. Pharm. Pharmacol. 23, 90. Barlow, R. B., Scott, K. A., and Stephenson, R. P. (1963). Brit. J. Pharmacol. Chemother. 21, 509. Barlow, R. B., Franks, F. M., and Pearson, J. D. M. (1972a). Brit. J . Pharmacol. 46, 300. Barlow, R. B., Franks, F. M., and Pearson, J. D. M. (1972b). J. Pharm. Pharmacol. 24, 753, Bebbington, A., and Brimblecombe, R. W. (1965). Advan. Drug Res. 2, 143. Bebbington, A., Brimblecombe, R. W., and Rowsell, D. G. (1966). Brit. J. Pharmacol. Chemother. 26, 66. Beckett, A. H., Harper, N. J., and Clitherow, J. W. (1963). J. Pharm. Pharmacol. 15, 362. Belleau, B., and Pauling, P. (1970). J. Med. Chem. 13, 737. Biggs, D. F., Casy, A. F., and Jeffrey, W. K. (1972). J. Med. Chem. 15, 506. Bignami, G. (1967). In “Neuropsychopharmacology” (H. Brill, ed.), Vol. IV, p. 819. Excerpta Med. Found., Amsterdam.

140

T. D. INCH AND R. W. BRIMBLECOMBE

Bignami, G., and Rosic, N. (1970). Psychopharmacologia 17, 203. Boissier, J. R., Tardy, J., and Diverres, J. C. (1960). Med. Exp. 3, 81. Botkin, S. (1862). Arch. Pathol. Anat. Physiol. Klin. Med. 24, 83. Bowden, K., and Young, R. C. (1970). J. Mad. Chem. 13, 225. Bowman, W. C., Rand, M. J., and West, G. B. (1968). “Textbook of Pharmacology.” p. 724. Blackwell, Oxford. Bradley, P. B. (1968). Znt. Rev. Neurobiol. 11, 1. Bradley, P. B., and Key, B. J. (1958). Electroencephalogy Clin. Ncurophysiol. 10, 97. Brimblecombe, R. W. (1970). In “Drugs and Cholinergic Mechanisms in the CNS” (E. Heilbronn and A. Winter, eds.), p. 52 1. Forvarets Fonkningsanstalt, Stockholm. Brimblecombe, R. W., and Buxton, D. A. (1972). Progr. Brain Rcs. 36, 115. Brimblecombe, R. W., and Everett, S. D. (1969a) Brit. J . Pharmacol. 36, 172P. Brimblecombe, R. W., and Everett, S. D. (1969b). J . Physiol. (London) 203, 19. Brimblecombe, R. W., and Everett, S . D. (1970a). Brit. J . Pharmacol. 40, 45. Brimblecombe, R. W., and Everett, S. D. (1970b). Brit. J . Pharmacol. 40, 57. Brimblecombe, R. W., and Green, D. M. (1967). J. Physiol. (London) 194, 16. Brimblecombe, R. W., and Green, D. M. (1968). Znt. J . Neurofiharmacol. 7, 15. Brimblecombe, R. W., and Inch, T. D. (1970). J. Pharm. Pharmacol. 22, 881. Brimblecombe, R. W., and Pinder, R. M. (1972). “Tremors and Tremorogenic Agents.” Scientechnica, Bristol. Brimblecombe, R. W., Green, D. M., Stratton, J. A., and Thompson, P. B. J. (1970a). Brit. J. Pharmacol. 39, 822. Brimblecombe, R. W., Green, D. M., and Inch, T. D. (1970b). J . Pharm. Pharmacol. 22, 951. Brimblecombe, R. W., Inch, T. D., Wetherell, J., and Williams, N. (1971a). J. Pharm. Pharmacol. 23, 649. Brimblecombe, R. W., Green, D. M., Inch, T. D.,. and Thompson, P. J. (1971b). J. Pharm. Pharmacol. 23, 745. Brodie, B. B., Kurz, H., and Shanker, L. S . (1960). J. Pharmacol. Exp. Ther. 130, 20. Brown, B. B., and Wernder, H. W. (1949). J . Pharmacol. Exp. Ther. 97, 157. .Brown, D. M., Hughes, B. O., and Mehta, M. D. (1969). Nature (London) 223, 416. Brown, D. R., Lygo, R., McKenna, J., McKenna, J. M., and Hutley, B. G. (1967). J . Chem. Soc. B 1184. Buckett, W. R., and Haining, C. G. (1965). Brit. J . Pharmacol. Chemother. 24, 138. Bulbring, E. (1946). Brit. Pharmacol. Chemother. 1, 38. Bur&, J. (1968). Progr. Brain Res. 28, 61. Burger, A., ed. (1970). “Medicinal Chemistry,” Vol. 2. Wiley (Interscience), New York. Butler, T. C. (1953). J . Pharmacol. Exp. Ther. 108, 474. Cahn, R. S., Ingold, C. K., and Prelog, V. (1956). Experientia 21, 81. Canon, J. G., and Long, J. P. (1967). In “Drugs Affecting the Peripheral Nervous System” (A. Burger, ed.), p. 133. Arnold, London. Carlton, P. L. (1963). Psychol. Rev. 70, 19. Carlton, P. L. (1968). In “Psychopharmacology, A Review of Progress 1957-1967” (D. H. Afron et al., eds.), p. 125. American College of Neuropsychopharmacology and Psychopharmacology Research Branch, Washington, D.C. Casy, A. F. (1970). In “Medicinal Chemistry”, (A. Burger, ed.), Vol. 1, p. 81. Wiley (Interscience), New York. Chang, K. J., Deth, R. C., and Triggle, D. J. (1972). J. Med. Chem. 15, 243. Cho, A. K., Haslett, W. L., and Jenden, D. J. (1962). J . Pharmacol. Exr. Ther. 138, 249. Cloetta, M. (1911). Arch. Exp. Pathol. Pharmakol. 64, 427.

ANTIACETYLCHOLINE DRUGS

141

Coleman, D. W., Little, P. E., and Grant, G. A., (1961). Can. J. Biochem. Physiol. 39, 351. Coleman, I. W., Little, P. E., and Bannard, R.A. B. (1962a). Can. J. Biochem. Physiol. 40, 815. Coleman, I. W., Little, P. E.,and Bannard, R. A. B. (1962b). Can. J. Biochem. Physiol. 40, 827. Coleman, I. W., Little, P. E., and Barnard, R. A. B. (1963). Can. J. Biochem. Physiol. 41, 2479. Connor, J . D., Rossi, G. V., and Baker, W. W. (1966). Int. J. Neuropharmacal. 5, 207. Cox, B., and Hecker, S . E. (1971). Brit. J . Pharmacol. 41, 19. Cox, B., and Potkonjak, D. (1969). Brit. J. Pharmacol. 35, 521. Craig, P. N., Caldwell, H. C., and Groves, W. G. (1970). J. Med. Cham. 13, 1079. Cushley, R. J., and Mautncr, H. G. (1970). Tetrahedron 26, 2151. Cushny, A. R. (1903). J. Physiol. (London) 30, 176. Cushny, A. R. (1921). J. Pharmacol. Ex$. Ther. 17, 41. Cushny, A. R. (1926). “Biological Relations of Optically Isomeric Substances”. Baillikre, London, 1926. Cushny, A. R., and Peebles, A. R. (1905). J. PhyJiol. (London) 32, 501. Dahlbom, R., Karlen, B., Ramsby, S., Kraft, I., and Mollberg, R. (1964). Actu Pharm. Suecica 1, 237. Dale, H. H. (1914). J. Pharmacol. Ex#. 77ter. 6 , 147. Dale, H. H., and Gaddum, J. H. (1930). J . Physiol. (London) 70, 109. Decsi, L., Varxzegi, M. K., and Mehes, J. (1963). Arch. In#. Pharmacodyn. Ther. 144, 399 D6da, M., Gyorgy, L., and Nador, K. (1963). Arch. Int. Pharmacodyn. Ther. 145, 264. Domino, D. F., and Hudson, R. D. (1959). J. Pharmacol. Exp. Ther. 127, 305. Duffin, W. M., and Gfeen, A. F. (1955). Brit. J. Pharmacol. Chemother. 10, 383. Dutta, N. K. (1949). Brit. J. Pharmacol. Chemother. 4, 197. Eliel, E. L. (1962). “Stereochemistry of Carbon Compounds,” p. 83. McGraw-Hill, New York. Ellenbroek, B. W. J., Nivard, R. J. F., van Rossum, J. M., and Ariens, E. J. (1965). J. Pharm. Pharmacol. 17, 393. Evertsbusch, V., and Ceiling, E. M. K. (1956). Arch. Znt. Pharmacodyn. Ther. 105, 175. Fleisher, J. H., Harris, L. W., Miller, G. R., Thomas, N. C., and Cliff, W. J. (1970). Toxicol. Appl. Pharmacol. 16, 40. Fodor, G. (1967). Alkaloids 9, 269. Fodor, G., and Csepreghy, G. C. (1961). J. C h m . Soc., London p. 3222. Friedman, A. H., and Everett, G. M. (1964). Aduan. Pharmacol. 3, 83. Fromherz, K. (1933). Naunyn-Schmcidebergs Arch. Ex$. Pathol. Pharmakol. 173, 86. Fujita, T., and Hansch, C. (1967). J. Med. Chem. 10, 991. Gabel, N. W., and Abood, L. G. (1965). J. Med. Chem. 8, 616. Gabourel, J. D., and Gosselin, R. E. (1958). Arch. Znt. Pharmcodyn. Ther. 115, 416. Gaddum, J. H. (1957). Pharmacol. Rev. 9, 2 11. Giarman, N. J., and Pepeu, G. (1962). Brit. J . Pharmacol. Chemother. 19, 226. Gill, E. W., and Rang, H. P, (1966). Mol. Pharmacol. 2, 284. Glick, D., and Glaubach, S. (1941). J. Gen. Physiol. 25, 197. Golikov, S. N., and Pechenkin, V. A. (1963). Bull. [email protected] i d . Med. (USSR)56, 1249., Gosselin, M. D., Gabourel, J. D., and Wills, J. H. (1960). Clin. Pharrnacol. Thw. 1, 597. Gosselin, R. E., Gabourel, J. D., Kalser, S. C., and Wills, J. H. (1955). J. Pharmacol. Exp. Ther. 115, 217. Haffner, F. (1914). Arch. Znt. Phurmacodyn. Thsr. 24, 547. Hansch, C. (1967). Annu. Rep. Med. Chem. p. 347.

142

T. D. INCH AND R. W. BRIMBLECOMBE

Hansch, C. (1968). Annu. Rep. Med. Chem. p. 348. Hansch, C. (1969). Accounts Chem. Res. 2, 232. Hansch, C., and Anderson, S. M. (1967). J . Med. Chem. 10, 745. Hansch, C., Steward, R., Anderson, S. M., and Bentley, D. L. (1967). J . Med. Chem. 11, 1. Hansch, C., Lien, E. J., and Helmer, F. (1968). Arch. Biochem. Biophys. 128, 319. Harris, L. S. (1961). Fed. Proc., Fed. Amer. SOC.Exp. Biol. 20, 395. Harris, L. W., Fleisher, J. I-I., Vick, J. A., and Cliff, W. J. (1969). Biochem. Pharmacol. 18, 419. Harvey, A. M. (1939). Bull. Johns Hopkins Hosp. 65, 223. Heilbronn, E., and Tolagen, B. (1965). Biochem. Pharmacol. 14, 73. Herz, A., Teschemacher, H., Hofstetter, A., and Kurz, H.(1965). Int. J . Neuropharmacol. 4, 207. Holmstedt, B., and Lundgren, G. (1965). In “Mechanism of Release of Biogenic Amines,” Proc. Int. Wenner-Green Symp., p. 439. Pergamon, Oxford. Holmstedt, B., Lundgren, G., Schuberth, J., and Sundwall, A. (1965). Biochem. Pharmacol. 14, 189. Hunt, R. J., and Robinson, J. B. (1972). J . Pharm. Pharmacol. 24, 324. Inch, T. D., and Brimblecombe, R. W. (1971). J . Pharm. Pharmacol. 23, 813. Inch, T. D., Ley, R. V., and Rich, P. (1968), J . Chem. Sac. Cpp. 1683 and 1693. Inch, T. D., Chittenden, R. A., and Dean, C. (1970). J . Pharm. Pharmacol. 22, 954. Inch, T. D., Green, D. M., and Thompson, P. B. (1973). J . Pharm. Pharmacol. 25, 359. Ing. H. R., Davies, G. S., and Wajda, I. (1945). J . Pharmacol. 85, 85. Innes, I. R., and Nickerson, M. (1970). In “The Pharmacological Basis of Therapeutics” (L. S. Goodman and A. Gilman, eds.), 4th. ed., p. 524. Macmillan, New York. Jaco, N. T., and Wood, D. R. (1944). J. Pharmacol. Exp. Ther. 82, 63. Janssen, P. A. J., Niemegeers, C. J. E., Schellekens, K. H. L., Demoen, P., Lanaerts, F. M., van Nueten, J. M., van Wijngaarden, I., and Brugmans, J. (1971). Arzneim. Forsch. 21, 1365. Kalser, S. C., and McLain, P. L. (1970). Clin. Pharmacol. Ther. 11, 214. Kalser, S. C., Wills, J. H., Gabourel, J. D., Gosselin, R. E., and Epes, C. F., (1957). J . Pharmacol. Exp. Ther. 121, 449. Karczmar, A. G., and Long, J. P. (1958). J . Pharmacol. Exp. Ther. 123, 230. Karltn, B., and Jenden, D. J. (1970). Res. Commun. Chem. Pathol. Pharmacol. 1, 471. Karltn, B., Traskman, L., and Sjoqvist, F. (1971). J . Pharm. Pharmacol. 23, 758. Kato, G., and Yung, J. (1971). Mol. Pharmacol. 7, 33. Kato, G., Yung, J., and Ihnat, M. (1970). Mol. Pharmacol. 6 , 588. Kleinwachter, L. (1864). Berlin Klin. Wochenscher. 1, 369. Kojima, M., Nose, T., Shintomi, K., and Yoneda, N. (1971). Jap. J . Pharmacol. 21, 276. Kords. H., Lullmann, H., Ohnesorge, F. K., and Wasserman, 0. (1968). Eur. J . Pharmacol. 3, 341. Lands, A. M. (1951). J . Pharmacol. Exp. Ther. 102, 219. Lendle, L., and Paul, H. A. (1964). Naunyn-SchmiedebergsArch. Exp. Pathol. Pharmakol. 249, 295. Leo, A., Hansch, C., and Elkins, D. (1971). Chem. Rev. 71, 525. Leonard, F., and Simet, L. J. (1955). J . Amer. Chem. SOC.71, 2855. Leslie, G. B. (1969). J . Pharm. Pharmacol. 21, 248. Leslie, G. B., and Conway, G. E. (1970). Pharmacol. Res. Commun. 2, 201. Lien, E. J., Hansch, C., and Anderson, S. M . (1967). J . Med. Chem. 11, 430. Lindgren, S., Lindqvist, A., Lindeke, B., Karlen, B., Dahlbom, R., and Blair, M. R. (1970). J . Pharm. Pharmacol. 22, 707.

ANTIACETYLCHOLINE DRUGS

143

Long, J. P., Luduena, F. P., Tullar, B. F., and Land, A. M. (1956). J. Pharmacol. Exp. Ther. 117, 29. Longo, V. G. (1966). Pharmacol. Rev. 18, 965. Lukas, G., Brindle, S. D., and Greengard, P. (1971). J. Pharmacol. Exp. Ther. 178, 562. McFarland, J. W. (1970). Drug. Res. 15, 123. Madill, H. D., Stewart, W. C., and Savoie, M. L. (1968). Can. J . Physiol. Pharmacol. 46, 559. Marshall, P. B. (1955). Brit. J . Pharmacol. Chemother. 10, 354. Martindale, W. (1968). “Extrapharmacopea,” 25th ed., p. 149. Mayer, O., and Michalek, H. (1971). Biochem. Pharmacol. 20, 3029. Meier, R., and Hoffmann, K. (1941). Helv. Med. Acta 7, Suppl. VI, 106. Meyerhoffer, A., and Carlstrom, D. (1969). Acta Crystallogr., Sect. B 25, 1119. Meyers, B., and Domino, E. F. (1964). Arch. Znt. Pharmacodyn. Ther. 150, 525. MiloSevic, M. P. (1970). Brit. J. Pharmacol. 39, 732. Moffett, R. S., and Aspergren, D. B. (1957). J. Amer. Chem. SOC.79, 4451. Moran, J. F., and Triggle, D. J. (1969). In “Fundamental Concepts in Drug-Receptor Interaction” (J. F. Danielli, J. F. Moran, and D. J. Triggle, eds.), pp. 133-176. Academic Press, New York. Natoff, I. L. (1970). J. Pharm. Pharmacol. 22, 133. Nogrady, T., and Algieri, A. A. (1968). J . Med. Chem. 11, 212. Oelkers, H. A., Raetz, W., and Rinteln, K. (1932). Arch. Pharm. ( Weinheim) 275, 820. Oliverio, A. (1967). Psychopharmacologia 11, 39. Ostfeld, A. M., and Aruguette, A. (1962). J . Pharmacol. Ex). Ther. 137, 133. Parkes, M. W. (1955). Brit. J . Pharmacol. Chemother. 10, 95. Parkes, M. W. (1965). Psychopharmacologia 7, 1. Paton, W. D. M., and Rang, H. P. (1965). Proc. Roy. Soc., Ser. B 163, 1. Pauling, P. J., and Petcher, T. J. (1969). Chem. Commun. p. 1001. Pauling, P. J., and Petcher, T. J. (1970). Nature (London) 228, 673. Pennington, J. T., Beckett, L., Bentley, D. L., and Hansch, C. (1969). Mol. Pharrnacol. 5, 333. Pfeiffer, C. C. (1956). Science 124, 29. Pomeroy, A. R., and Roper, C. (1971). Brit. J. Pharmacol. 41, 683. Pratesi, P., Villa, L., Ferri, V., Grana, E., and Sossi, D. (1969). Farmaco. Ed. Sci. 24, 313. Pyman, F. L. (1917). J . Chem. SOC.,London p. 1103. Ramachandran, B. V. (1966). Biochem. Pharmacol. 15, 1577. Ramachandran, B. V. (1967). Biochem. Pharmacol. 16, 2061. Rama Sastry, B. V. (1970). Zn “Medicinal Chemistry” (A. Burger, ed.), Vol. 2, p. 1544. Wiley (Interscience), New York. Randall, L. O., Benson, W. M., and Stafko, P. L. (1952). J. Pharmacol. Exp. Ther. 104, 284. Rang, H.P. (1966). Proc. Roy. SOC.Ser. B 164, 488. Roche Products (1955). British Patent 728, 579, Chem. Abstr. 50, 5773 (1956). Sanderson, D. M. (1965). J. Pharm. Pharmacol. 17, 124. Schild, H. 0. (1947). Brit. J. Pharmacol. Chemother. 2, 189. Segwa, T., Kojima, M., and Takagi, H. (1967). Jap. J . Pharmacol. 17, 465. Smythies, J. R. (1971). Eur. J . Pharmacol. 14, 268. Spek, A. L., Peerdeman, A. F., van Wijngaarden, I., and Soudijin, W. (1971). Nature (London) 232, 575. Spencer, P. S. J. (1965). Brit. J. Pharmacol. Chemother. 25, 442. Stubbins, J. F., Hudgins, P. M., Murphy, D. C., and Dickerson, T. L. (1972). J . Pharm. Sci. 61, 470.

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Thron, C. D. (1972). J. Pharmacol. Exp. Ther. 181, 529. Thron, C. D., and Waud, D. R. (1968). J. Pharmacol. Exp. Ther. 160, 91. Tonnesen, M. (1950). Acta. Pharmacol. Toxicol. 6, 147. Triggle, D. J. (1971). “Neurotransmitter-Receptor Interactions.” Chapter 4. Academic Press, New York. Thutt, C. C., and Bottini, A. T. (1968). J. Amsr. Chem. Soc. 90, 4752. van Wijngaarden, I. (1969). Lifc Sci. 8, 517. van Wijngaarden, I. (1970). Lye Sci. 9, 489. van Wijngaarden, I., and Soudijn, W. (1968). Lifc Sci. 7, 225. van Wijngaarden, I., Soudijn, W., and Van der Eycken, C. (1970). Lifc Sci. 9, 1289. von Braun, J., Braunsdorf, O., and Rath, C. (1922). Ber. Deut. Chem. Ges. 55, 1666. Wagman, W. D., and Maxey, G. C. (1969). Psychopharmacologia 15, 280. Werner, von G., and Schmidt, K-H. (1968). Hoppe-Syler’s Z . Physiol. Chem. 349, 741. Wiechowski, W. (1901). Arch. Exp. Pathol. Pharmakol. 46, 155. Wikler, A. (1952). Proc. Soc. Exp. Biol. Med. 79, 261. Wikler, A. (1954). J. Ncrv. Ment. Dis. 120, 157. Wilen, S. H. (1971). Top. Stereochem. 6, 107. Wills, J. H. (1963). I n “Handbuch der Experimentellen Pharmakologie,” Part XV, Chapter 20. Springer-Verlag, Berlin and New York. Zejmal, E. V., and Votava, Z. (1961). Experientia 17, 471.

KRYPTOPYRROLE AND OTHER MONOPYRROLES IN MOLECULAR NEUROBlOLOGYl By Donald

G. Irvine2

Saskatchewan Hospital Research Unit, North Battleford Saskatchewan, Canada

.

I. Introduction 146 11. Molecular Structures and Context 147 A. Molecular Structures 148 B. The Molecular Biological Context 149 C. Acute Intermittent Porphyria as a Model for the Molecular Biology . . of Major Psychiatric Disorders 151 151 111. Chemistry and Chromatography . 151 A. The Chromatography of Kryptopyrrole . 153 B. Kryptopyrrole Oxidation Products . . C. Chromatographic Anomalies Attributable to Interconversion of Oxypyrrole Lactams during Extraction and Chromatography . 153 156 D. Parallel Reactions of Propentdyopents . E. Structural Analogies between Pyrrole Oxidation Products and Biologically Active Molecules . 157 . 158 IV. Biochemistry A. New Analytical Techniques . . 158 . . 159 B. Pyrroles in Blood C. Pyrroles in Cerebrospinal Fluid, and the Blood-Brain Barrier . . 160 D. Relation to Porphyrias and to Bile Pigment Metabolism . . 160 E. Relation to Pyrrolooxygenases . . 162 F. Biological Sources of Excreted Kryptopyrrole . . 163 V. Pharmacology . . 165 A. Toxicity . . 165 B. Eye Effects . . 166 C. Anticonvulsant Action . . 167 D. Neuroelectrical Effects . 167 VI. Behavioral Pharmacology . . 168 A. General Effects . 168 B. Effects on Fixed-Ratio Responding for Food Reinforcement . 169 . C. Effects on Discriminated Avoidance . 170 VII. Neurobiology of Other Pyrroles . 170 A. Pyrrolic Pheromones . . 170 B. Batrachotoxin . . 172 . C. Porphobilinogen and Acute Intermittent Porphyria . 172 Supported by the Psychiatric Services Branch of the Saskatchewan Department of Public Health, and in part by grants from the Schizophrenia Biological Research Foundation, and from the Medical Research Council of Canada. Present address: Psychiatric Research Unit, University Hospital, Saskatoon, Saskatchewan, Canada. 145

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D. In Hyperprolinemia Type I1 and Other Human Diseases E. Indoles Are Pyrroles . VIII. Relationship to Clinical Psychiatry . . References

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173 175 176 179

1. Introduction

Over the past 15 p a r s there has been a rapid growth of interest in the monopyrroles in psychiatry and neuropharmacology, with the detection of three such metabolites in neuropsychiatric disorders, with the development of behaviorally active pyrrolic drugs, and with the realization that all indoles (with their rich and diverse associations in neurobiology) are but a subclass of pyrroles (Irvine, 1973a). The molecular structures involved will be considered in detail later, but it may be noted that the “parent” monopyrrole nucleus is an extremely small and very simple one, consisting of just four carbon atoms, five hydrogen atoms, and one nitrogen atom. Such simplicity may be an advantage in probing the neurobiological significance of these molecules. Historically, neurobiological interest in the monopyrroles had its first upsurge with the discovery of the pyrrolic nature of the abnormal metabolite characteristic of the hereditary neuropsychiatric disorder, acute intermittent porphyria (Cookson and Rimington, 1954). Some evidence for a second monopyrrole associated with other psychiatric disorders was provided somewhat later (Irvine, 1961), and this material was identified, essentially, as kryptopyrrole (Irvine et al., 1969b; Sohler et al., 197b). Many clinical correlates of this substance have been described from the laboratories of Sohler, Pfeiffer, Osmond, Hoffer, and O’Reilly and from our own; certainly its general association with psychotic states has found confirmation in several centers (see Irvine, 1973a). Quite recently, this second pyrrole, or one indistinguishablefrom it, has been found in large amounts and in a very high proportion of cases, in acute intermittent porphyria (Irvine and Wetterberg, 1972). The confirmed toxicity, behavioral activity, neuroelectrical effects, and anticonvulsant activity of this pyrrole will be reviewed below, Currently, evidence is accumulating (Applegarth et al., 1974) that a third monopyrrole may be associated with another human disease affecting the central nervous system (CNS), namely hyperprolinemia type 11. Our chemical studies on this new metabolite, alluded to by Applegarth et al. (1974), will also be reviewed here. While the exact nature of the relationships between such natural monopyrroles and neuropsychiatric disorders remains unclear, the reproducibility of the clinical associations, along with the neurotoxicity of many pyrroles suggest some possible etiological relevance, and in any case provide a challenging background for studies on the basic neurobiology of these relatively little-studied compounds. Considerable emphasis is placed on the known

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molecular biology of the disordered pyrrole metabolism characteristic of the hereditary neuropsychiatric disease acute intermittent porphyria, as a heuristic model for such common psychiatric disorders as schizophrenia, where genetic predisposition presupposes a molecular-biological mechanism, but where this mechanism is still almost totally obscure. The purpose of this review is to extend an earlier one (Irvine, 1973a), by bringing it up to date in regard to our own work, by including many of the significant advances recently made in other laboratories, and by continuing application of the principles of experimental zoology and molecular biology to place the new developments in a neurobiological context. While it is only a short time since the previous review on this subject was written, there were in the interval a number of important developments : more adequate chemical description of the pyrrolic metabolites (as lactam adducts) ; explanation of their anomalous chromatographic behavior ; discovery of the pyrrolooxygenases; detection of a clinical correlation between pyridoxine, zinc, and kryptopyrrole; demonstration of a close connection between acute intermittent porphyria and kryptopyrrole; and confirmation of the behavioral, EEG, and anticonvulsant activities of this pyrrole. Since the previous review covered many clinical correlates (Irvine, 1973a), the emphasis here will be on the biochemistry and pharmacology of kryptopyrrole and related substances. More effort will be expended on being up-to-date than on being comprehensive in coverage. Only the simple monopyrroles will be covered, thus excluding tetrapyrroles (such as the porphyrins, common bile pigments, heme, and vitamin B,,), and indoles (such as serotonin, LSD, psychotomimetic tryptamine derivatives, and skatole metabolites). Most pyrrolic drugs will also be excluded since this review will concentrate on monopyrroles occurring naturally, particularly in man. While these excluded, more complex, pyrroles constitute a large number of substances relevant to neurobiology and psychiatry, they are compounds more frequently reviewed elsewhere. II. Molecular Structures and Context

The available data on kryptopyrrole and other monopyrroles relevant to mental health will be reviewed in the context of biology and molecular biology, rather than medicine or pure chemistry. Some advantages of this approach have been suggested (Irvine, 1961). I t emphasizes, in addition to general biochemistry and metabolism, molecular architecture, molecular pharmacology, genetics, metabolic regulation, and especially the interplay between these factors, and the interactions between them and the environment (Irvine, 1972b,c). This section will review some of the molecular structures and molecular biological context pertinent to the neurobiology of the monopyrroles.

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A. MOLECULAR STRUCTURES The monopyrroles are compounds of the heterocyclic series, containing one ring made up of four carbon atoms arranged as in butadiene, and one nitrogen atom forming a bridge between the first and last carbons of the chain (Fig. la). Kryptopyrrole (Fig. lb) is a naturally occurring member of this class of pyrroles; it constitutes the basic structure of the urinary metabolite, formerly known as “mauve factor” and associated with psychotic disorders, certain psychiatric symptoms, and a poor prognosis. Porphobilinogen (PBG), the characteristic pyrrolic metabolite of patients suffering from acute intermittent porphyria, is depicted in Fig. lc. With the identification of such monopyrroles associated with neuropsychiatric disorders, it becomes possible to rule out some artifactual sources, suggest their origins, study their metabolism directly, test them for pharmacological, electrophysiological, and behavioral activity, and relate their molecular structures to the molecular architecture of drugs and receptors relevant to neurobiology and mental health. Striking molecular relationships between kryptopyrrole and various psychoactive or psychiatrically relevant compounds have been delineated

H’

on

I

Pyrrole nucleus /-O

Kryptopyrrole

Porp hobi linogen

FIG.1. (a) The pyrrole nucleus, (b) kryptopyrrole, and (c) porphobilinogen.

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already (Irvine et al., 1969b). The structurally related compounds noted previously included :the bile pigments of kernicterus, the porphyrins of acute intermittent porphyria, the transmitter serotonin, the psychotomimetic tryptamine derivatives, LSD, the antidepressant hematoporphyrin, the skatole derivatives excreted by many psychotic patients, and the cobalamins (vitamin B12). While this list is remarkable for the large number of psychiatrically relevant substances it includes, it is not exhaustive, and additional structures related either to the parent kryptopyrrole itself (Irvine and Zdanivsky, 1971a) or to the newly discovered oxy lactams derived from it (Irvine et al., 1973a,b) have been reported. The general similarity between various psychotomimetic tryptamines and synthetic kryptopyrrole was pointed out earlier, it may be significant that two of the simpler indolic psychotomimetics (which are not tryptamine derivatives) have precisely the same side chains as kryptopyrrole, on one side of the parent nucleus. These compounds are medmain and nitroserotonin, described in the literature of antiserotonin psychoactive substances (Woolley, 1960). The same identity of side chains on the pyrrole nucleus is seen in the newer antipsychotic agent Molindone, an apparently effective agent in the clinic (Sugerman and Herrmann, 1967;Simpson and Krakov, 1968).The structure of this drug has a special significance, in that its only conjugated unsaturated (or “aromatic”) ring is the pyrrole nucleus. Of all the compounds we have considered so far, with the exception of Molindone, all contained more than one pyrrole nucleus, or contained in addition a fused benzene ring, or other aromatic groups. It would seem now, however, that for this series, psychoactivity may reside primarily in the pyrrole nucleus. This impression is strengthened by the structural analogies between kryptopyrrole and the monocyclic psychoactive agents chlormethiazole (Mouriquand et al., 1958; Gershon, 1968), and the Russian tranquilizer 4-ethylpyrazole, or T-53 (Anonymous, 1964). In addition, from the work on pheromones, bactrachotoxin, and pyrrolizidine alkaloid metabolites, it is becoming clear that certain other neurobiological activities also can be based on the monopyrrole nucleus devoid of additional aromatic or directly attached rings (see Sections VII A, B, D). The molecular parallels between oxykryptopyrrole lactams (rather than kryptopyrrole itself) and- other substances of neurobiological interest will be found in Section 111, E.

B. THEMOLECULAR BIOLOGICAL CONTEXT While an explicit molecular biological understanding of major psychoses (including schizoprenia) remains out of reach, a biological (and implicitly molecular-biological) approach is appropriate. In the rare hereditary neuropsychiatric disorder, acute intermittent porphyria, an explicit and detailed

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Medmoin

Nitroserotonin

Chlormethiozole ( hemineurin)

Mo lindone

4 - Ethylpyrarol

FIG.2. Additional substances of neurobiological interest structurally related to kryptopyrrole : (a) medmain, a psychotomimetic; (b) nitroserotonin, a halucinogenic depressant; (c) Molindone, an antipsychotic; (d) chlormethiazole, an anticonvuhant tranquilizer; and (e) 4-ethylpyrazole (T-35), a Russian tranquilizer.

molecular-biological model is already available, and therapeutically useful (Irvine, unpublished data) and this particular disease may provide a valuable model for the commoner but less understood major psychiatric disorders. Different approaches have been applied to the study of kryptopyrrole, in different laboratories, ranging from the clinical “malvaria” hypothesis of Hoffer and Osmond (1963), to a particularly empirical biological approach (Irvine, 1961, 1963, 1966). Recently, more molecular mechanisms have been integrated into the existing biological context (Irvine, 1973b), and the

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interaction between environmental and genetic factors in producing attacks of metabolic and neuropsychiatric disorder, has been emphasized (Irvine, 1972b,c).The interaction was diagrammed in terms of specific environmental actions at the levels of DNA (induction, repression, predisposing genes, transcription), RNA (translation, assembly of enzymes or structural proteins), enzyme function (coenzymes, inhibition, substrate levels), and tissue assembly (Irvine, 1972c, 1973b).Molecular biology clearly showsjust how the different elements of the environment interact sequentially with the primary hereditary material (DNA) and its subsequent products, and use of such interaction diagrams may deter any simplistic interpretation or premature application of results in biochemical or molecular psychiatry.

C. ACUTEINTERMITTENT PORPHYRIA AS A MODEL FOR

THE

MOLECULAR

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The theoretical importance of acute intermittent porphyria (AIP) in reasoning about the biochemistry of schizophrenia has long been emphasized (Irvine, 1961; 1963). AIP was cited as an hereditary metabolic disease which in spite of its single-gene basis, was clinically very diverse, failed to develop before adulthood, had an intermittent course, and could be precipitated by drugs. With the demonstration (Irvine et al., 1969a) that “mauve factor” (a frequent correlate of psychoses) was a pyrrole structurally similar to the porphyrin precursor PBG, this type of porphyria took on practical as well as theoretical significance for kryptopyrrole research. With the rapid growth of knowledge concerning the molecular biology of AIP, this model is becoming more incisive. A significant development which assists in teasing apart the threads of cause and effect in relating kryptopyrrole to clinical psychiatry, is the finding (Irvine and Wetterberg, 1972), of an exceptionally high incidence and concentration of kryptopyrrole in the hereditary neuropsychiatric disease AIP, in which the specific gene is known to disturb the metabolism of another endogenous monopyrrole, porphobilinogen. The hereditary nature of the PBG pyrrole dysmetabolism in this illness provides a benchmark for establishing causal directions: the PBG dysmetabolism in this case cannot be primarily an environmental effect. Some details and implications of this AIP model will be covered in Sections IV, D and VIII. 111. Chemistry and Chromatography

A. THECHROMATOGRAPHY OF KRYPTOPYRROLE Even before 1961, it had become apparent that natural kryptopyrrole (then called mauve factor) was heterogeneous and sensitive to mild extraction

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DONALD G. IRVINE

procedures (Irvine, 1961). Its heterogeneity was apparent even on unidimensional paper chromatograms (Irvine, 1961; Heacock, 1962; Sohler et a/., 1967) using such solvent systems as isopropanol/ammonia/water. With unidinietisional systems, resolution was inadequate, and much better separation of the component zones, and other advantages, were realized when hybrid two-dimensional (autotransfer) chromatography (Irvine and Anderson, 1965; Irvine et af., 1967; Irvine and Bayne, 1969) was applied. By means of this technique, components of a sample are first classified (primarily by adsorption) on a thin-layer plate, then transferred at right angles and further separated (primarily by solubility) in a paper partition system. This combination of dissimilar systems increases not only the resolution, but also the interpretability of the a, coordinates in terms of functional groups and other chemical characteristics (Irvine and Bayne, 1969). For resolving natural kryptopyrrole, a combination of thin-layer chromatography (TLC) on silicic acid/diethyl ether, transfer with isopropanol-water, and paper chromatography using Reio’s Solvent D (Reio, 1958, 1960) proved very effective. This chromatographic system yielded the characteristic pattern of five chromatographic spots (“compnents 1, 2, 3, 4, and 5”; Irvine and Bayne, 1969; Irvine et al., 1967, 1970) which led to the identification of kryptopyrrole in urine from psychiatric patients. While our previous work made it clear that all five of these Chromatographic components were attributable to kryptopyrrole, it was pointed out (Irvine el al., 1969b, 1970) that it was scarcely possible that all five could be identical with unchanged kryptopyrrole (2,4-dimethyl-3-ethyIpyrrole), and it was indeed necessary to process the kryptopyrrole in dilute solutions in order to obtain the pattern typical of the natural material. From one of the five chromatographic components of the natural material a n oxygen-containing derivative of kryptopyrrole was isolated, and characterized using high-resolution mass spectrometry (Irvine et al., 1969b). Further consideration of the two-dimensional hybrid chromatographic coordinates of the natural kryptopyrrole components suggested that they contained amide (or lactam) linkages and/or hydroxyl groups (Irvine et al., 1967; Irvine, 1973b, p. 163). I n the latter review, the situation was summed up as follows: “Another important early difficulty was the virtual impossibility of demonstrating that mauve factor was a simple pyrrole. This was due to (1) the alteration of kryptopyrrole’s chromatographic properties by extremely mild pretreatments, and (2) the movement of kryptopyrrole exposed to water to a chromatographic position in Reio’s systems that was characteristic of highly polar amides, glycols, and enols. This position was distant from that occupied by the simple neutral indoles, aromatic hydrocarbons, and expected for simple pyrroles. The reasons for this highly anomalous position of kryptopyrrole ‘component 1’ are still conjectural, but it is now known that

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this component is the ether formed by a reversible reaction between two molecules of kryptopyrrole and ~ a t e r . ” ~ While the precise mass analysis of this isolated component of natural kryptopyrrole was consistent with dikryptopyrrole ether structure, the autotransfer chromatogram (ATC) coordinates for this component did not suggest an ether, but some much more polar and/or hydrogen-bonding structure. Consequently, it was necessary to determine just what was occurring chemically, and Dr. Lightner’s group at UCLA was encou.raged to synthesize and characterize some oxidation products of kryptopyrrole to serve as model compounds for further clarification of the chemical and chromatographic anomalies.

B. KRYPTOPYRROLE OXIDATION PRODUCTS Following the above leads, which suggested the presence of oxygen in the natural products derived from kryptopyrrole, and taking into account a suggestion (S. F. MacDonald, personal communication, 1972) that the autooxidation products of kryptopyrrole be examined, collaborative studies were undertaken with Lightner and Crandall at UCLA, who added kryptopyrrole to the series of monopyrroles and dipyrroles that group was subjecting to sensitized photooxidation (Quistad and Lightner, 1971a,b). Their latest work (Lightner and Crandall, 1973) has resulted in the unambiguous identification of four different dioxy products from kryptopyrrole (KP). These are illustrated in Fig. 3. A simplified terminology for these compounds will be adopted here, although it lacks rigor. The synthetic model compounds (numbered with Roman numerals to distinguish them from the chromatographic components) are, with synonyms: (I) 3-OH-KPL or 8-OH-KPL, 4-ethyl-3-hydroxy-3,5-dimethyl-A4pyrrolin-2-one (11) 5-MeO-KPL or a’-MeO-KPL, 4-ethyl-5-methoxy-3,5-dimethylA3-pyrrolin-2-one (111) MEM, methylethylmaleimide (IV) 5-OH-KPL or a‘-OH-KPL, 4-ethyl-5-hydroxy-3,5-dimethyl-A3-

p yrrolin-2-one where L stands for “-lactam-2,” and the first prefix may be the numeral or the corresponding Greek letter. TO INTERCONVERSION OF C. CHROMATOGRAPHIC ANOMALIES ATTRIEIUTABLE OXYPYRROLE LACTAMS DURING EXTRACTION AND CHROMATOGRAPHY The synthetic work of Lightner and Crandall provided, for the first time, the essential model compounds for comparison with the natural

From “Orthomolecular Psychiatry: Treatment of Schizophrenia” (D. Hawkins and L. Pauling, eds.,). Freeman, San Francisco. Copyright 1973.

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DONALD G . IRVINE

4-Ethy1-3-hydroxy-3.5- dimethyl-A4-pyrrolin-2-one ;3-Hydroxykryptopyrrolone-2)

4-Ethyl-~methoxy-35t~l-A3-~ro~2-on (5-Methoxykryptopyrolone-2)

H

im) Methylethylrnaleirnlde

I

(El

\A

OH

4-Ethyl-5-hydroxy-3.5-d~methyl-A3-pyrrdm-2-one

(5-Hydroxy krypto~rrolone-2) FIG.3. Photooxidation products from kryptopyrrole.

substances attributable to kryptopyrrole. Their chromatographic behaviors were monitored, and cochromatography was performed with authentic components from aqueous solutions of synthetic KP, or from clinical specimens (Irvine e! al., 1973a,b). O n direct chromatography of these pure oxidation products, all three of the lactams reacted with Ehrlich’s reagent exactly like natural kryptopyrrole, while the imide (HI) did not react at all. Two of the lactams were positional isomers, with a hydroxyl group at the p or a’ position, (I and IV, respectively). By direct testing in multiple chromatographic systems, the 8-hydroxy lactam (I) did not correspond to any component of natural kryptopyrrole, while the a’-hydroxy lactam (IV) corresponded perfectly with component 1. The remaining lactam model (XI) had a methoxy group in the a’-position, and by direct testing, it yielded chromatographic components 2, 3, and 5 (in the

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presence of formic acid-containing solvent systems, e.g., Reio’s System D), or just component 3 (when formic acid or other organic acids were removed from those solvent systems). Our studies utilizing diagonal chromatography (Hais, 1970) established that “component 2” was really identical with component 1, being produced by the acid vapor acting upon component 3, during equilibration preceding chromatography. The results of diagonal chromatography further suggested that “component 5” was also identical to component 1, but was produced by the action of acid in the liquid phase rather the vapor phase, during the chromatographic run. None of the model compounds yielded any component 4,when tested directly. Consequently, only two compounds are responsible for four out of the five “component spots” on ATC’s of natural kryptopyrrole solutions, and one of these (11) is the methyl ether of the other (IV). Further work (Irvine et al., 1973a,b) showed that despite the clear, specific relationships between structure and chromatographic behavior noted when these model compounds were tested directly, a remarkable “scrambling” takes place if any of these lactams is passed through the routine extraction procedure (Irvine, 1961) before chromatography. No matter which lactam (I, 11, or IV) is so processed, a l l j v e components are seen on the chromatogram. In spite of the effectiveness of this analytical procedure in both qualitative (Irvine et al., 1969b) and quantitative (Sohler et al., 1970) work, clearly it is capable of converting one positional isomer into another (I+IV), of exchanging methoxy and hydroxy substituents in the “5” position, and of producing component 4 from a variety of lactams not identical with it. Now, the two compounds (I1 and IV) responsible for four out of the five component spots” on ATC’s of kryptopyrrole solutions are easily interconverted using water or methanol, especially in the presence of a catalyst. Since one of these (the methoxy compound 11) depends on the presence of methanol in the extraction or processing system, it too is essentially a product of the analysis, and the main component is therefore No. 1 (compound IV). That this compound is the basic or “parent” compound of the series is supported also by additional evidence cited in Section IV. Here, however, we are concerned with the chromatographic properties and chemical nature of this component. <(

‘ ~ ~ ~ k r ~ p tEther” opy~~l Earlier work (Irvine et al., 1969b) involving high-resolution mass spectrometry identified the compound actually eluted from component spot 1 as dikryptopyrryl ether, seemingly only distantly related to compound IV, the molecule which, when chromatographically tested, yields component 1. However, the ATC coordinates of component 1 are not those expected for

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ethers, and are typical instead of multifunctional polar, neutral compounds, particularly amides and dihydroxy compounds (Irvine et al., 1970). Perhaps this paradox is not entirely surprising, however, because ever since “dikryptopyrryl ether” was first reported in the chemical literature its structure has been questioned (Fischer and Hartmann, 1934), and recently such structures have been revised (Hoft et al., 1967). It now seems clear that “dikrytopyrryl ether” is actually the a’-kryptopyrryl adduct of compound IV, i.e., a’(C,)-kryptopyrrylkryptopyrrole-a(CJ-lactam. This clears up entirely the “anomalous” position of the component spots on autotransfer chromatograms-the responsible substances are all lactams, not simple pyrroles or ethers, and have R, coordinates which would be predicted for such structures. O n the other hand, the mass spectral evidence (Irvine et af., 1969b) indicating a dipyrrolic or dimeric structure for component 1 may seem difficult to reconcile with the appearance of this component in the direct chromatographic tests of (mononuclear) compound IV. However, early work reported in the German literature (Fischer et al., 1932a,b; Fischer and Hartmann, 1934; Fischer and Orth, 1934; Metzger and Fischer, 1937) emphasized the difficulties and uncertainties encountered with these compounds, including their tendency to dimerize and rearrange, and it has been suggested that “dikryptopyrryl ether” can readily split into free kryptopyrrole and a monokryptopyrrole-lactam (Fischer and Hartmann, 1934). A reexamination of the chemistry of this entire class of pyrrolic lactams may be overdue, and many of the problems encountered in previous purely chemical work may be resolved by closer attention to the lactam structures, the remarkable reactivity of C-5 in the unsaturated lactam-2-ring7 and the corresponding “adduct” formation and exchange in these molecules (Hoft el al., 1967,1968; von Dobeneckand Brunner, 1969; Irvine, unpublished data). Whatever the mechanism, if the sort of splitting referred to above is reversible (as is hydroxylation of C-5 in the monokryptopyrrole-lactam) , then there will be little real discrepancy between the production of component 1 by compound IV and finding that the eluted component, on mass spectral analysis, consists instead of the lactam isomer of dikryptopyrryl ether.

D. PARALLEL REACTIONS OF PROPENTDYOPENTS The ready interconversion of components of natural kryptopyrrole has been attributed to the oxy lactam structures, interacting with water or solvents. While it is very unusual for simple dissolution in water or in methanol at room temperature to effect incorporation of these solvents into a molecule via covalent bonds, and for these groups to be readily exchanged by changing solvents, this type of chemical behavior has been independently

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observed (Bonnet et al., 1968; von Dobeneck and Brunner, 1969) as a characteristic of propentdyopent bile pigments. This parallel pattern of unusual chemical reactivity has now been explained (Irvine et al., 1973a,b) in terms of a molecular structure which is common to both the monopyrrole (oxy) lactams, and to the propentdyopents (Fig. 4). One half of the propentdyopent molecule is essentially identical to the entire KP-lactam, and the “water adduct” and “methanol adduct” of the propentdyopent involves the same carbon atom (C-5) as that involved in the facile exchanges of OH and M e 0 groups in the lactams making up natural kryptopyrrole. Since the propentdyopents also readily add -SH, -NH, or active -CH groups at C-5, it is likely that the monokryptopyrrole-lactam will do the same, and consequently will form covalent bonds with a wide variety of compounds in the body. The possible biological implications of such alkylations will be dealt with later.

E. STRUCTURAL ANALOGIES BETWEEN PYRROLE OXIDATION PRODUCTS AND BIOLOGICALLY ACTNEMOLECULES The structural similarities between the parent pyrrole (2,4-dimethyl-3ethylpyrrole, = kryptopyrrole) and neurobiologically relevant compounds were reviewed in a previous section, and the demonstration that natural kryptopyrrole consists of oxy lactams rather than the pyrrole itself suggests

(01

tCH3OH

water propcnldyopent

methanol proprnldyoprnl

Mnpawnl s w

&aIrr

kryplopyrrolonr

componrnt 3, or =d-methanol

hryplopyrrolm

FIG.4. Parallel structures and reactions of (a) propentdyopents, and (b) kryptopyrrole lactams (mauve factor).

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an examination of the molecular structures and biological activities related to such lactams. The molecular similarities, expected biological activities, and probable mechanisms of action of the kryptopyrrole lactams will be presented in detail elsewhere. Here it may be pointed out that these substances are analogous with a large series of unsaturated lactones noted for their antibiotic and other pharmacological properties. Being oxo-derivatives of a simple heterocycle, these lactams are also rather similar to a large series of anticonvulsant drugs (Irvine et al., 1973c) and analgesics. Other parallels may be seen in the structures of most benzodiazepine tranquilizers, the psychotomimetic agents in Amanih muscan’a, and the psychoactive components of kava. In chemical terms the kryptopyrrole lactams, especially the main one, 5-OH-KPL, also resemble (a) some of the target reagents recently employed as probes and modifiers of molecular structures, (b) catalysts for macromolecular synthesis, and ( c ) transition state analogs (Lienhard, 1973) for inhibition of enzymes. These points, and their mechanistic implications for any biological activities of the KP lactams, will be detailed elsewhere (Irvine, unpublished data). IV. Biochemistry

A. NEWANALYTICAL TECHNIQUES

It is relatively easy to extract natural kryptopyrrole from urine, but it is much more difficult to separate it from interfering substances and to measure it specifically. All methods reported to date use the very nonspecific Ehrlich’s aldehyde, and hence, to be specific for the KP compounds, must first remove all the indoles, resorcinols, and chromogenic phenothiazine metabolites. The selectivity ofthe original analytical method (Irvine, 1961) has been confirmed (Sohler el of., 1970). However, some of the simplified analytical procedures (e.g., Ellman et al., 1968) have been incapable of distinguishing the pyrroles from the phenothiazines (Sohler et uf., 1970; Irvine, 1973a). The most reliable technique so far available for distinguishing natural kryptopyrrole from all other metabolites is autotransfer chromatography, as described above. Combining this type of hybrid 2-dimensional chromatography, with selective extraction procedures (Irvine, 1961) ensures that it is the pyrtole lactams which are being measured. The adsorption/elution/ extraction method reported in 1961 gives excellent recovery (Sohler et al., 1970). Other details of the basic analytical procedure are referred to in the previous review (Irvine, 1973a, pp. 169 and 170). Recently, the ATC components, visualized with Ehrlich’s aldehydic reagent, have been quantitated by integrating densitometry (Irvine et al., 1971; Irvine, 1972a; Irvine and Wetterberg, 1972). In this procedure, it is

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important to carry the standards through the entire extraction as well as the chromatographic procedure. With the identification of natural kryptopyrrole as oxypyrrole-lactams, efforts have been directed toward (1) specific ultramicro analysis of the lactams, and (2) their measurement in tissues and body fluids other than urine. An application of the integrated ion current (IIC) method of massspectral quantification (Majer and Jenkins, 1967; Boulton and Majer, 1970) is under development to increase the sensitivity' beyond that provided by Ehrlich's chromogenic agent. Consideration should be given to radioimmunoassay as well, now that LSD has been analyzed successfully with this method, down to the picogram range (Taunton-Rigby et al., 1973).

Blood and Tissue Methods Recently, a method for determining PBG in serum was described (Miyagi et al., 1971), and its levels were related to porphyric disorders. Two methods for extracting the pyrrolic metabolites of pyrrolizidine alkaloids from tissues were presented in another recent paper (Mattocks and White, 1970). Until this year, no procedures were available for the extraction and determination of tissue KP. Reviewing the new findings on the ready chemical reaction between methanol and the kryptopyrrole hydroxy lactams, the difficulties experienced in attempting methanol extraction of kryptopyrrole from tissues, as well as the recent success of Mattocks and White in using ethanol for extracting pyrroles from tissues, we tried ethanol to extract our compounds. When ethanol, with or without diethyl ether, was used (Irvine et al., 1973a, Table 1) to extract kryptopyrrole (lactams) from blood or tissues of rats given intraperitoneal injections of synthetic KP, satisfactory analyses were achieved by subsequent ATC densitometry.

B. PYRROLES IN BLOOD Porphobilinogen exists in human blood, and its quantitative relationships to attacks of acute intermittent porphyria were recently examined (Miyagi et. al., 1971). Kryptopyrrole (or its p-sidechain isomer) also exists in some specimens of blood, at least as the highly characteristic lactam components on ATC's. By the use of the ethanol extraction procedure, a good yield of component 1 was obtained (Irvine et al., 1973a). The only other component found (and then only occasionally) was component 4. Components 3, 2, and 5 were uniformly absent, which is consistent with their likely dependence upon added methanol. A series of parallel determinations of natural kryptopyrrole in corresponding blood and urine specimens (n = 12) revealed that the pyrrole

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(lactam) was uniformly present in blood from persons positive for KP in the urine, and undetectable in the blood of those who did not excrete detectable amounts of the natural kryptopyrrole (Irvine et al., 1973a).

C. PYRROLES IN CEREBROSPINAL FLUID,AND

THE

BLOOD-BRAIN BARRIJIR

It is quite important for the neurobiological assessment of KP and its lactams, to know whether this material exists in cerebrospinal fluid, crosses the blood-brain barrier, and/or is present in brain tissue. Few answers are yet available in this area, and a definite statement concerning this permeability of the blood-brain barrier may have to await testing of the synthetic lactams, and use of the Oldendorf (1971) method. However, when nontoxic doses of KP are given to rats intraperitoneally, and the brain is removed after saline exchange transfusion (to remove blood from brain) under anesthesia, KP component 1 is demonstrable in brain tissue (D. G. Irvine and H. Miyashita, unpublished work, 1973). The anticonvulsant activity and the EEG effects of KP, described below, also suggest that this pyrrole or its lactam(s) enter the brain from the circulation. By direct test, using the new ethanol/ether extraction method cerebrospinal fluid from a psychiatric patient with natural KP in his urine and blood, also yielded a substantial amount of KP component 1 (Irvine et af., 1973a). This result and the blood results just described, represent the first demonstration of (at least chromatographically characterized) natural kryptopyrrole in biological sources other than urine. Also for the first time, natural component 1 is chromatographically characterized not simply as a metabolite “attributable” to kryptopyrrole or as a form of kryptopyrrole, but rather as the specific molecule which bears a hydroxy group in the a’ position, and a lactarn carbonyl corresponding to the original free a position of kryptopyrrole.

D. RELATION TO PORPHYRIAS AND

TO

BILEPIGMENT METABOLISM

Now that some data have been presented on the natural KP components in blood and cerebrospinal fluid, and the chemistry of natural KP has been reviewed, it may be profitable to consider just what molecular structure(s) of natural KP should be tested for any functional biochemical relationship with the known intermediary metabolism of pyrroles and tetrapyrroles, whether these be porphyrins, precursors, or bile pigments. Taking into consideration all the available experimental evidence, the key substance in naturally occurring kryptopyrrole would seem to be the a’-hydroxy-a-lactam, because (1) it is the most abundant component; (2) it is the most frequent component; (3) it is the least method-dependent component (components 2 and 5 depend on added organic acids; component

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3 depends upon added methanol) ; (4) it is the predominant one of the two components found in the blood, and the one so far detected in cerebrospinal fluid; (5) it is readily formed from the other compounds by action of water at room temperature, and, since tissues and body fluids are predominantly water, this form is the likely one in such hydrophilic environments. In consequence, it is concluded that the key structure among the components of naturally occurring kryptopyrrole is 5-OH-KP-lactam-2 or, more formally, 4-ethyl-5-hydroxy-3,5-dimethyl-A3-pyrrolin-2-one. While the ready interconversion of the different oxy derivatives of kryptopyrrole, e.g., during its analysis, makes it difficult to determine the exact form(s) of kryptopyrrole present in the native state in body fluids or tissues, the available evidence indicates that “natural kryptopyrrole” isolated from body fluids is essentially one or more adducts of the lactam attributable to kryptopyrrole. There remains a possibility, however, that the side chains attached at the and /3‘ positions may be interchanged in some or all of the naturally occurring kryptopyrrole, and this would relate any such isomeric portion of the natural product, more closely to hemopyrrole (Irvine and Wetterberg, 1972), and hence to the porphobilinogen structure. Because natural KP exists as the lactam and its adducts (i.e., pyrroles with oxygen atoms directly attached to the ring), there is a more immediate structural similarity to the bile pigments (which are similarly “oxidized”) than to the porphyrins (which are not). The molecular similarity of the parent KP to typical tetrapyrrolic bile pigments has been depicted (Irvine et al., 1969b; Irvine, 1973a), and the similarity is enhanced in a comparison of the pigments with the lactam structure instead of the parent pyrrole. When the dipyrrolic bile pigments are considered, the parallel is even closer, and the strict molecular homology (Irvine et al., 1973a) between half of the propentdyopent water adduct and the hydroxy-lactam form of natural KP has been presented above (Fig. 4). The naturally occurring bile components, the promesobilifuscins (Siedel et al., 1948) are also closely similar to the KP lactam structure. Pigments of the bilifuscin or dipyrrolic type have been reported as elevated in some porphyrias (With, 1968; Schmid, 1972; Russell, 1972, p. 279), providing a potential link between these two main divisions of pyrrole metabolism. While the porphyrins are less like the lactams, disorders of porphyrin metabolism are often associated with psychiatric disorders, whereas disorders of bile pigment metabolism are more frequently related to mental retardation. Also, in the inborn error of metabolism AIP, there is typically a marked excretion of kryptopyrrole (HuszSk and Durk6, 1971) and/or of KP lactams or their isomers (Irvine and Wetterberg, 1972). In view of this, and the greater importance of porphyrin precursors rather than the preformed porphyrins in AIP, additional studies were conducted to explore the link

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between porphyrin intermediary metabolism and K P lactams. First a blind study of the correlation between urinary levels of K P and porphyrin precursors was conducted (Irvine, 1972a) in which a significant positive association was found between S-aminolevulinic acid (ALA) and K P excretion (though not between PBG and KP). These results indicated closer ties between natural K P (i.e., K P lactams) and porphyrin precursor metabolism but the introduction of the oxygen atoms remained entirely unexplained until the work of the Frydmans on pyrrolooxygenases.

E. RELATION TO

PYRROLOOXYCENASES

The structure given in Fig. 3 for the main component of natural kryptopyrrole, 5-O#-KPL, was completely independently established only a few months ago, by the Frydman team in Buenos Aires, for the sole product of pyrrolooxygenase action on synthetic kryptopyrrole (B. Frydman, personal communication). Consequently, it is likely that the biosynthesis of the key component of naturally occurring kryptopyrrole involves the action of these newly discovered pyrrolooxygenase enzyme(s) . In a systematic series of investigations, the Frydmans’ group (Frydman et al., 1972a,b,c; Frydman and Frydman, 1973) have discovered a new series of enzymes, the pyrrolooxygenases, have worked out methods for purifying them, separating different members, establishing substrate specificities, cofactors, and inhibitors, and have established structures of the metabolic products formed. These enzymes attack pyrrole nuclei, not only those in monopyrroles like kryptopyrrole or PBG, but also those which form part of the indole nucleus of naturally occurring indolic compounds (including skatole and simple derivatives of tryptophan) and even attack the pyrrole ring of the indole nuclei constituting side chains essential for the activity of certain proteins. M o s t significantly, pyrrolooxygenase not only attacks such crucial indole side chains in PBG-deaminase (thus partially inhibiting the conversion of PBG to uroporphyrinogen) but also oxidizes PBG, to form the corresponding lactam. By reference to a detailed metabolic chart of the “porphyrin” metabolic pathway (Irvine, 1972c, 1973b), the effects of a functional excess of pyrrolooxygenase can be predicted quite readily. First, by decreasing functional PBG-deaminase (thereby derepressing heme-regulated aminolevulinic acidsynthetase formation) it should increase the levels of PBG and probably ALA, without significantly increasing the various porphyrinogens or porphyrins; this corresponds with what is found basically in the disease AIP. Second, while the attack of pyrrolooxygenase upon PBG-deaminase will predictably raise the level of PBG, the same class of enzyme is known to oxidize PBG itself, and ring-oxidized PBG should accumulate. Third, the newly discovered pyrrolooxygenase provides, for the first time, an alternative pathway from

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PBG, and any deviation of PBG along this branch should further frustrate the normal control mechanism. These mechanisms can function adequately only if there are no significant branch points between the final product (heme) and the initial rate-controlling step (aminolevulinic acid synthetase). A multifactorial working hypothesis regarding 5-QH-KP-lactam, and incorporating these mechanisms, will be summarized in Section VIII and will appear in detail elsewhere.

F. BIOLOGICAL SOURCES OF EXCRETED KRYPTOPYRROLE The question of the source of excreted kryptopyrrole can be considered a t four general levels: possible artifactual origin; species producing it, and its induction in those which do not; the organs, tissues, and subcellular structures involved; and the precursors and enzymes directly responsible for its synthesis, Any simple artifactual origin for excreted KP has been ruled out (Sohler et al., 1970; Irvine, 1973a). Its structure, as well as the many and varied experiments 'described in the papers cited, make it difficult indeed to accept the suggestion (Ellman et al., 1968) that the excreted K P might consist of phenothiazine drug metabolites. Nevertheless, it is equally difficult to rule out every possible source of artifact. I n this connection it is interesting to note that despite the rich mixture of porphyrins and apparently monopyrrolic metabolites found in routine urine extracts we have prepared from induced porphyrias, or from the porphyric species Sciurus niger, and especially from a case of bovine congenital porphyria, no kryptopyrrole components have been detected (Irvine et al., 1971). Failure to find even traces of kryptopyrrole in the bovine porphyrin urine shows once again that the kryptopyrrole is not the result of a simple breakdown of porphyrins after they are excreted. Similarly, 41 of schizophrenic patients selected as excretors of deviant porphyrins fail to excrete detectable amounts of kryptopyrrole (HuszPk et al., 1971, 1973). As for the species producing K P naturally, there seems to be only oneman. Although fascinating, this situation poses many practical problems. I t would be most convenient for experimental work if an animal excreting kryptopyrrole could be found or alternatively if the condition could be induced. Consequently the North Battleford laboratory has taken several approaches to this end (Irvine, 1972a), extracting and chromatographing urine specimens from the following sources: (a) the usual range of laboratory animals; (b) domesticated animals; (c) selected exotic species (through the courtesy of the Calgary Zoo) ; (d) a wide range of primate species under conventional laboratory conditions ; (e) from rhesus monkeys rendered behaviorally abnormal by restrictive rearing (through the courtesy of the Madison, Wisconsin, Primate Research Center).

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All five of these approaches yielded negative results. But because of the correlation between certain porphyric diseases in humans and the excretion of kryptopyrrole, we extended this chemical screening to: (f) groups of rats rendered porphyric with such chemicals as allylisopropylacetamide, hexachlorobenzene, and DDCP (a collidine derivative) ; (g) rats given 8-ALA; (h) a bull with hereditary erythropoietic porphyria; (i) fox squirrels (Sciurus niger), a species virtually unique in having 100% incidence of porphyria (Turner, 1937; Levin and Flyger, 1971); (j) mice of strain PRO/Re, with hereditary dysmetabolisrn of pyrrolidines (Blake and Russell, 1972). All the induced rats became porphyric, but none produced kryptopyrrole. The precursor-loaded rats, as well as the porphyric bull gave negative results, and, although fox squirrels were clearly porphyric by the red fluorescence of their calcified structures, they nevertheless failed to excrete appreciable KP. Only the hyperprolinemic (PRO/Re) mice excreted any kryptopyrrole detectable on the chromatograms, and this only initially or sporadically in longitudinal studies. This result was encouraging and suggested that perhaps the right type of stress, with the right genetic/biochemical constitution, might produce KP, even in animals. There have been repeated findings of a relationship between strcss and natural kryptopyrrole excretion, in the research laboratories of the New Jersey Neuropsychiatric Institute (A. Sohler, unpublished reports), although we have had little success in demonstrating such a relationship in the strains of animals used in North Battleford. There is no doubt, nevertheless, that stress does have a predictable enhancing effect upon the levels of various other Ehrlich-positive metabolites (Mandell et al., 1961a,b, 1963; Bliss et al., 1972). Very recently, the Frydman group has found that certain steroids markedly induce pyrrolooxygenase activity (B. Frydman, personal communication, 1973). There would appear to be increasing evidence that one or more steroids or their metabolites are involved in either an obligatory or a facultative way, in the metabolic derangements leading to the excretion of “natural kryptopyrrole.” Even more recently, we have for the first time, repeatedly produced significant excretion of natural kryptopyrrole in experimental animals, and this again has been possible only through the use of steroid compounds (Irvine, 1973b). The steroid treatment has now caused excretion of the hydroxy kryptopyrrole lactam both in Sprague-Dawley rats, and in female PRO/Re mice. Now that induction of K P in experimental animals appears possible, progress can be expected in elucidating the anatomical, histological, and ultrastructural substrata for its production. Since the last review, very, little progress has been made in this area. There remains the evidence for involvement of the intestinal tract (Irvine, 1973a) ; and the new associations with

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AIP, bile pigment (lactam) structures, and pyrrolooxygenase, suggest that the liver may be significant, but no experimental evidence is yet available on this point. At the level of intermediary metabolism the source of K P again has been difficult to determine, as experimental animals failed to produce it. Now with the new method to cause its production in animal species (Irvine, 1973b), isotopic tracer studies become much more feasible. However, before this latest development, a number of possible endogenous sources for natural K P were advanced: from amino sugars, amino acids plus carbohydrates, or N-acetylneuraminic acid (Sohler et al., 1967, before its identification) ; from bile pigments, broadly defined and perhaps related to porphyrin precursors (Huszbk et al., 1973); from intermediates between PBG and uroporphyrinogen I11 (Russell, 1972); from dipyrrolic and other bile pigments, porphyrins, PGB, hemes, other sources of preformed pyrrole rings, or simpler molecules such as aminoacetone and pentanone-2 (Irvine, 1973a). In a more experimental vein, loading studies in a man excreting natural K P have shown no increase of its excretion with oral hemoglobin or glycine but a highly significant rise with ingestion of 8-ALA (Miyashita and Irvine, 1972). This result was consistent with a naturalistic study (Irvine, 1972a) in which a significant association between 8-ALA and K P excretion was found. Failure of PBG levels to correlate with those of K P may indicate that ALA or PBG is being metabolized by a branch pathway, rather than or in addition to the usual pathway through the porphyrinogens to heme. The only known branch pathway from PBG (the first product from ALA) is provided by pyrrolooxygenase (Frydman et al., 1972c), which also, of course inactivates the porphobilinogen deaminase enzyme essential for conversion of PBG to the first porphyrinogen in the normal metabolic path (Frydman and Frydman, 1973). It may not be coincidental that the sole product of pyrrolooxygenase action on synthetic kryptopyrrole is identical with the 5-OH-KP-lactam directly responsible for the main component of natural kryptopyrrole. This seems to be the first real clue to the probably enzymatic source of natural kryptopyrrole. It ties in well with the accumulating clinical evidence, as well as our current working hypothesis (Section VIII) involving the possible origin of natural kryptopyrrole in relation to a disordered control of the porphyrin metabolic pathway. V. Pharmacology

A. TOXICITY The toxicity of pyrroles has long been recognized. As a class, pyrroles have been called “nerve poisons” (Corwin et al., 1960), and the sensitivity of the nervous system to such substances, demonstrated in early studies

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DONALD G. IRVINE

(Saccardi, 1922; Rabbeno, 1929, 1930; Rezek, 1937) has since been confirmed (Moffett, 1968; Irvine and Zdanivsky, 1971b; Wetterberg, 1972). Moffett (1968) showed that the toxicity of KP was unusually high among a series of related pyrroles; he found a screening LB,, of 77 mg/kg in the mouse, while in other mice Wetterberg (1972) found an LD,, near 120 mg/kg, and Irvine and Zdanivsky (1 97 1b) reported a n LD,, of 57 mg/kg in the Wistar strain of rat. Quarrtitativrly, chickrns and frogs are relatively resistant to KP, while dogs and cats may be more sensitive to it than are mice or rats. The toxicity of this compound is most obvious in the central nervous system, the lung and pleural cavity, although the eye and orbital region is profoundly affected also (Irvine and Zdanivsky, 1971b; also see below). From the toxicological trials conducted in rats, cats, and dogs (Irvine, 1973a, p. 171), it has become obvious that kryptopyrrole in toxic doses produces both an early and a delayed phase of intoxication, the delayed toxicity being associated with a marked accumulation of fluid in the pleura1 cavity. This pattern is reminiscent of the delayed toxicity and pleural edema found in pyrrolizidine alkaloid poisoning (hlattocks, 1968) which is also associated with monopyrrolic metabolites (Mattocks, 1968, 1969). Other parallels exist between the effects of KF' and those of unsubstituted pyrrole itself, although the latter is 50 to 1000 times less toxic (Irvine and Zdanivsky, 1971b). Most of the literature on the pharmacology of simple pyrroles has concentrated upon systems other than the CNS, and emphasized toxicology. Nevertheless, there is, in psychiatry, a long history of workers seeking an endogenously prodticcd toxic substance which might be implicated in the genesis of schizophrenia or other psychoses, and i t may be worthwhile to take a close lmk at the toxicity of kryptopyrrole, even though we may not share this particular research orientation. An important next step in the basic neurobiology of' KP will be to determine the exact mechanisms underlying thc toxicity. An important new facet of this question is whether the lactam adducts may be t h r toxic agents, rather than K P itself. First indications are that the K P lactams do produce toxic effects (L. Wetterberg, personal communication, 1972).

B. EYEEFFECTS Two of the most striking effccts of even low doses of kryptopyrrole (intraperitoneal) are thc narrowing of the palpebral fissures and the sinking in of the eye (enophthalmos). If the K P is given by the intracarotid route, there is a predominantly unilateral enophthalmos and pupillary paralysis (Irvine, 1972a). Detailed observations on these phenomena have been made (N. P. V. Nair and D. G. Ifvine, unpublished) and are to be reported elsewhere. Recently, in joint work (A. Heller and D. G. Irvine, unpublished pilot study,

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1972) using Heller's methods (Brownstein and Heller, 1968) for decentralizing and stimulating the superior cervical ganglion in rats and monitoring the resultant enophthalmia/exophthalmia, it was observed that up to 20 mg/kg K P failed to block this ganglion, and that the degree of palpebral narrowing and enophthalmia produced by this dose of K P was greater than that produced by entirely depriving the ganglion of its central afferents surgically. Much more work is required to fully understand and exploit this clue to the mechanism of action of kryptopyrrole.

ACTION C. ANTICONVULSANT From an early report on the parent compound, pyrrole (Rezek, 1937), and from a report that porphyrin precursors may have epileptogenic activity (Kosower and Rock, 1968), one might expect kryptopyrrole to induce or simulate epilepsy. However, in 1948 Wfoffett produced evidence that K P was, instead, an anticonvulsant. His test system was electroshock seizures in rats. A more recent study (Irvine et al., 1973c) sought to determine whether the anticonvulsant effect of this pyrrole would be sufficiently general to block seizures provoked through a different sensory modality, in a different class of animal, and where there is a marked genetic susceptibility (seizure-prone homozygous Fayoumi fowl). As predicted, nontoxic, nonsedative doses of kryptopyrrole did significantly block the induction of seizures in this species as well. Comparison with EEG results from parallel studies utilizing known anticonvulsants and seizure-prone chickens, suggested a close similarity to the neuroelectrical effects of Diazepam. As mentioned earlier in this review, there are structural similarities between diazepam (or other benzodiazepine lactam drugs) and natural kryptopyrrole. With KP, priming doses or a series of doses appear to enhance the anticonvulsant activity of a subsequent dose. These points are consistent with a possible in vivo conversion of K P into adducts of the K P lactam, with a mechanism of action perhaps akin to that of the benzodiazepine lactams.

D. NEUROELECTRICAL EFFECTS Since some of the compounds structurally similar to kryptopyrrole lactams are anticonvulsants, while kryptopyrrole itself blocks electroshock-induced (Moffett, 1968) or photically provoked (Irvine et al., 1973c) seizures, and because kryptopyrrole is associated with abnormal or unusual features of the EEG (Keogh, 1964), it seems reasonable to look for neuroelectrical effects in experimental animals. Much of the work done in this area has been summarized (Irvine, 1973a, p. 173), but several studies were too recent to be covered there. The generalized slowing of the EEG (with or without superimposed'beta activity), and

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the inhibition of single neurons in the cat’s cochlear nucleus, now have to be related to results (a) from quantitative integrated (Drohocki, 1948) EEG’s from surface and implanted electrodes in cats (I. Huszik, unpublished data) and hippocampal electrodes in rats (Irvine’s group) ; (b) from EEG studies in sedated rabbits (Sohler ct of., 1970); and (c) from EEG monitoring of the anticonvulsant effects of this pyrrole in chickens (Irvine el al., 1973~).These newer studies confirm the neuroelectrical activity of K P in nontoxic doses, and further indicate the nature of this activity. T h e work in New Jersey showed that K P in low doses deepened the sedation of rabbits. T h e Hungarian study revealed the regional differences in sensitivity of brain structures to KP (with the greatest effect in the hippocampus and amygdala), generally confirmed the slowing, the increase in beta amplitude, and quantitated the decrease in integrated mean electrical content. The Saskatchewan studies confirm and extend the unpublished integrated EEG studies of Huszik, especially in regard to the hippocampal region, looking more closely a t the coefficient of variability (Goldstein et ol., 1965; Marjerrison et af., 1968), and examine EEG parallelisms between KB and drugs affecting the limbic system. While we are encouraged by the varied experimental evidence that KP possesses neuroelectrical activity, and that this is somewhat specific, and while there are parallels between the experimental findings and the human electrophysiological correlates of endogenous K P in psychiatric patients, speculation on this would still be premature. VI. Behavioral Pharmacology

A. GENERAL EFFECTS While the obvious neurotoxisity of the neutral monopyrroles is of some interest in neurology and psychiatry, the behavioral pharmacology of these substances is more relevant. Moffett (1968) first pointed out the “depressant” action of kryptopyrrole, and this has been confirmed in a general way by Irvine and Zdanivsky (1971a), who noted huddling and hypoactivity of rats in their homc cages, reduction of exploratory behavior, and less running in an activity whccl. Most recently, Wrtterberg (1972) has reported a progressive sedation, and sleep- or torpor-inducing action, on increasing the dosage of kryptopyrrole administered to mice. At moderate dosage levels, the mice showed less spontaneous activity and less than usual response to touch and sound. Such “depressant” effects were difficult to discern, however, in a stimulating environment, e.g., a programmed “Skinner box” (Irvine and Zdanivsky, 1971a), and animals “depressed” by nontoxic doses of KP were surprisingly easily alerted. Until EEG studies are completed, it is uncertain whether the behavioral sleep seen with the higher doses of KP is normal sleep (Wetterberg, 1972).

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I n mice given high doses of KP, ataxia is accompanied by marked hypothermia and probably some analgesia. The mechanisms are unknown (Wetterberg, 1972). A variety of other, more clearly behavioral effects of K P have been observed in pilot experiments meant to probe the breadth of this substance’s action spectrum : disruption of DRO discrimination, inhibition of acquisition of a stable FR30 for food reinforcement, yet failure to disrupt performance of an overlearned maze task (Irvine and Zdanivsky, 1971a). Besides the established general behavioral toxicity and the more tentative results of several behavioral probes with KP, quantitative behavioral studies under controlled conditions have been conducted. These involve studies of the disruption of established performance on FR schedules of positive reinforcement (Section VI, B), and on discriminated avoidance schedules (Section VI, C). ON FIXED-RATIO RESPONDING FOR FOODREINFORCEMENT B. EFFECTS

Appel (1968) has emphasized the value of simple FR schedules in quantitatively assessing behavioral responses of rats to psychotomimetics and related compounds. Consequently, Irvine and Zdanivsky (1971a,b) measured the behavioral effects of kryptopyrrole upon fixed-ratio behavior in rats. These studies indicted that kryptopyrrole disrupted a stable fixed-ratio pattern of responding for food, specifically lengthening post-reinforcement pausing, and reducing the overall rate of responding so that fewer reinforcements were obtained, especially toward the end of each experimental session. Under KP, rats which previously responded consistently on fixed-ratio schedules for food reinforcement no longer behaved effectively. This disruption was greater with increasing dose, and with increasing schedule demand. Direct observation and supplementary experiments showed that K P did not alter food consumption, that any food pellets supplied during periods of operant nonresponding were immediately consumed, that the rats were not satiated, sedated, nor ataxic, and were active. Normal patterns of behavior were reestablished within 24 hours after KP. Appel et al. (1970) reported that d-LSD combined with p-chlorophenylalanine (PCPA), a depletor of brain serotonin, also produced a very similar pattern of effects upon simple fixed ratios and so did I-LSD, the little-studied optical isomer of d-LSD (Appel and Freedman, 1965). I n view of these parallels between K P and LSD effects on the pattern of fixed ratio responding, and in view of the early history of K P research (Irvine, 1961, 1973a), it would be interesting to know whether kryptopyrrole’s behavioral activity might resemble that of hallucinogens as a class. Naturally it was not possible from the fixed ratio study alone to determine which class of drugs it resembled in its behavioral activity.

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C. EFFECTS ON DISCRIMINATED AVOIDANCE Ncgatively reinforced operant behavior, as contrasted with positively reinforced behavior, might be affected differently by KP, and the comparison might help decide the nature of its action. A recent study (Zdanivsky and Irvine, I972 ;and unpublished data) determined the effects of kryptopyrrole upon bar-pressing to avoid electric shock, using a discriminated avoidance procedure, and analyzing the results in terms of Bovet-Gatti profiles, which, according to Smythies et af. (1969), are capablc of distinguishing the class of rieuropharmacological agents to which an unknown belongs, For the same strain of rats in the same equipment, higher doses of KP were required to disrupt performance on the avoidance task than on the fixed-ratio foodreinforced task. Moderately high doscs (e.g., 30 mg/kg) produced disruption characterized by a profile (Bovct and Gatti, 1964) intermediate between the amine-depletor and psychotomimctic profiles. With higher doses on an acute basis, severe toxic reactions or death might be expected ; however, as noticed in the fixed-ratio (G. B. Zdanivsky and D. G. Irvine, unpublished observations), repeated injections with KP lead to physical tolerance, and thc lethal dose can rise by a factor of 3 or more. While behavioral tolerance to KP was not seen in the fixedratio experiments, a form of it did appear in the avoidance series. In the KP-tolerant rat, very high doses produced avoidance disruption patterns (Bovet-Gatti profiles) identical with those of the psychotomimetics dLSD, N,N-dimethyltryptamine, and mescaline. This phenomenon, while interesting and possibly related to the metabolism of KP, is not yet understood. VII. Neurobiology of Other Pyrroles

No attempt is being made in this section to be at all comprehensive in coverage; its purpose is simply to point out some neurobiologically interesting pyrroles which are not usually drawn together on the basis of a single pyrrole nucleus as the common denominator. A. PYRROLIC PHEROMONES I n Fig. 5a is depicted the molecular structure of a substance involved in communication between certain butterflies (Lycorea ceres c e m ) during courtship (Pliske and Eisner, 1969), minute amounts evoking marked electrical responses in the antenna1 olfactory receptors (Schneider and Seibt, 1969). Like kryptopyrrole, this behaviorally active natural substance is a trisubstituted monopyrrole with one a position free. Butterflies are among the very few species besides the human to produce monopyrroles other than the

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ca,

H , C -

-

171

2,3 Di hydro -7-me1 hyl- 1H-

-

pyrrolizin 1- one

I

CHI

I

-C R

Indoles

0s

pyrroles

Pyrrolizidine alkaloid rneiabolites

FIG. 5. Other natural monopyrroles: (a) pyrrolic pheromone of certain butterflies; (b) batrachotoxin, a neuromuscular toxin; (c) pyrrolizidine alkaloid metabolites (liver and lung toxins); and (d) indoles.

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ubiquitous porphobilinogen (Meinwald and Meinwald, 1966; Meinwald al., 1969).

et

B. BATRACHOTOXIN In Fig. 5b is presented the structure of batrachotoxin (Tokuyama cf al., 1968), a naturally occurring potent neuromuscular blocking agent. This toxin is produced by certain species of frogs from Central and South America, is used as an arrow poison by tribes indigenous to the area, and is perhaps the most powerful animal toxin known (Daly et al., 1965). While the molecular structure is quite complex, being a pyrrole carboxylic acid in ester linkage with a highly modified steroidal alcohol, the more complex nonpyrrolic portion is only 1/500th as toxic as the whole molecule (Warnick et al., 1971). The pyrrole moiety, which is of crucial importance for the toxicity of batrachotoxin, is, like kryptopyrrole, a trisubstituted monopyrrole, with one aposition free. Pharmacologically, batrachotoxin (BTX) interferes with muscle contraction and neuromuscular transmission in such a way as to (a) decrease the tension of, and then completely block, the indirectly or directly elicited muscle twitch, (b) irreversibly depolarize the muscle membrane, and (c) produce a biphasic muscle contracture (Warnick et al., 1971). The BTX membrane depolarization was blocked by tetrodotoxin, as were the BTX contractures. BTX contractures were also blocked by lowering sodium or raising calcium levels, and were blocked by prior depolarization with potassium. BTX in turn blocked the induction of contractures by caffeine or potassium. T h e effects of BTX were accompanied by ultrastructural changes in the sarcotubular system (Warnick et al., 1971). Electrophysiologically, BTX produced in muscle a two-phase rcsponsc : first a high-frequency discharge of miniature end-platc potentials (MEPP) and an increase in hZEPP and end-plate potential (EPP) amplitudes, and then a decrease in MEPP and EPP amplitudes and failure of transmission. The transmission block was shown to be of presynaptic origin. From the *detailed electrophysiological findings in the presence and absence of inhibitors and modified levels of Na, K, or Ca (Albuquerque et al., 1971), it was concluded that BTX acts by selectively increasing sodium permeability, thereby depolarizing the membrane and transicntly incrcasing both spontaneous and evoked release of transmitter. Basically similar results were obtained for the action of batrachotoxin on Purkinje fibers of the heart (Hogan and Albuquerque, 1971 ) .

C. PORPHOBILINOGEN AND ACUTEINTERMITTENT PORPHYRIA Unlike monopyrroles in general, porphobilinogen (PBG) is included frequently in reviews on porphyrias, metabolic regulation, and the biosynthesis

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of heme. Here PBG will be considered rather selectively, only in relation to AIP and in relation to molecular structural or pharmacological similarities to other pyrroles of neurobiological interest. Structurally, PBG (Fig. lc) is quite similar to KP, being a trisubstituted monopyrrole with one free ci position. Its substituents are on carbons 2, 3, and 4,as in kryptopyrrole; but unlike KP, PBG is a readily ionizable compound, bearing one primary amino group and two carboxyls. Since a high level of PBG is the most characteristic nonenzymatic biochemical sign of the hereditary neuropsychiatric disorder AIP, and since the syndromes seen in this disorder all appear to be ascribable to some neurotoxic metabolite (Taddeini and Watson, 1968), it was natural to suspect PBG to be the causal agent. After several studies in various laboratories over more than a decade, there seems to be a consensus that PBG is relatively inert pharmacologically, particularly in vivo. It is noteworthy, however, that under favorable conditions in vitro at least, PBG is a fairly potent inhibitor of neuromuscular transmission (Feldman et al., 1971). This inhibitory effect of PBG was manifested as (a) decreased tension of indirectly stimulated muscle twitches, (b) inhibition of potassium-induced depolarization and stimulation, and (c) decreased frequency of MEPP’s in the presence of excess K. The significance of these in vitro results is difficult to assess, but in view of their potential importance, and ‘the fact that an oxidation product of PBG, porphobilin, is 10 times as potent as the parent compound (Feldman et al., 1971), parallel studies on structurally similar kryptopyrrole and its oxidation products may be worthwhile. D. INHYPERPROLINEMIA TYPE I1 AND OTHER HUMAN DISEASES It is either established or extremely probable that monopyrroles are intimately associated with hyperprolinemia type I1 (with febrile convulsions), some collagen diseases, Chiari’s syndrome,Jamaican “venoocclusive disease,” and Dzhalangarsk encephalitis. In hyperprolinemia type 11, characterized by febrile convulsive tendencies and abnormal EEG with or without mental retardation, there is a unique Ehrlich-reactive metabolite (Selkoe, 1969; Applegarth et al., 1971). Recent preparative chromatography and mass spectral examination of this material (D. G. Irvine and J. R. Majer, unpublished work; D. G. Irvine and D. A. Durden, unpublished work) revealed a mixture of apparently pyrrolic metabolites whose high-resolution mass spectra revealed ion peaks characteristic of pyrrole-2-carboxylic acid, and others corresponding with amino acid amides of this pyrrole (reported in Applegarth et al., 1974). The significance of these pyrroles remains unknown; they are unlike kryptopyrrole in that the pyrrole nucleus apparently is only monosubstituted.

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The Wakayama group (Yamaguchi et d., 1966; Nishimura et al., 1966, 1967) has reported that a pair of disubstituted monopyrroles is associated with certain cases of collagen disease. These urinary metabolites, pyrrole-l,2dicarbimide and pyrrole- 1,2-dicarbaniide, are structurally related to the pyrrole-2-carboxamide derivatives tentatively identified in hyperprolinemia type 11, and are perhaps more like kryptopyrrole a t least in having a disubstituted pyrrole nucleus, but there is no known connection between this interesting pair of metabolites and neurobiological functions. More closely related to kryptopyrrole’s molecular structure are the pyrrolic metabolites of many pyrrolizidine alkaloids. Ingestion of various pyrrolizidine alkaloid-containing seeds or plants has caused serious disease and loss of livestock in various countries, and these natural products have been considered to be causally involved in such human diseases as Chiari’s syndrome (Selzer and Parker, 1951), Jamaican venoocclusive disease (Bras and McLean, 1963), Jamaican cirrhosis (Bras el d., 1961), some tropical cancer (Schoental, 1968), and Dzhalagarsk encephalitis (Shtenberg and Orlova, 1955). Whatever the degree of involvement of the pyrrolic metabolites of pyrrolizidine alkaloids in the etiology of these diseases, it is established through extensive experimental studies in animals that the pyrrolizidine alkaloids produce liver disorders, histological changes in liver and lung, pleural effusion, hepatic carcinoma, mutations, and chromosomal damage (Butler et at., 1970; Schoental, 1968; Clarke, 1959, 1960; Avanzi, 1961). While the anticholinergic activity of some pyrrolizidine alkaloids is quite high, there is no correlation with hepatotoxicity or mutagenic potency. The latter activities are associated with an unsaturated (pyrroline) ring in the parent alkaloid, difficult hydrolyzability of the ester groupings, and a good yield of pyrrolic metabolite in vivo. It used to be considered that the very complex structures of the acids esterifying the hydroxylated pyrrolizidine rings were important in determining biological potency in this series of alkaloids, but it now appears quite definite that they act primarily by ensuring the protection of the parent compound from esterases, thereby allowing time for the pyrrolizidine ester to be converted to the corresponding biologically active pynole. Indeed, not only is a complex acid not necessary for biological activity, but neither is the secondary ring normally present in these alkaloids and their pyrrolic metabolites (Mattocks, 1971). The pyrrolizidine alkaloids are metabolized in viuo into one of three classes of compounds: the trisubstituted monopyrroles (which biologically are very active) the nonpyrrolics (inactive), and thedisubstituted monopyrroles (somewhat active). Except for this reviewer’s emphasis on the number of substituents attached to the pyrrole ring, this classification is from Mattocks and White (1971), who emphasized instead the presence or absence of a n allylic acyloxy grouping (i.e., esters in conjugation with the pyrrole N). Whatever

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the correct interpretation of the structure/activity correlations among pyrrolizidine alkaloid metabolites may be, it is clear that the most biologically active ones belong to the trisubstituted monopyrrole group (Fig. 5c) and hence are most similar to kryptopyrrole. This KP-like structure is also shared by a new, totally synthetic model possessing many of the biological properties of the natural alkaloid metabolites, but lacking the second ring characteristic of the pyrrolizidine metabolites. The biological activity, then, does reside in the pyrrole structure of the alkaloid metabolites. Theoretically, these pyrroles could be oxidized to the corresponding &unsaturated lactams, and their water adducts (as occurs for kryptopyrrole) ; such reactive compounds may mediate the ultimate attack of these compounds on sensitive tissue sites to produce such parallel results as the delayed lethal effect accompanied by massive pleural effusion, seen both with KP (Irvine and Zdanivsky, 1971b) and with pyrrolizidine alkaloids (Barnes et al., 1964) or their pyrrolic metabolites (Mattocks, 1968; Butler et al., 1970), or related simpler synthetic pyrrole esters (Mattocks, 1971). Finally, as a special case among the pyrrolizidine alkaloid intoxications, there is “Dzhalangarsk encephalitis” or “Trichodesma toxicosis,” after the causal agent, the seeds of Trichodesma incanum, a good source of the pyrrolizidine alkaloids trichodesmine and incanine. This disorder is of particular neurobiological interest since in this case the alkaloids produce not just human disease, but specifically a neurological disease. As with the majority of the biological actions of such alkaloids, it is most probable that pyrrolic metabolites of trichodesmine and/or incanine are the actual toxic agents. Both of these alkaloids would produce pyrroles of the kryptopyrrole-like trisubstituted class. Why these particular examples of the pyrrolizidine series should affect the CNS so profoundly, whereas most others attack mainly the liver, lungs, and pleura, is not known, although the esterifying acids in the parent compounds are among the least polar commonly found in the pyrrolizidine alkaloid series, and enhanced lipoid solubility might enhance CNS uptake of the corresponding pyrrolic metabolites. The neurotoxins deriving from T. incanum deserve renewed attention in neurobiology laboratories.

E. INDOLES AREPYRROLES We have by no means exhausted the list of nonindolic monopyrroles having biological activities and/or occurring naturally (e.g., the antibiotic pyrrolonitrin, the hydroxyproline metabolite pyrrole-2-carboxylic acid, and the simple pyrroles of coffee and tobacco), but only a few of these are neurobiologically active, and none of them belong to the trisubstituted class most closely resembling KP, PBG, bactrachotoxin, and the postulated neurotoxin

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of “Dzhalangarsk encephalitis.” Consequently, i t may be more profitablc to again note (Irvine, 1973a) that all indoles are pyrroles (Fig. 5d) and hence the majority of statements in the neurobiology literature relating psychoactivity or other neural effects to the indole nucleus, are equally true of the (contained) pyrrole nucleus. In general, the pyrrole nucleus in the indoles of neurobiological interest is also of the 2,3,4-trisubstituted type, with one a position free, just as in kryptopyrrole, although drugs of the reserpine, harrnine, ibogaine, and yohimbine series have, in addition, a substituent at the 5 position, and hence lack a free u position. VIII. Relationship to Clinical Psychiatry

Considerable and understandable enthusiasm for the earliest possible clinical application of basic research findings in this area of monopyrrolcs in neurobiology has been manifest in several ccntcrs. Whether this applied work has always maintained a suitable sense of proportion with the hard facts from the basic research laboratories can be debated, but not here. As the editors of this series of reviews put it (Pfeiffer and Smythies, 1964), “Progress in neurohiological research must maintain a dclicatc balance between the fascination of basic explanation of clinical and physiological phenomena by means of chemical and physical concepts on the one hand and the pressing needs for the development of new and effective treatments of disease on the other. Advances in basic biochcmistry and biophysics often givc rise to developments in the clinical field, but maturc judgement is requircd to select from the vast detail of biochemistry and biophysics, those parts which are likely to apply to human disease.” Without inappropriately presuming to review the clinical psychiatric aspects of monopyrrolc neurobiology critically, it is possible to review the findings from the point of view of biology, and molecular biology. Modern biology has rendcrcd any simple naturc/nurture controversy anachronistic (Irvine, 1972b,c). While it has become popular to state that it is the interaction between hereditary and environmental factors which determines the state of the organism at a given time, even in psychological terms (Anastasi, 1958), molecular biology has revealed that this intcraction is concretely multilevel, orderly, and open to experimental “dissection” (Irvine, 1 9 7 2 ~ )Interactive . causation is no longer a vague and hence weak concept, but can be applied both theoretically and practically, in favorable circumstances. As can be depicted in a general genelenvironment interaction diagram (Irvine, 1972c, 1973b), the only things inherited are DNA moleculescertainly not a disease. In an orderly series of stcps (with each step vulnerable to modulation or distortion, blockade, ctc.), the coded information is transcribed from DNA into RNA (if not repressed by metabolites, drugs, or diet), the RXA message is interpreted and executed to form proteins (unless amino

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acids are missing, or the step is blocked) and the proteins are structural, hormonal, or enzymes. The enzymes will convert substrates to appropriate products (if the substrate is present, any needed vitamin or mineral cofactor is present, and it is not inhibited). At this point, and no sooner, can the first truely metabolic abnormality appear, which might exert some neurobiological action, including perhaps the development of psychiatric maladaptation. Thus, the expression of a gene in the simplest case allows ample opportunity for involvement of the environment : repressor substances, other metabolites, dietary components, drugs, etc. Application of this basic scheme in detail to the major neuropsychiatric disorders (e.g., schizophrenia) is not yet possible. However, AIP, while very rare, is the neuropsychiatric disease about which we know the most at the level of molecular biology, and this can be used as a very real model for the more common but less understood mental disorders (Irvine, 1972b,c). AIP is an intermittent disorder, even though its basic “cause” is a single dominant gene which upsets the regulatory mechanism normally limiting the rate of formation of the first enzyme in the “porphyrin pathway.” Excess synthesis of this enzyme results in abnormal accumulation of two early intermediates in this pathway, and this is accompanied by neurotoxic symptoms. This does not occur before sexual maturation, however, and attacks may be related to menstrual cycles, pregnancy, and stress, suggesting that steroidal induction of the first enzyme is involved. Attacks of symptoms are also precipitable by alcohol, sulfa drugs, and barbiturates. Attacks can be treated or prevented by glucose supplements, which repress transcription of the AIP gene controlling synthesis of the first enzyme. This type of interactive analysis of AIP (particularly as a model for major mental disorders) will be presented in detail elsewhere (Irvine, unpublished data), but the point to be stressed here is that a simplistic approach to the question of the psychiatric relevance of such metabolic abnormalities is no longer adequate, as more powerful approaches are available. The concept of “one psychiatric diagnosis/one metabolic abnormality” is clearly inadequate, as a further examination of AIP will demonstrate. Many AIP cases present as hysterics, but the same individual at another time, or other cases may present as psychotics, or as paralytics without noteworthy psychiatric problems. And although many porphyrics are “hysterics,” the great majority of hysterics are certainly not porphyrics. Glucose treatment of hysterics in general is as unlikely to benefit the group as phenylalanine deprivation is unlikely to benefit all mentally retarded children as a group. It is only when the PKU child is distinguished from the rest of the retarded individuals, by molecular means, that a cause-directed therapy can be usefully applied. Similarly it is likely that only the schizophrenic or paralytic who is etiologically a porphyric will benefit from glucose supplements.

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While it is not the purpose of this review to examine the therapeutic implications of the finding of elevated levels of certain monopyrroles in body fluids of patients, attempts to segregate schizophrenic patients into different biochemical types, and then design specific therapies for these subtypes have met with some success (see Pfeiffer’s work). From the parallels between the recent work in Princeton (Pfeiffer and Iliev, 1973; and unpublished data) and in Szeged (Huszdc and Durko, 1964, 1968a,b, 1970), it appears that further attention to the interrelationships between kryptopyrrole, zinc, pyridoxine, and porphyrin intermediary metabolism may be rewarding. On a more general level, a working hypothesis aimed at facilitating basic neurobiological work on monopyrroles relevant to neuropsychiatry has been presented in preliminary form (Irvine, 1973b) and will appear in detail elsewhere, but may be summarized in three statements : 1. Excessive or deviant pyrrolooxygenase activity underlies the significant excretion of natural (oxidized) kryptopyrrole * and of the skatole metabolite m-sulfatoxy-o-aminoacetophenone,both being associated with psychiatric disorders. 2. This pyrrolooxygenase activity is inducible by human steroids, including metabolites of corticosteroids and hence by stress, and may result in parallel oxidation of PBG, oxidative inactivation of PBG deaminasc, and release of C-2 from tryptophan to the 1-C pool, conceivably increasing methylation of a variety of substrates. 3. Pyrrolooxygenase products from monopyrroles (usually related to faulty regulation of the porphyrin metabolic pathway) may be involved in the neurotoxic effects at least in acute intermittent porphyria (AIP), and perhaps in other cases of neuropsychiatric disease where oxidized monopyrroles are prominent. In the latter, the AIP gene need not necessarily be involved.

While this schema tends to integrate a variety of previous observations, it is emphasized that this is a working hypothesis, whose utility must be assessed through appropriate experiments. Whatever may be the eventual clinical import of kryptopyrrole or the other monopyrroles, their unusual chemical reactivities and varied biological actions suggest that further neurobiological research in this area will be rewarding.

‘

The “natural kryptopyrrole” referred to in this hypothesis is the a’-hydroxy a-lactam of kryptopyrrole and/or the corresponding B,p side-chain positional isomers, together with very closely related minor adduce.

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REFERENCES Albuquerque, E. X., Warnick, J. E., and Sansone, F. M. (1971). J . Pharmucol. Exp. Ther. 176, 511. Anastasi, A. (1958). Psychol. Rev. 65, 197. Anonymous (1964). Unlisted Drugs, 16, 41. Appel, J. B. (1968). In “Psychopharmacology: A Review of Progress 1957-1967” (D. H. Efron, ed.), pp. 1211-1222. US Govt. Printing Office, Washington, D.C. Appel, J. B., and Freedman, D. X. (1965). Lijie Sci. 4, 2181. Appel, J. B., Lovell, R. A., and Freedman, D. X. (1970). Psychopharmacologia 18, 387. Applegarth, D. A., Ingram, P., Hingston, J., Sturrock, S., and Hardwick, D. F. (1971). Proc. Int. Congr. Pediat., 13th, 1971 5, 249. Applegarth, D. A., Ingram, P., Hingston, J., and Hardwick, D. F. (1974). Clin. Biochem. 7, (1). Avanzi, S. (1961). Caryologia 14, 251. Barnes, J. M., Magee, P. N., and Schoental, R. (1964). J . Pathol. Bacteriol. 88, 52 1. Blake, R. L., and Russell, E. S. (1972). Science 176, 809. Bliss, E. L., Thatcher, W., and Ailion, J. (1972). J . Psychiat. Res. 9, 71. Bonnet, R., Dimsdale, M. J., and Stephenson, G. F. (1968). Chem. Commun. p. 1121. Boulton, A. A., and Majer, J. R. (1970). J . Chromatogr. 48, 322. Bovet, D., and Gatti, G. L. (1964). PTOC.Int. Pharmucol. Meet., 2nd, I963 1, 75. Bras, G., and McLean, B. (1963). Ann. N . Y. Acad. Sci. 111, 392. Bras, G., Brooks, S. E. H., and Watler, D. C. (1961). J . Pathol. Bacteriol. 82, 503. Brownstein, M. J., and Heller, A. (1968). Science 162, 367. Butler, W. H., Mattocks, A. R., and Barnes, J. M. (1970). J . Pathol. 100, 169. Clarke, A. M. (1959). Nature (London) 183, 731. Clarke, A. M. (1960). 2. Vererbungdehre 91, 74. Cookson, G. H., and Rimington, C. (1954). Biochem. J . 57, 476. Corwin, A. M. et al. (1960). Encycl. Brit. 18, 801. Daly, J. W., Witkop, B., Bommer, P., and Biemann, K. (1965). J . Amer. Chem. SOC.87, 124. Drohocki, Z. (1948). Reu. Neurol. 8, 619. Ellman, G. L., Jones, R. T., and Rychert, R. C. (1968). Amer. J . Psychiat. 125, 849. Feldman, D. S., Levere, R. D., Lieberman, J. S., Cardinal, R. A., and Watson, C. J. (1971). PTOC.Nut. Acad. Sci. US.68, 383. Fischer, H., and Hartmann, P. (1934). Hoppe-Seyler’s 2.Physiol. Chem. 226, 116. Fischer, H., and Orth, H. (1934). “Die Chemie des Pyrrols,” Vol. 1, p. 328. Akad. Verlagsges., Leipzig. Fischer, H., Baumgartner, H., and Plot,, E. (1932a). Justus Liebigs Ann. Chem. 493, 1. Fischer, H., Yoshioka, T., and Hartmann, P. (1932b). Hopbe-Syler’s 2.Physiol. Chem. 212, 146. Frydman, R. B., and Frydman, B. (1973). Biochim. Biophys. Acta 293, 506. Frydman, R. B., Tomaro, M. L., and Frydman, B. (1972a). Biochim. Biophys. Acta 284,63. Frydman, R. B., Tomaro, M. L., and Frydman, B. (1972b). Biochim. Biophys. Acta 284,80. Frydman, R. B., Tomaro, M. L., Wanschelbaum, A., and Frydman, B. (1972~).FEBS Lett. 26, 203. Gershon, S. (1968). Psychiat. Res. Rep. A m r . Psychiat. Ass. 24, 166. Goldstein, L., Sugerman, A. A., Stolberg, H., Murphree, H. B., and Pfeiffer, C. C. (1965). Electroencephafogr. Clin. Neurophysiol. 19, 350. Hais, I. M. (1970). J . ChTOmUtOgT. 48, 200. Hoffer, A., and Osmond, H. (1963). Acta Psychiat. Scand. 39, 335. Haft, E., Katritzky, A. R., and Nesbit, M. R. (1967). Tetrahedron Lett. p. 3041.

180

DONALD C . IRWNE

Hoft, E., Katritzky, A. R.,and Nesbit, M. R. (1968). Tetrahedron Lett. p. 2028. Hogan, P. M.,and Albuquerque, E. X . (1971). J. Pharmacof. Exp. Thm.176, 529. HuzAk, I., and Durk6, I. (1964). Acta Ewchim. Pol. 11, 389. HuszAk, I., and Durk6, I. (1968a). “A porphyrin anyagcsere zavara schizophreniaban” (Disturbances of Porphyrin Metabolism in Schizophrenia), Ideg&g3jaszati Szmnlc (Nrrue Specialrsts’ Reuiew) 11, 157. Huszbk, I., and Durk6, I. (1968b). Proc., WorfdCongr. Pfychiat., 4th, 1966 Int. Congr. Ser. No. 150, p. 2931. HuszAk, I., and Durk6, I. (1970). Acta Physiof. 38, 77. Huszik, I., and Durk6, I. (1971). Abstr. I d . Meet. In,. Soc. N e u r o h n . , 3rd, 1971 p. 183. Huszhk, I., Durk6, I., and Karsai, K. (1971). Abstr. fnt. Meet. Int. Soc. Neurochem., 3rd, 1971 p. 183. Huszhk, I., Durk6, I., and Karsai, K. (1973). Acta Physiol. (in press). Irvine, D. G. (1961). J . Neuropsychiat. 2 , 292. Irvine, D. G. (1963). J . Cfin.Chem. 9, 444. Irvine, D. G. (1966). Proc., Id.G n g r . Cybmrct. Med., 4th, 1966 p. 535. Irvine, D. G. (1972a). “Kryptopyrrole: Some Clinical Metabolic and Pharmacological Correlates,” Proc. Sask. Psychiat. Res. Meet., p. 171. Irvine, D. G. (1972b). “Use of Marker Genes in Transcultural Psychiatric Research,” Inaug. Meet. Can. Chapt. Int. Ass. Cross-Cult. Psychol. Brock University, St. Catherines, Ontario. Irvine, D. G . (1972~). “Nature, Nurture, and Inferaction,” Inaug. Meet. Can. Chapt. Int. Ass. Cross-Cult. Psychol. Brock University, St. Catherines, Ontario. Irvine, D. G. (1973a). In. “Orthomolecular Psychiatry” (D. Hawkins and L. Pauling, eds.), Chapter 8, p. 146. Freeman, San Francisco, California. Irvinc, D. G. ( 1973b). “Pyrrolooxygenases and Psychiatric Disorders: a Ncw Hypothesis,” Proc. Sask. Psychiat. Res. Mcet. (in press). Irvine, D. G., and Anderson, M. E. (1965).J. Chromafop. 20, 541. Irvine, D. G., and Bayne, W. (1969). Enzymol. B i d . Clin. 10, 361. Irvine, D. G., and Wetterberg, L. (1972). Lancet 2, 1201. Irvine, D. G., and Zdanivsky, G . B. (1971a). Abstr., Int. Meet. I d . Sac. Neurochem., 3rd, 1971 p. 182. Irvine, D. G., and Zdanivsky, G. B. (1971b). Proc., World Congr. Psychiat., Sth, 1971 Abstracts, p. 352. Irvine, D. G., Bayne, W., and Anderson, M. E. (1967). J t . Annu. Meet. Can. Soc. Cfin. Chtm., Ilih, p. 13. Irvine, D. G., Bayne, W.. and Miyashita, H. (1969a). Enzymol. Eiof. Cfin. 10, 398. Irvine, D. G., Rayne, W., Miyashita, H., and Majer, J.R.(1969b). Nature (London) 224,811. Irvine, D. G., Bayne, W.,and Majer, J. R . (1970). J. Chromatogr. 48,334. Irvine, D. G., Bayne, W., and Miyashita, H. (1971). “Massive Urinary Excretion of a Kryptopyrrolc-like substance in an Inherited Neuropsychiatric Disorder: Acute Intermittent Porphyria,” Proc. Sask. Psychiat. Ra. Meet., p. 86, ’Table 1. Irvine, D. G., Bayne, W., and Miyashita, H. (1973a). “The Main Form of NaturallyOccurring Kryptopyrrole: Its 5-OH-2-Lactam, a Product of Pyrrolooxygenase,” Proc. Sask. Psychiat. Res. Meet., Fig. 2. Irvine, D. G., Bayne, W., and Miyashita, H. (1973b). Proc. Can. Fed. Biol. Soc. 16, 125. Irvine, D. G., Ell, D., and Crichlow, E. C. (1973~). fnt. Meet. Int. Soc. Neurochem., 4th, 1973 p. 359. Keogh, R. P. (1964). Spzkc B W a v e J . 13, 2 Koaower, N. S., and Rock, R. A. (1968). Nature (London) 217, 565.

KRYPTOPYRROLE AND OTHER MONOPYRROLES

181

Levin, E. Y., and Flyger, V. (1971). Science 174, 59. Lienhard, G. E. (1973). Science 180, 149. Lightner, D. A., and Crandall, D. C. (1973). Experientia 29, 262., Majer, J. R., and Jenkins, A. G. (1967). Talanta 14, 777. Mandell, A. J., Slater, G. G., and Mersol, I. (1961a). Science 133, 1832. Mandell, A. J., Slater, G. G., and Mersol, I. (1961b). Arch. Gen. Psychiat. 5, 234. Mandell, A. J., Slater, G., Geertsma, R. H., and Mersol, I. (1963). Arch. Gen. Psychiat. 9, 89. Marjerrison, G., Krause, A. E., and Keogh, R. P. (1968). Electromephalogr. Clin. Neurophysiol. 24, 35. Mattocks, A. R. (1968). Nature (London) 217, 723. Mattocks, A. R. (1969). J . Chem. Soc., C p. 1155. Mattocks, A. R. (1971). Nature (London) 233, 451. Mattocks, A. R., and White, I. N. H. (1970). Anal. Biochem. 38, 529. Mattocks, A. R., and White, I. N. H. (1971). Nature (London), New Biol. 231, 114. Meinwald, J., and Meinwald, Y. C. (1966). J . Amer. Chem..Soc. 88, 1305. Meinwald, J., Meinwald, Y. C., and Mazzocchi, P. H. (1969). Science 164, 1174. Metzger, W., and Fischer, H. (1937). Justus Liebigs Ann. Chem. 527, 1. Miyagi, K., Cardinal, R., Bossenmaier, I., and Watson, C. J. (1971). J. Lab. Clin. Med. 78, 683. Miyashita, H., and Irvine, D. G. (1972). “Source of Kryptopyrrole: Quantitative Loading Studies with Certain Unlabelled Substrates,” Proc. Sask. Psychiat. Res. Meet., p. 242. Moffett, R. B. (1968). J. Med. Chem. 11, 1231. Mouriquand, G., Lechat, P., Chareton, J., Edel, V., and Chighizola, R. (1958). C. R. Acad. Sci. 246, 668. Nishimura, N., Mori, Y., Matsunaka, M., Kawasaki, H., Matsukawa, S., and Yamaguchi, M. (1966). Wakayama Med. Rep. 11, 15. Nishimura, N., Mori, Y., Matsunaka, M., Matoba, S., Kawasaki, H., Yamaguchi, M. and Matsukawa, S. (1967). J . Chronic. Dis. 20, 55. Oldendorf, W. H. (1971). Amer. J. Physiol. 221, 1629. Pfeiffer, C. C., and Iliev, V. (1973). Fed. Proc., Fed. Amer. SOG.Exp. Biol. 32, 276 (Abstr. No. 350). Pfeiffer, C. C., and Smythies, J. R. (1964). Znt. Rev. Neurobiol. 6, vii. Pliske, T. E., and Eisner, T. (1969). Science 164, 1170. Quistad, G. B., and Lightner, D. A. (1971a). Chem. Commun. p. 1099. Quistad, G. B., and Lightner, D. A. (1971b). Tetrahedron. Lett. No. 46, 4417. Rabbeno, A. (1929). Arch. Znt. Pharmacodyn. Ther. 35, 377. Rabbeno, A. (1930). Boll. Soc. Ztal. Biol. Sper. 5, 324. Reio, L. (1958). J. Chromatogr. 1, 338. Reio, L. (1960). J . Chromatogr. 4, 458. Rezek, P. (1937). Z . Gesamte. Ex@. Med. 101, 358. Russell, C. S. (1972). J. Theor. Biol. 35, 277. Saccardi, P. (1922). Biochem. Z . 132, 443. Schmid, R. (1972). I n “The Metabolic Basis of Inherited Disease” ( B. Stanbury, B. Wyngaarden, and D. S. Fredrickson, eds.), 3rd ed., Chapter 46, esp. p. 1147. McGrawHill, New York. Schneider, D., and Seibt, U. (1969). Science 164, 1173. Schoental, R. (1968). Cancer Res. 28, 2237. Selkoe, D. J. (1969). Neurology 19, 494. Selzer, G., and Parker, R. G. F. (1951). Amer. J. Pathol. 27, 885.

182

DONALD 0. XRVINE

Shtenberg. A. E., and Orlova, N. V. (1955). Vop. Pifan. 14, 27. Sicdel, W., Stich, W., and Eisenrcich, F. (1948). Nahawirsenschaftcn 10, 316. Simpson, ti. M., and Krakov, L. (1968). Cun. Thn. Res. 10, 41. Smythies, J. R.,Johnston, V. S.,and Bradley, R.J. (1969). Brit. J. Psych&. 115, 55. Sohler, A., Rcnz, R. H., Smith, H., and Kaufman, J. (1967). Int. J. Neuropsychiat. 3, 327. Sohlcr, A., Beck, R.,and Noval, J. J. (1970). Nature (Inndon) 228, 1318. Sugerman, A. A., and Herrmann, J. (1967). Clin. Pharmacol. 7%. 8, 261. Taddeini, L., and Watson, C. J. (1968). Semin. Hcmatol. 5, 335. Taunton-Rigby, A,, Sher, S. E., and Kelley, P. R. (1973). Science 181, 165. Tokuyama, T., Daly, J., and Witkop, B. (1968). J. A m . Chem. Soc. 91, 3931. Turner, W. J. (1937). J . Eiol. Chem. 118, 519. von Dobeneck, H., and Brunner, E. (1969). 2. Klin. Chcm. Klin. Biochcm. 2, 113. Warnick, J. E., Albuquerque, E. X.,and Sansone, F. M. (1971). J. Pharmacol. Exp. Ther. 176, 497. Wetterberg, L. (1972). O r f h o m o h l . Psychiat. 1, 141. With, T. K. (1968). “Bile Pigments: Chemical, Biological, and Clinical Aspects,” csp. pp. 48 and 97. Academic Press, New York. Wolley, D. W. (1960). Dir. Nnu..Sqst. (Suppi. 2) 21, 87. Yamaguchi, M., Mori, Y., and Nishimura, N. (1966). W d q a m a Med. Rep. 11, 119. Zdanivsky, C. B.,and Irvine, D. G. (1972). “The EKect of Kryptopyrrole upon a Standardized Discriminated Avoidance Task,” Proc. Sask. Psychiat. Res. Meet., p. 188. Sask. Dept. Public Health, Regina, Canada.

RNA METABOLISM I N THE BRAIN By Victor E. Shashoua McLean Hospital Research Laboratory, Harvard Medical School, Belmont, Massachusetts

I. Introduction . 11. RNA Content of Brain as Compared to Other Organs . A. Total RNA Content . B. Comparative Metabolic Rates of Brain and Other Organs . . C. Comparison of Base Composition Measurements of Brain RNA with Other Organs . 111. Anatomical and Subcellular Distribution of Brain RNA A. RNA in Different Cell Types . B. RNA in Axons . . C. RNA Transport in Axons D. RNA in Brain Subcellular Fractions . IV. RNA Changes in Single Cells Produced by Physiological and Electrical Stimulation . V. RNA Changes in the Brain during Development . . Correlation of Brain RNA Content with Age . VI. Types of Brain RNA . A. RNA Turnover Rates . B. Neuronal and Glial RNA Metabolism . C. Pattern of RNA Synthesis . D. Base Compositions E. Metabolic Pools . F. Special Classes of RNA Molecules . VII. Brain Function and RNA Metabolism . A. General Stimulation Studies on RNA Metabolism . B. Nonlearning Behavioral and Physical Effects on RNA Metabolism . . C. RNA Changes and Learning . VIII. Biogenic Amine Effects on Brain RNA Metabolism A. Studies with Acetylcholine . B. Serotonin Level Changes . C. Cyclic AMP Effects on Brain RNA Metabolism . D. Norepinephrine Effects on Brain RNA Metabolism IX. Drug Effects on RNA Synthesis . X. A Theoretical Model for Information Storage . References

.

.

.

.

.

183 184 184 185 187 189 189 191 192 194 197 199 199 204 204 204 204 205 206 206 206 206 208 2 10 217 217 218 219 219 219 222 228

1. Introduction

The brain is a dynamic organ. Over 98% of its proteins have a half-life of 14 days (Lajtha, 1964). This rapid protein turnover requires an equally 183

184

VICTOR E. SHASHOUA

fast rate of RNA synthesis. I n the past ten years, the distribution, synthesis, and turnover rate of brain RNA have been correlated with a wide variety of functions and a number of reviews have dealt with certain aspects of this subject (Hyden, 1960; Glassman, 1969; Rose, 1970; Jarvik, 1972). This review will discuss RNA metabolism in terms of the basic cellular metabolic rate of brain as a tissue and in terms of its involvement with certain specialized brain activities, such as electrical processing, axonal transport, neuronal regeneration, and drug interactions. In addition, the effects of behavior and learning on RNA metabolism will also be discussed. Whenever possible, the material for the review has been selected to emphasize those features that distinguish brain from other tissues. II. RNA Content of B n i n as Compared to O t h e r Organs

A. TOTAL RNA CONTENT

A variety of methods have been used to determine brain RNA content for comparison with other organs. The results, in large measure, depend upon what parameters are used as a reference for expressing the data. Measurements of RNA using a given wet weight of tissue give deceptively low values for brain white matter and higher values for gray matter. The measurements that report the total RNA content in a given cell type may be unreliable since i t is difficult to dissect out the cells of the brain without eliminating a large proportion of their processes. Thus, such measurements of RNA are biased toward the low side. When total protein content is used as a reference, a value of 25 pg/mg, is obtained for the RNA content of rat brain tissue (see Table I). This result classifies the brain among tissues with the lowest RNA content. Thus, liver, spleen, and skeletal muscles come at the high end of the list with 36-38 pg of RNA per milligram of protein and brain at the low end along with kidney at 25. Also biasing these analyses toward giving low values for brain, is the fact that the brain, unlike other tissues, has a large proportion of white matter. This gives a large contribution to the protein content but contains little or no RNA in the axonal fibcrs. Perhaps the most informative measure of RNA levels is the ratio of RNA to DNA content. As shown in Table I, this ratio is highest for liver at 5.14, compared to the intermediate value of 1.18 for brain, and a low value of 0.14 for rat thymus. It is important to recognize that the use of DNA as a reference also presents some difficulty since not taken into account are those categories of neuronal cells that have a tetraploid DNA content. I n addition the ribonuclease activity of each tissue varies substantially and it is difficult to assess how much of the RNA is destroyed during its isolation. However, provided these reservations are kept in mind, it would appear

185

RNA METABOLISM IN THE BRAIN

TABLE I COMPARISON OF THE RNA CONTENT OF TISSUES OF ADULTRAT

Tissue Liver Pancreas Kidney Brain Spleen Bone marrow Heart Thymus

RNA (pglmg protein) 36.3

27.3 25.4 36.4 -

-

DNA (pglaverage cell)

RNA DNA

Reference

1.14 0.70 0.65 0.61 0.63 0.67 0.62 0.72

5.14 4.1 2.4 1.18 1.0 0.97 1.03 0.14

Mandel et al. (1950) Schneider (1946) Mandel et al. (1950) Mandel et al. (1950) Rambach et al. (1952) Rambach et al. (1952) Rambach et al. (1952) Schneider and Klug (1946)

that the brain as a tissue has an intermediate level of RNA content as compared to other tissues. Table I1 shows the RNA content of brains from different species. With the exception of the lower values for carp and duck brains, the RNA/DNA ratio seems to be quite constant. In terms of protein content, however, the values for man and tortoise seem to be 2.5-3.0 times higher than for rat. From these observations the significance of the brain RNA content as an index of the rate of metabolism becomes questionable, since such data can give only the base-line levels without any reference to turnover rates.

B. COMPARATIVE METABOLIC RATESOF BRAINAND OTHERORGANS A number of studies have used radioactive RNA precursors to measure the rates of turnover of RNA in brain as compared to that in other tissues. TABLE I1 BRAINRNA CONTENT IN DIFFERENT SPECIES~

Species Carp Tortoise Duck Rat Guinea pig Cat Dog Man

RNA (Pgglmg protein)

RNA (pglcell)

DNA (pglcell)

20.6 29.6 10.6 9.7 18.5 8.9 8.2 24.4

1.28 3.26 1.69 1.18 2.33 1.14 1.18 3.87

0.45 1.63 0.44 0.72. 1.61 0.79 0.79 2.63

Data from Bieth and Mandel (1953).

RNA DNA 0.35 0.5 0.26 0.61 0.69 0.69 0.65 0.68

186

VICTOR E. SHASHOUA

Schaer et (11. (1969) classified tissues into three types according to their proliferation rate. The brain and liver have the lowest rate, kidney is intermediate, and spleen has the highest rate. These experiments compared the incorporation of [5-3H]uridine into the total nucleic acids for various organs of the mouse. Table I11 shows the results obtained from such a study. TABLE 111 [5-3H]URIDINE NUCLEIC ACIDS"

INCORPORATION OF

INTO

Organ Brain Liver Kidney Spleen

[5-3H]Uridine in total nucleic acids (pCi) 34 415

870 69 1

a Data from Schaer cf nl. (1969). Measurements are for mouse at 120 minutes after injection of the label.

The amount incorporated into brain was over 10 times lower than for liver, which in turn was about 2 times lower than for kidney. These data, however, do not take into account thc distribution of the RNA precursor (uridine) into the various tissues. Even though the organs from the same animal are compared, the nucleic acid pools and the distribution of label varies quite widely in each tissue. Thus, one cannot assume that a low level of radioactive uridine incorporation signifies a low rate of RNA synthesis and turnover. Bondy and Roberts (1968) used the techniques of hybridization for comparing the RNA turnover rates for rat liver and brain. I n this technique (Gillespie and Spiegelman, 1965) thc denatured DNA (i.e., single-stranded) is adsorbed onto some inert matrix (usually cellulose nitrate). T h e labeled RNA is then incubated for a period of 1 hour with the bound DNA. T h e nucleotides of the single-stranded DNA molecules form double strands with the complementary base sequences of the RNA. T h e unbound RNA is then removed with ribonuclease treatment, and the bound radioactivity, corresponding to the complementary sequences, is measured as bound radioactivity in the sample. Table IV summarizes the hybridization experiments, expressed as the percent of the total radioactivity hybridized from each of the RNA samples. The results indicate that the nuclear RNA from both brain and liver hybridize to a greater extent than the RNA derived from the cytoplasmic fraction. I n all cases, however, brain RNA hybridized higher than liver RNA

187

RNA METABOLISM IN THE BRAIN

TABLE IV HYBRIDIZATION OF PULSE-LABELED CEREBRAL AND HEPATIC RATRNAQsb

Tissue Brain

Liver

Percent radioactive RNA hybridizing

Time after pulse label

Nuclear RNA

4 Min 20 Min 50 Min 2 Days 4 Min 20 Min 50 Min 2 Days

18.5 19.5 14.9 4.1 10.3 10.1 9.1 1.7

Cytoplasmic RNA 15.3 13.3 0

9.4 8.1 0.7

Data from Bondy and Roberts (1968). Methods: RNA fractions were isolated from brain a t various intervals after intracervical injection of 1 mCi of [53-H]uridine and from liver after intraperitoneal administration of 100 pCi of [6-14C]orotic acid. (Incubations were carried out for 18 hours with 3.8 pg of RNA and 30-50 pg of denatured rat liver DNA.) The values are the averages of 3-5 duplicate measurements. a

by a factor of about 2. The authors suggested that a larger proportion of RNA synthesis in the brain is directed towards mRNA production. Pulselabeled cerebral nuclear RNA hybridized to a greater extent than cytoplasmic RNA for at least a week after administration of labeled precursor, and this suggested that the nuclei contain hybridizable components that are not transferred to the cytoplasm. These findings support the notion that the rate of RNA synthesis in the brain is at least equivalent to that in liver and may be as much as 2 times larger. Additional corroborating evidence was obtained by Bondy and Roberts (1969) in studies of RNA synthesis in cellfree systems. Nuclear chromatin from brain was found to incorporate 20'7, more nucleic acids into RNA than hepatic chromatin.

C. COMPARISON OF BASECOMPOSITION MEASUREMENTS OF BRAINRNA WITH OTHER ORGANS Table V summarizes the base composition measurements reported for nervous tissue from a variety of species. The analyses show no distinctive features that can be attributed specifically to brain tissue. The base composition of purified brain ribosomal 18 S and 28 S RNA was found to be essentially identical for brain and liver (Schneider and Roberts, 1968). However, the base composition measurements of the RNA derived from brain subcellular fractions do show some brain-specific characteristics, Table VI shows the base compositions of newly synthesized RNA labeled

TABLE V COMPARISON OF BASECOMPOSIT~ON OF BRAINRNA

WITH

OTHEROROANS

~

Source of RNA Rat braina Rat brain Chick sensory gangliab Goldfish Mauthner cellC (I

Rat brain"

Rat liverd Rat brain" Rat liver"

a

~

~~~~

~

Type of RNA

CMP

AMP

GMP

UMP

Total Hybridizable fraction Total Perikaryon Axonal Schwann Cerebral Nuclear Ribosomal Nuclear 28 s 18 s 28 s 18 s

20.6 f 0.5 21.7 f 1.7 31.8 29.7 26.3 24.1 22.8

19.8 f 0.7 21.7 f 1.7 19.8 22.3 18.2 19.9 16.3

26.9 f 0.8 28.3 f 1.4 26.4 30.0 35.9 37.9 35.3

32.7 & 1.2 28.3 k 0.9 22.3 18.3 19.5 18.1 25.7

27.6 25.3 28.9 f 0.4 25.8 f 0.4 28.9 f 0.2 26.6 0.4

17.0 17.2 16.6 19.8 16.8 20.1

33.9 35.8 36.0 31.4 35.5 31.0

21.5 21.3 19.0 22.9 18.8 22.5

Bondy and Roberts (1968). Burdmann (1969). Edstrom (1956). Schneider and Roberts (1968).

k 0.3 f 0.2 f 0.2

f 0.3

k 0.5 f 0.4 f 0.3 f 0.6

f 0.3 f 0.2 f 0.1 f 0.2

~~

Labeled precursor

3=p 3=P A + C None None None None None None

0

w

M

E

X 0

F

189

RNA METABOLISM IN THE BRAIN

with Ps2 (Bondy and Roberts, 1967; Balazs and Cocks, 1967). The measurements for cerebral nuclear and ribosomal RNA are substantially different from the values for liver when the ratio of uridine to cytidine is used as an index for comparison. Thus, the U/C ratio is 1.10 for the cerebral nuclear RNA as compared to 0.8 for liver. The corresponding values for ribosomal RNA were 0.77 and 0.69, respectively. The U/C ratio changes arise from an increased U and a decreased C content. This ratio can be used (Shashoua, 1971) as an index of increased mRNA and nuclear heterodisperse RNA synthesis. Similar analyses for the RNA bound to polysomes and for the nuclear hybridizable RNA gave U/C ratios of 1.6 and 1.3, respectively TABLE VI BASEC O M P O S I ~METHOD ON

U

RNA type

25.7 Cerebral nuclear 21.4 Cerebral ribosomal 21.3 Liver nuclear 19.9 Liver ribosomal 22.5 Cytoplasmic 24.8 Purified nuclear mRNA (polysomal) 30.4 21.5 rRNA Hybridizable nuclear 28.3 Rat liver DNA 28.5(T)

C

A

23.1 27.6 25.3 28.8 27.4 24.8 19.1 27.6 21.7 21.6

16.2 17.0 17.7 17.8 22.4 25.0 23.3 17.0 21.7 28.8

G

U/C

A/G

Reference

35.3 1.11 0.46 BondyandRoberts (1967) 33.9 0.77 0.50 BondyandRoberts (1967) 34.9 0.84 0.51 BondyandRoberts (1967) 33.7 0.69 0.53 BondyandRoberts (1967) 27.7 0.83 0.81 BalPzsandCocks (1967) 25.6 1.00 0.98 Balhzs and Cocks (1967) 27.3 1.6 0.85 Zomzelyetal. (1970) 32.4 0.78 0.52 Zomzelyctal. (1970) 28.3 1.30 0.77 Zomzelyctal. (1970) 21.5 1.32(T/C)1.34Zomzelyctal.(1970)

(Zomzely et al., 1970). These values represent a shift to a more DNA-like T/C ratio. Thus, by this criterion the RNA synthesized in brain has an increased nuclear heterodisperse RNA and mRNA content than liver. 111. Anatomical and Subcelluar Distribution of Brain R N A

A. RNA

IN

DIFFERENT CELLTYPES

A number of reviews have summarized the pattern of distribution of RNA in different cell types of the nervous system (HydCn, 1959a, 1960). Through the use of the microdissection method (Hydtn, 1959b, J. E. Edstrom, 1964); the quantity of RNA present in neuronal perikarua is found to be quite large, varying from 20 pg per cell for small cells, such as the retinal ganglion cells of the rabbit, up to the level of 10,000 pg per cell for the large Mauthner cell of the goldfish (A. Edstrom, 1964). These values correspond to about 5-10x of the cell dry weight. Table VII lists a number of the reported findings for different neuronal cell types. The only other cells that are known to contain comparable quantities of RNA are those of the secretory glands,

190

VICTOR E. SHASHOUA

TABLE VII RNA CONTENT FOR DIFFERENT TYPES OF NEURONS Cell type Retinal ganglion cells (rabbit) Spinal ganglion cells (rabbit) Ganglion cells supraoptic nucleus (rabbit) Hypoglossal cells (rabbit) Anterior horn cells (rabbit) Anterior horn cells (man) Deiters’ cells (rabbit) Lateral vestibular cells (rabbit) Mauthner cell (goldfish)

RNA content (pglcell) 2&100 1,070 70 200 530 670 1,140 700 10,000

Reference

EdstrGm and Eichner (1957) Edstrom and Pigon (1958) Hydln (1959a) H y d h (1959a) Edstrom (1956) HydCn (1959a) Hydln (1959a) HydCn (1959a) A. Edstrom (1964)

such as the cells of the pancreas (Hydtn, 1955). These large amounts of RNA are suggestive of a high metabolic rate for neuronal cells. From studies of the quantity of RNA present in cells of different sizes [i.e., the spinal motor horn cells (Edstrom, 1956), the retinal ganglion cells (Edstrom and Eichner, 1957), and the spinal ganglion cells (Edstrom and Pigon, 1958)], Edstrom suggested that there is a direct relationship between the surface area of a neuron and its RNA content. An observation of this kind is consistent with the notion that a major function of the cell metabolic machinery is to establish and maintain membranes in the neuron. The relationship of glial to neuronal cells has been widely investigated. As early as 1968, HydCn and Egyhizy pointed out the interrelationships of neurons to glia in terms of RNA synthesis during their various functional states. In 1966 Daneholt and Brattgard and Volpe and Giuditta (1970) reported that the rate of synthesis of RNA in glial cells is about twice as high as that in neurons. In another study Flangas and Bowman (1970) used homogenization and fractionation techniques to isolate neuron-enriched and glia-enriched fractions of the nervous tissue. The fractions with a higher content of glial cells were identified by the presence of a 10-fold higher carbonic anhydrase enzyme activity while those with enriched neuronal content had larger cell nuclei. Neuron-rich fractions in this study were found to have twice the rate of incorporation of RNA precursors into macromolecules as glial fractions. In another study, Lsvtrup-Rein and Grahn (1970) measured the incorporation of RNA precursors into neuronal, microglial, and astroglial cell nuclei. This was accomplished by fractionating prelabeled tissue into the nuclei of different cell types, according to the method of LsvtrupRein and McEwen (1966). The RNA associated with neuronal and astrocyte nuclei was found to have the same specific activity, whereas oligodendroglia and microglia cells have a much lower amount of

191

RNA METABOLISM IN THE BRAIN

TABLE VIII COMPARISON OF RNA SYNTHESIS RATES IN NEURONAL AND GLIAL CELLNUCLEI~ Specific activity (cpmlpg RNA)

Cell type Neurons

79 f 2.8

Astrocytes

74 f 2.3

Oligodendrogliaand microglial

19 f 0.2

Ratios Astrocytes 0.94 Neurons Glial 0.24 N x n s

Data from Lovtrup-Rein (1970).

labeled RNA (see Table VIII). The incorporation ratio for astrocytes to neurons was about 1, whereas the corresponding ratio was 1:5 for the amount incorporated into microglia compared to neurons. Such studies clearly emphasize that brain tissue is a complex mixture of many cell types with metabolic rates that can either function independently or can become coupled in certain circumstances. Thus a specific RNA synthesis rate for all types of brain cells cannot be assumed.

B. RNA

IN

AXONS

In recent studies more evidence has been obtained to indicate the presence of RNA in axons. The early work of A. Edstrom (1964) suggested that RNA is present in special cell types, such as the Mauthner neuron of the goldfish. This neuron has its soma in the rostra1 part of the medulla oblongata while its axon spans the whole length of the spinal cord. There are only two such neurons per animal. Edstrom developed the technique of isolating the Mauthner cell by freehand dissection, Analysis of its axonal RNA content after removal of the myelin sheath gave a value for the concentration of RNA in the axon equal to 1/40of the amount present in the soma. Since the volume of the axoplasm of the Mauthner cell axon is much higher than that of the soma, the total quantity of RNA present in the Mauthner axon becomes 5 times as high as that in the soma. Similar studies by Koenig in 1965 show that the RNA concentration in axons of the eleventh nerve of the cat is 1/500 to 1/1000 that of the cell body. Figure I shows a pIot of the RNA content per section of the Mauthner cell axon as a function of the position of the axon in the spinal cord. The quantity of RNA per segment is higher at the proximal end of the axon, i.e., 0.05% ; it then decreases to 0.03% in the middle region and finally increases again to a high level of 0.077, at the caudal tip of the axon. These values are 20 times lower than what is normally found in perikaryon, i.e., 1 % wlv. This type of axonal RNA distribution was considered to arise from a bimodal distribution of the mitochondria in the axon.

192

VICTOR E. SHASHOUA

I

5

10

IS

20

25

30

35

LO

L5

50

mm

FIG.1. The concentration of RNA (w/v) in the Mauthner axon as a function of distance along the spinal cord. Vertical bars indicate the standard deviation. From A. Edstrom (1964).

In 1969, Edstrom ct al. studied the pattern of incorporation of uridine into RNA of the Mauthner cell axon in vitm. Analysis of the sucrose density gradient patterns after a 4-hour labeling time gave a maximum distribution of label in the 45 S RNA and tRNA region together with some ribosomal RNA. After 24 hours the label was largely in the transfer RNA region. Similar types of findings have been reported by Lasek (1972) for the squid giant axon. Again the RNA present seems to be largely in the transfer RNA region of the gradient. From the measurements of the short 4-hour labeling time, rRNA and tKNA seem to be synthesized and present in the axons, but at the 24-hour labeling only tRNA is present (Edstrom et al., 1969). T h e function of this type of RNA in these special types of axons has yet to be identified.

C. RNA TRANSPORT IN AXONS Peterson ct a!. (1 968,1970) reported the formation of RNA and its transport down the sciatic and optic nervesof the chick embryo following an injection of labeled orotic acid into the spinal cord and the posterior chamber of the eye. Autoradiographic studies demonstrated that both the sciatic nerve and the optic nerve contained labeled granules in their axoplasm as well as in the Schwann cell sheath of the axon. Further work on the transport of labeled RNA in optic nerve axons of the chick (Bondy, 1971) using direct isolation of the transported products showed that after a n 18-hour labeling time more

193

RNA METABOLISM IN THE BRAIN

labeled RNA was present in the contralateral (C) optic lobe than in the ipsilateral lobe (I) following an injection of tritium-labeled uridine into the posterior eye chamber. Table IX shows that the C:I ratio increased to a TABLE I X AXONAL TRANSPORT OF LABELED RNA INTO CHICK OPTIC LOBES (L

9

Time after injectionb 1 Hour 6 Hours 18 Hours 3 Days 6 Days

Acid soluble (cpmlmg tissue) CC IC C:I 370 380 1,490 560 -

381 359 980 455 -

0.97 1.05 1.52 1.23

-

RNA bound (cpmlmg R"4) C I C:I 510 1,370 3,720 12,550 20,500

520 1,360 1,840 1,880 2,020

0.98 1.01 2.02 6.68 10.05

Data from Bondy (1971). Injection of 10 pCi [5-3H]uridine into one eye. C = Optic lobe contralateral to injected eye; I = lobe ipsilateral to injected eye. (L

maximum of about 1O:l after 6 days. This suggests a considerably slower rate of transport for RNA than has been reported for proteins in axons (Ochs, 1972). Analysis of the transported RNA showed that it contained ribosomal and tRNA components as well as a component associated with the mitochondrial particulate fraction. More recent studies (Ingoglia et al., 1973) indicate that RNA is also transported in goldfish optic nerve and in the rat nervous system (Ochs, 1972). I n an interesting series of experiments, Amaldi and Ruska (1970) demonstrated the presence of RNA in growing nerve fibers. Dissociated cells from sensory ganglia of 8-day-old chicks were incubated with nerve growth factor in the presence of radioactive uridine as the RNA precursor. It was found that rapid outgrowth occurred and that all the neurites growing during a 6-hour and a 24-hour labeling time were filled with granules that were radioactive and observable by autoradiographic methods. I n separate experiments, nerve growth factor was shown to stimulate only the neuronal growth, not the proliferation of glial processes. When actinomycin D was added to the cultures prior to labeling, no labeled RNA could be found in the processes. The results could not, however, rule out the possibility that the RNA was present in the mitochondria. The observations on the presence of RNA in axons are consistent with the notion that certain specialized cells and neurons that are undergoing rapid growth contain axonal RNA. The results with the goldfish optic nerve (Ingoglia et al., 1973), however, are suggestive of a more general pattern for the distribution of the RNA in axons.

194

VICTOR E. SHASHOUA

D. RNA IN BRAWSUBCSLLULAR FRACTIONS BalAzs and Cocks (1967) studied the incorporation of C14-labeled orotic acid into rat brain and analyzed its distribution in subcellular fractionations. The methods of Whittaker (1959) were used to separate synaptosomes from the nuclear, cytoplasmic, and microsomal fractions. A substantial part of the newly synthesized KNA was found to be present in the synaptosomal fraction. This constituted about 16% of the newly synthesized RNA. Table shows TABLE X

THEDISTRIBUTlON PAITERN OF INCORPORATION OF [l*C]OROTIC ACIDINTO RATBUN SVBCBLLULAR FRACTIONS~

RNA content (’7,) Fraction

Based on CS

Based on S

~~

Crude synaptosomal fraction Microromal (0.3-0.8 M sucrosc layer) Synaptosmal fraction (0.8-1.2 M sucrose) Mitochondria1 fraction (pellet in 1.2 M sucrose) supernatant Synaptic vesicles Microsoma1 Synaptosome ghosts Synaptowme ghosts Damaged synaptmmes Mitochondria a

100 16.4 37.3 46.3

100

1.1 1.2 0.6 1.7

8.3 32 55

Data from BalQzsand Cocks (1967). 5 synaptosomd fraction.

cs = crude synaptosomal; s

the results of one such experiment in which the crude synaptosomal fraction was further separated into purified synaptosomes, mitochondria, and microsomes. About one-third of the RNA present in this fraction was found to be in the pure synaptosomes. Further fractionation of synaptosomesresulted in a separation of synaptosomal membranes from synaptic vesicles and mitochondria, again according to the methods of Whittaker (1959). About 10% of RNA was found to be bound to synaptosomal membranes and 55y0 was associated with the mitochondria; the vesicles and microsomal components contained minor amounts of about 1 yo.The base compositions of the RNA in the subsellular fractions are shown in Table XI. Only the nuclear RNA had a high U/C ratio of 0.9 indicating a “DNA-like” base composition.The analytical data for the other fractions suggest the presence of largely ribosomal and transfer RNA components due to a low U/C ratio of about 0.7 (Shashoua, 1971).

195

RNA METABOLISM IN THE BRAIN

TABLE XI BASECOMPOSITION OF RNA I N SUBCELLULAR FRACTIONS OF RATBRAIN' ~

AMP Fraction Homogenate Crude nuclear Mitochondria1 Microsoma 1 Supernatant Purified nuclear

+ GMP

CMP

AMP

GMP

UMP

CMP+UMP

UMP/CMP

29.3 25.3 27.8 29.7 29.8 24.8

20.7 22.0 21.1 20.8 20.4 25.0

30.8 30.3 31.3 29.6 30.3 25.6

19.0 22.7 19.9 19.8 19.6 24.8

1.06 1.09 1.10 1.02 1.03 0.95

0.66 0.90 0.72 0.66 0.66 1.o ~~

a

Data from BalPzs and Cocks (1967).

Similar studies of the distribution of RNA in subcellular fractions of goldfish brain (Shashoua, 1973a) showed the presence of a substantial amount of the newly synthesized RNA in the synaptosomal fraction. Table XI1 shows a time course of the pattern of labeling with uridine. The amount of RNA in the crude synaptosomal fraction was essentially constant at about 16-18X after 1 hour and at 24-hour labeling times, whereas the nuclear RNA component started at 73y0of the total incorporated in 1 hour and decreased to around a level of 50% after 24 hours. During the same period the cytoplasmic fraction increased from 11% to a high of 40y0 in 3 hours and then gradually decreased to 31 in 24 hours (see Table XII). Further fractionation of the crude synaptosomal fraction showed very little RNA to be present in the microsomal or myelin fraction, less than 1%, whereas the purified synaptosomal fraction contained 5% of the total label and the mitochondria1 fraction contained 12.2%. The presence of RNA in the synaptosomal fraction does not distinguish whether the RNA is in axonal or dendritic elements of the synaptosomes. TABLE XI1 RNA DISTRIBUTION IN SUBCELLULAR FRACTIONS OF GOLDFISH BRAINa Labeling timeb ~

Fraction Nuclear Cytoplasmic Crude synaptosomal Myelin and microsomal Purified synaptosomal Mi tochondrial yo Recovery a

Data from Shashoua (1973a) Label: [5-3H]uridine.

1 Hour

3 Hours

4 Hours

24 Hours

73 11 16

42.4 39.7 17.9

- -

-

51 31 10 -

<

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46.8 35.5 17.7 -

4.5 12.2 104

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FIG. 2. Sucrose density gradient sedimentation patterns for goldfish brain WNA, showing the incorporation of [5-3H]uridine as a func, in tion of time into cytoplasmic (C), synaptmomal (S), and nuclear (N) RNA at 1.5 hours, 3 hours, and 21 hours; - - - - -UJ4C trichloroacetic acid precipitate, , absorbance at 260 nm. From Shashoua (1973a).

RNA METABOLISM IN THE BRAIN

197

Figure 2 shows sucrose gradient patterns as a function of labeling time for the various classes of RNA molecules present in subcellular fraction of goldfish brain (Shashoua, 1973a). The optical density profiles of the nuclear and synaptosomal fractions have optical density ratios of 0.9 to 1.0 for the 18 S to 28 S components. The labeling profile of the synaptosomal fraction, however, resembles data for the cytoplasmic component. I n all three fractions the classes of labeled RNA present after a 24-hour labeling time are the ribosomal and tRNA components sedimenting at 28 S, 18 S, and 4 S. The optical density profile of cytoplasmic fraction shows the 2:l ratio for 28 S and 18 S ribosomal RNA's. The RNA associated with the synaptosomal fraction appears to be quite sensitive to changes of patterns of behavioral inputs (Shashoua, 1974b). IV. RNA Changes in Single Cells Produced by Physiological and Electrical Stimulation

Studies of the pattern of RNA synthesis in single cells offer, in principle, a good method for determining the effects of stimulation without the complicating factors of a whole nervous system. I n 1950, Liu et al. found that antidromic stimulation of motor neurons resulted in no RNA changes. Similar effects were observed when the crustacean stretch receptors were activated by a repeated muscle stretch (Grampp and Edstrom, 1963) or when the cells of the dorsal root ganglia were activated by electrical stimulation of their own axons (Liu et al., 1950). I n 1965, Jakoubek and Edstrom ' used a more physiological method for neural stimulation. Goldfish were rotated at 60 rpm for 90 minutes to induce lateral line inputs to the vestibular nuclei and the Mauthner neuron. This method essentially drives the Mauthner cell to exhaustion, and the RNA content of the cell decreases by about 40%. Moreover, the recovery to its original FNA content requires 3 hours after stimulation is discontinued. I n more recent experiments (Berry, I969 ; Peterson and Kernell, 1970), the incorporation of RNA precursors into the R2 cell of the abdominal ganglion of Aplysia californica was studied. The effects of electrical stimulation via one of the input axons was compared to that of electrical stimuli delivered at the soma, where there are no synapses. Figure 3 shows a histogram of the relative amounts of RNA label incorporated into the stimulated cell in experimental ganglia as compared to controls. Only synaptic stimulation via one of the axons of the connectives produced an increase of RNA synthesis-about %fold. This increase occurred even though fewer spikes were delivered to the cells via the axon than when stimulation was directly at the soma, i.e., nonsynaptic. I n addition, the amount of incorporation was found to be directly proportional to the number of synaptic stimuli. The

198

VICTOR E. SHASHOUA

I

31

stim

sllm.

sttrn.

No

Oir.

Syn.

stim.

stim.

stim

FIG. 3. RNA labeling pattern of the R2 neuron of Ajlysia califmica with [5-3H]uridine. Panel A shows ratios of incorporation of label into RNA for stimulated (E) to control neurons (C). The number of spikes delivered in each experimental group is shown in B, Direct stimulation (Dir. Stim.) was via intracrllular electrodes (n = 6); synaptic stimulation (Syn. Stim.) delivered via stimulation of ganglionic nerves (n = 4). NO stimulation (NoStim.) n = 5. Vertical b a n give thc standard error of the mean values. From Kernell and Peterson (1970).

results, however, do not distinguish between possibility of differential breakdown rates of the RNA and increases in the precursor pools (Kernell and Peterson, 1970). In more recent studies, Berry and Cohen (1972) demonstrated that the R2 cell of the abdominal ganglion of Aplysia incorporated 67y0more uridine into RNA after 3000 impulses were delivered to the cell via synaptic inputs. The amount incorporated was proportional to the number of stimuli, and no increased uridine uptake occurred; thus the RNA changes could not be due to a pool-labeling phenomenon. Autoradiographic methods showed that the stimulation effects produced largely a neuronal change; the incorporation into glial cells could not account for the data.

RNA METABOLISM IN THE BRAIN

199

The RNA studies in single cells present two conflicting findings: (1) The data from the exhaustive physiological stimulation of the Mauthner neuron give a decrease in RNA content (Jakoubeck and Edstrom, 1965). (2) The synaptic stimulation data of Berry (1969) and Kernel1 and Peterson (1970) indicate increased RNA synthesis. This conflicting evidence can be resolved if we consider the two aspects of RNA synthesis and RNA content as separate phenomena. Thus, stimulation initiates a higher rate of synthesis in response to an increased demand arising from an increased use. In exhaustive stimulation the higher synthetic rate cannot keep up with the rate of use, so that the total content decreases. The fact that it takes 3 hours after physiological stimulation before the rate of synthesis is able to restore the cell to its initial RNA content supports this notion. Thus, during stimulation it is possible in principle to have a higher rate of synthesis with an increased amount of label incorporated together with a net decrease in the RNA content. Such a concept is supported by the findings of Gisiger (1971), who measured the specific activity of RNA after synaptic stimulation of the superior cervical ganglion of the rat at 32/sec. There was an initial decrease of 28y0 after 1 hour, but after 3 hours there was a net increase of about 30%. Thus, an initial depletion may have triggered the shift of the metabolism to a higher level. V. RNA Changes in the Brain during Development

The studies of brain metabolism during development offer the possibility of detecting critical phases that require specific proteins for restructuring and organizing the nervous system. For example, at the onset of myelination there is a sudden demand for the basic protein (Shashoua and Wolf, 1971) and 'for the proteolipid protein; such a change should be preceded by an abrupt synthesis of the messenger RNA molecules coding for these proteins. Similar requirements may also occur during embryogenesis,as in the process of initiation of axonal transport, which occurs in the chick brain embryo abruptly at age 13 days (Bondy and Madsen, 1971). The information presently available in the literature does not yet allow us to confirm such hypotheses. However, there are experimental data that show changes in the brain RNA content and in its synthesis rate as a function of age. OF BRAIN RNA CONTENT WITH AGE CORRELATION

Dellweg et al. (1968) measured the changes in the amount of ribosomal RNA and transfer RNA in the developing rat brain (see Table XIII) for animals 29 days up to 146 days old. During this period the weight of rat brain increases from 1.4 gm to 2 gm while the body weight increases by about a factor of 10. The amount of transfer RNA and ribosomal RNA in the brain

200

VICl'OR E. SHASHOUA

.4MOUNT OP

Age (days)

Body weight (gm)

TABLE XI11 RIBOSOMAL AND tRNA IN EVELO LOPING RAT BRAIN'.^ Brain weight (gm)

rRNA &/brain

tRNA

p G / g wet wt. FG/brain pC/g wet wt.

. _ ~

29 34 37 41 46 50 54 62 68 73

78 124 146 a

61 78 95 118 141 164 195 244 26 1 290 312 416 512

1.4 1.6 1.9 1.9 1.9 1.9 2.o 2.0 2.0

2.0 2.0 2.0 2.0

988 1420 1532 1580 1621 1638 1646 1652 1649 1656 1644 160.5 1586

705 887 806 831 854 862 823 826 825 828 822 803 793

192 276 294 290 310 305 319 320 315 310

318 309 303

137 172 154 152 163 I60 150 160 158 155 159 154 151

Data from Dellweg ct 01. (1968). Average ratio of rRNA/tRNA = 5.22.

increases by 60%. As shown in Table X I I I , if the results of brain RNA content are expressed as micrograms of RNA per gram wet weight of brain, then a maximum increase of about 25y0 occurs between ages 29 and 34 days. After 34 days the ratio of RNA to brain weight and the ratio of ribosomal RNA to transfer RNA become and remain constant. T h e increase in the quantity of brain RNA after age 34 days merely reflects an increase in the weight of the tissue with age. I n younger animals, however, Sung (1969) found that large variations in the rate of DNA and RNA synthesis can occur in the developing rat brain. In these experiments the rate of incorporation of radioactive label into RNA and DNA precursors was studied in brain tissue slices from cortex and cerebellum (see Table XIV). From age 2 days up to 10 the quantity of RNA synthesized per milligram DNA decreases quite markedly. There is a 50y0 decrease by 10 days in the cerebellum. I n the cortex, however, the quantity of RNA synthesis increases by about 25%. If the results are expressed as micrograms per gram per tissue, then the decrease is less pronounced in the cerebellum and the cortex shows a marked decrease (- 50%) instead of the 25% increase relative to DNA content. These findings are clearly related to the proliferation rates of the tissue. In another study with brain tissue slices, Guroff ct al. (1971) reported the incorporation of labeled RIVA precursors into tissue slices from cerebellum, cortex, and diencephalon regions of rat brain. Figure 4 shows a 3-fold decrease in the amount of uridine incorporated per gram wet weight

201

RNA METABOLISM IN THE BRAIN

DNA Age (days) 2 6 10 2 6 10

AND

TABLE XIV RNA SYNTHESIS IN DEVELOPING RATBRAIN‘

DNA synthesisb (patom 14C incorporated per hour) Per gm tissue

RNA synthesisb (patom 14C incorporated per hour)

Brain

Per mg DNA

Per gm tissue

Per mg DNA

region

2.94 2.76 1.51 0.45 0.49 0.3 1

28.2 25.6 21.2 13.4 11.2 7.9

4.55 3.32 2.05 4.16 5.25 5.34

Cerebellum Cerebellum Cerebellum Cortex Cortex Cortex

16.9 22.5 14.7 1.35 1.13 0.44

,

Data from Sung (1969).

* DNA synthesis-incorporation

of [2-14C]thymidine in tissue slices. RNA synthesis-incorporation of [2-14C]uridine in tissue slices.

of tissue with age. The cerebellum slices showed a much higher level of incorporation than either the cortex or the diencephafon slices. Guroff et al. suggested that these changes in the incorporation of nucleotides into brain tissue with age are related to the production of uridine nucleosidase in the tissue. It is seen that there is an inverse relationship of the quantity of nucleosidase activity with age (see Fig. 4). This finding, however, does not

1

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u’ 5 W

3 a za

- 0.5 0

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20

3

120

FIG. 4. The pattern of incorporation of [5-3H]uridine into rat brain cortical slices The slices were incubated in Kreb’s ringer as a function of age of the animal(.-.). + glucose for 20 minutes. The nucleosidase activity (0-0) was also measured as the amount of uracil formed per milligram of protein after a 2-hour incubation at 37°C. From Guroff et al. (1968).

202

VICTOR E. SHASHOUA

explain the observations of Bondy and Roberts (1969)) who demonstrated that nuclear chromatin derived from neonatal and fetal brains has a much higher rate for the cell-free synthesis of RNA. I n these experiments the quantities of the RNA precursors are controlled, yet the neonatal and fetal chromatin has RNA synthesis rates 60% greater than does nuclear chromatin from adult brains. Moreover, the quantity of label incorporated seems to be dependent on the region of origin of the tissue (see Table XV). The cereTABLE XV CHEMICAL COMPOSITION AND TEMPLATE ACTIVITYOF RATBRAINCHROMATIN^ Mass ratio relative to DNA

Fetal (5 days prenatal) Brain Liver Newborn Whole brain Cerebral cortex Cerebellum Thalamus-hindbrain Liver 14-Day-old Cerebral cortex Cerebellum Thalamus-hypothalamus Hindbrain-medulla Adults (6 weeks) Whole brain Cerebral cortex Cerebellum Thalamus-hypothalamus Hind brain-medulla Liver

RNA

Histone protein

Nonhistone protein

UMP incorporation"

0.07 0.05

0.85 0.76

0.8 1 0.57

1030 847

0.08 0.07 0.08 0.08

1.08 1.10 1.12 1.14

1.46 1.43 1.32 1.36

905 890 872 926 770

0.06 0.06 0.07 0.08

1.11 0.98 1.05 1.36

1.25 0.95 1.28 2.0

748 926 653 660

1.oo

0.98 1.15 0.90 1.25 2.31 0.79

680 545 872 552 578 695

0.08 0.105 0.08 0.18 0.07

0.98 0.94 1.10 1.03 0.97

-

~~

a

Data from Bondy and Roberts (1969). Picomoles incorporated after a 40-minute incubation at 37°C with 1.2 pg chromatin.

bellum, which matures at a slower rate than the rest of the rat brain, has a much higher rate of incorporation in the cell-free systems than does either the whole brain, cerebral cortex, thalamus, or hindbrain, even in the 6-weekold adult brain chromatin. One explanation for this type of observation is that nuclear chromatin from fetal and neonatal tissue has more regions of its DNA derepressed and ready for transcription.

203

RNA METABOLISM IN THE BRAIN

Another suggestion for the increased rate of RNA synthesis in neonatal brain has been put forward by Caldarera et al. (1969), who reported that spermine and spermidine are found at higher concentrations in actively proliferating tissues. Thus, the concentration of these molecules might control the rate of RNA synthesis during embryogenesis. Table XVI shows the TABLE XVI CONCENTRATIONS OF SPERMINE AND SPERMIDINE IN FETALBRAINAS A FUNCTION OF AGE^ Embryo age (day4

Spermine (pmoles/gm dry wt)

Spermidine pmoleslgm dry wt)

6 8 10 12 14 16 18 20

1.97 2.22 3.05 3.41 4.38 1.96 1.83 2.32

1 .oo 1.39 1.65

2.01 1.51 0.82 0.57 0.72

~

~

a

Data from Caldarera et al. (1969).

concentrations ofspermine and spermidine in chick fetal brain. I t is found that at an embryo age of 12-14 days the level of spermine and spermidine is at a maximum. I t decreases by 50% a t the hatching period of the embryo. This is a very interesting suggestion and requires additional confirmation. Johnson (1967, 1968) used brain cell suspensions (obtained by gentle homogenization of mouse brain tissue at room temperature) to study the rates of RNA synthesis. The incorporation of radioactive precursors (orthophosphate 32P)into ribonuclease hydrolyzable products was linear up to 60 minutes. The quantity of label incorporated per milligram of protein was found to decrease with the age of the tissue from which the cells were derived, i.e., from neonatal down to 10-day-old animals. The tissue extracts were found to be equally active in cell-free synthesis of proteins. This finding rules out the possibility that neonatal tissue may be less subject to damage in the homogenization step than adult cells. Yamagami and Mori (1970) isolated polysomes from rat brain of newborn up to 90-day-old rats. The quantity of polysomal RNA was found to increase with age, reflecting the increased weight of the brain. The incorporation of phenylalanine into protein, however, showed a maximum a t age 10 days and decreased by 50% to a steady level after age 30 days, indicating that there may be other controlling factors besides the quantity of polysomes present in the tissue.

204

VICTOR E. SHASHOUA

VI. Types of Brain RNA

A. RNA TURNOVER RATES Brain cells contain all the known types of RNA. The most abundant class of RNA molecules are the ribosomal RNA’s. The 28 S and 18 S components

in rat brain (Dawson, 1967) and goldfish brain (Shashoua, 1973a) are present in ratio of 2 to 1. The half-life of these two RNA molecules was determined as 12.5 days (Bondy, 1966) and 13-15 days (Kahn and Wilson, 1965). Dawson (1 967) reported a half-life of 6 days, which is similar to that for liver ribosomal RNA, i.e., 5-7.2 days (Loeb et al., 1965). The half-life of messenger RNA is estimated as 3 hours (Appel, 1967). From such measurements, Dutton et al. (1969) calculated that the rate of messenger RNA synthesis was 3 times faster than ribosomal RNA. Nuclear heterodisperse and cytoplasmic heterodisperse RNA are present in brain and have fast turnover rates (Dutton et al., 1969; Vesco and Giuditta, 1967).

B. NEURONAL AND GLIALRNA METABOLISM Several studies have attempted to distinguish between the rates of RNA synthesis in glial and neuronal cells. Daneholt and Brattgard (1966) found that glia synthesized RNA twice as fast as neurons. This, however, could not be observed in isolated nuclei from rat brain. Lwtrop-Rein (1970) separated nuclei according to their size, using centrifugation methods (Lsvtrop-Rein and McEwen, 1966), and measured the specific activity of the RNA in nuclei from neurons, astroglial cells, oligondendroglia, and microglia. Table VIII shows the results of such measurements for rat brains labeled in vivo prior to the isolation of the nuclei. The specific activity of the RNA in neurons was the same as for astroglial nuclei, whereas microglia and oligodendroglia had one fifth the activity of neurons. These results suggest that the pattern of incorporation of RNA precursors into differelit brain cell types can bc quite complex. Clearly the rate of uptake of label and the possibility of different metabolic pool sizes for different cell types must also be considered.

C . PATTERNOF RNA SYNTHESIS The pattern of transformation of large RNA macromolecules, synthesized in the nucleus, into ribosomal and mRNA has not been extensively studied in brain tissue. Gel electrophoretic analysis of the products of incorporation of orotic acid and 32P-labeled orthophosphate into rat brain nuclear RNA showed the presence of a 45 S and 3 1 S RISA peak (Tencheva and Hadjiolov, 1969). This is similar to the reported findings for other cell types (Perry, 1967). Additional RNA peaks corresponding to 24 S, 22 S , 14 S, 9 S, and 6 S

205

RNA METABOLISM IN THE BRAIN

were also detected by autoradiography of the electrophoretic separations of the RNA on agar gels. The 28 S and 18 S ribosomal RNA were present in a ratio of 2 :1 in 1he cytoplasmic RNA and 1 :1 in the nuclear RNA-components.

D. BASECOMPQSITIONS Base composition measurements have been reported for brain RNA from a variety of species (see Table V). Nuclear RNA has a base composition more similar to DNA than to ribosomal or transfer RNA. Zomzely et al. (1969) isolated the RNA from the nuclear, cytoplasmic, polysomal, and ribosomal fractions of rat brain. The base compositions of these molecules were determined (see Table VI). An analysis of the results shows that the ratio of the uridine to cytidine is about twice as high for the nuclear and for the polysomal RNA components as for the ribosomal RNA. The U/C ratio is a useful index foqdetermining large shifts in the pattern of RNA synthesis (Shashoua, 1971). Figure 5 shows the application of the method to study of the pattern of RNA synthesis in goldfish brain, using orotic acid as the labeled RNA precursor. A plot of the ratio of U/C incorporated into the RNA relative to the U/C found in the metabolic pool gives a high value a t short labeling times of 1 hour when the product of new RNA synthesis is largely nuclear heterodisperse RNA. This is followed by a decrease after 4 hours to a level which remains constant for 24 hours when the labeled RNA is largely 28 S and 18 S ribosomal RNA.

0

.

8

8

FIG. 5. The base composition of goldfish brain RNA as a function of labeling time. Labeled orotic a~id--5-~H was injected intracerebrally into goldfish, and the content of U and C label in the RNA was determined after various labeling times. The composition ratio reflects of the metabolic pool was also determined. The high (U/C)RNA/(U/C)pool the greatest amount of nuclear heterodisperse RNA present at short labeling times. The indicates that after 4 hours ribosomal RNA consticonstant ratio of (U/C)RN~/(U/C)pool tutes the major quantity of the RNA synthesized. From Shashoua, V. E. (unpublished data, 1972).

206

VICTOR E. SHASHOUA

E. METABOLIC POOLS Cain (1967) studied the uptake of RNA precursors into guinea pig cerebral cortex and observed a number of specific types of precursor-pool relationships. The uptake of uridine and UMP from the incubation medium by brain slices was 2.5 times higher than uracil or orotic acid, and these in turn were higher, by a factor of 2, than adenine. The incorporation of uridine and UMP from the pool into RNA were also found to be higher, by a factor of 10, than uracil and adenine. Addition of actinomycin D to the medium inhibited RNA synthesis, but only a small amount of the newly synthesized RNA was rapidly degraded in its presence.

F. SPECIAL CLASSES OF RNA MOLECULES Mandel et a/. (1967) demonstrated the synthesis of polycytidylic acid, poly(c), from CTP in the presence of a particulate fraction isolated from cell nuclei of rat brain tissue. The enzyme present in this fraction was previously described in thymus cell nuclei (Hurwitz et al., 1959). The enzyme activity was independent of the addition of actinomycin D, DNA, DNase, poly(C) , and inorganic orthophosphate, but it was inhibited by inorganic pyrophosphates and ammonium sulfate. In a more recent study, Ilravid et al. (1971) demonstrated the presence of a polyadenylic acid, poly(A) synthesizing enzyme in extracts of rat brain nuclei. This is similar to the enzyme reported by Edmonds and Abrahms (1963). A similar enzyme has also been found in the nonnuclear particulate fraction of rat brain (Gurgo et ul., 1970). The enzyme activity was not dependent on DNA. Poly(A) was capable of priming the incorporation of ATP into polyadenylate. GTP, CTP, and U T P inhibited the poly(A) synthesizing enzyme. The significance and function of these polynucleotides forming enzymes in brain is not yet clear. The possibility that poly(A) and poly(C), may be the storage forms for the nucleotides was suggested by Mandel et al. (1967). There has been no adequate demonstration, however, that these enzymes, obtained from nuclear tissue extracts, produce the same polymers in uiuo. The fact that the in uitro synthetic activity is inhibited by G T P and U T P suggests that other factors may be involved in its in uiuo function, since these nucleotide triphosphates are normally present in the metabolic pools within the cells. VII. Brain Function and R N A Metabolism

A. GENERAL STIMULATION STUDIES ON RNA METABOLISM The pattern of RNA changes in the brain during stimulation has been reviewed by Hyden (1967) and more recently by Jakoubek and Semiginovsky (1970). An analysis of the published data shows that mild stimulation of the

TABLE XVII GENERAL STIMULATION AND RNA CHANGES Stimulus Motor activity Running to exhaustion Cyclic rotation Vestibular stimulation (constant rotation, 90 minutes) Constant swimming, 5 hours Constant swimming, 4 hours

Animal

Label

Locus

RNA changes

References

Guinea pig Rat Goldfish

None None None

Spinal motor neurons Deiters’ cell vestibular N. Mauthner cell axon

Barracuda Goldfish

None Orotic acid

Spinal motor neurons Total brain

-

Hyden (1943) Hyden (1943) Jakoubek and - MY0 Edstrom (1965) +20% Hyden (1964) No base compo- Shashoua (1971)

+ 5 to 20y0

sition change Visual Intense light 10-Minute exposure of dark adapted

Frog Rats

Cytidine

-

Retinal rods

-

- 50%

+ 30 to 50y0on

Aplesia

Uridine

R2 Cell abdominal gang

+67y0

E

0

Ei?

2 4

Bok (1970) Appel (1967)

large polysomes Electrical 3000 Impulses through axonal inputs

w

F

8

I!

1:

Berry and Cohen (1972)

10 0

208

VICTOR E. SHASHOUA

nervous system causes an increase in RNA synthesis whereas intense stimulation to exhaustion results in a marked decrease in the content of RNA in the brain (see Table XVII). All types of sensory inputs can result in a slight increase in the RKA synthesis of the specific neuronal groups associated with the modality of the stimulated system. Thus, swimming causes an increase in RNA content (Hyden, 1964) of barracuda motor horn cells. Exposure of dark-adapted rats to even a brief 10-minute pulse of light causes an increase in the amount of amino acid incorporation onto polysomes in rat brain (Appel, 1967) ; thus, one would assume that the similar increase of high molecular weight RNA might be observed in the polysomes. Mild acoustic stimulation seems to present no definitive RNA changes whereas the stimulation of the vestibular system of rats by continuously rotating the animals causes an increase of the amount of RKA in the Deiters’ cell neurons by 5-25x (Hydkn and Egyhazi, 1962). T h e data for the effects of intense stimulation, however, give an entirely different type of a result. Intense motor activity such as running to exhaustion on a treadmill by guinea pigs was shown by Hydtn to give a decrease in the RNA content of spinal motor neurons. Similarly, Jakoubek and Edstrom (1965) showed that intense vestibular stimulation of goldfish caused a decrease of up to 407, in the RNA content of the Mauthner neurons. This decrease occurred in 90 minutes and required about 180 minutes for recovery to initial level. Intense visual stimulation of frog visual system resulted in a 50% decrease in the RNA content of material previously incorporated into retinal rods (Bok, 1970). The RNA changes obtained under mild stimulation conditions showed no base composition changes (Hyden, 1967). Similarly, in goldfish the incorporation of orotic acid into brain RNA during swimming in a whirlpool showed no base composition changes (Shashoua, 1971). I n addition, there was no increased formation of specific classes of RNA molecules in studies of the incorporation of uridine into RNA (Shashoua, 1974b). These findings suggest that mild stimulation which results in an increased use of the nervous system for a specific sensory modality can cause a shift of the rate of RNA synthesis to a higher level. This occurs for all types of RNA without increase in any specific molecules. However, when the system is driven to its exhaustive capacity, even the higher synthetic rate cannot keep up with the degradative processes, so that the net content of the brain RNA is decreased. AND PHYSICAL EFFECTSON RNA B. NONLEARNING BEHAVIORAL METABOLISM

Table X V I I I summarizes some of the observations reported on alterations in RNA metabolism induced by behavioral and physiological manipulations of the nervous system. Merritt and Sulkowski (1970) demonstrated that a circadian rhythm exists in rat brain, pineal, and heart tissue in terms of their

TABLE XVIII NONLEARKING BEHAVIORAL AND PHYSICAL EFFECTSON RNA METABOLISM Behavior

Animal

Label

Sleep vs wakefulness

Rabbits

Orotic acid

Circadian rhythm

Rat

KCI-induced convulsions Hibernation (cold induced) Cold-induced Epileptogenic foci

Goldfish Snail

Orotic acid

Rabbit

Uridine

Stress (passive response) Hypophysectomy

Goldfish Rat

Orotic acid Uridine

Axonal regeneration (after crush) Neuronal requirements (after axonal transection) Optic nerve transection

Rat and cat

Locus

RNA changes

Reference

+ In sleep for 28 to 50 S polysome region RNA Polymerase + 44y0 a t

Cortex

Mice

Uridine

Vitale-Neugebauer ct 01. (1970) Brain, pineal Merritt and heart midnight Sulkowski (1970) No base composition change Brain Shashoua (1971) Neurons in visceral - 70070 Holmgren and ganglion Holmgren (1 968) Cortex Decrease RNA in neuronal nuclei Engel and Morrell + increase in cytoplasm (auto- (1970) radiographic analysis) Brain No change in base composition Shashoua (1970a) liver Decrease in high molecular Brainstem Gispen ct al. (1970) weight RNA in polysomes Oderfield-Nowak and Sciatic nerve Increase due to Schwann cell proliferation Niemierko (1969) Hypoglossal neuron + 757’ after 3 days Haddad ct af. (1969)

Chicks

Uridine

Optic lobes

+

+ U p to 17 days

Bondy and Margolis (1970)

z

9

E

4

%

E 5

3

B

210

VICTQR E. SHASHOUA

RNA polymerase activities : a #yo increase was found during the periods of darkness (maximum at midnight) when these nocturnal animals are more active, as compared to the daylight level. Vitale-Neugebaur el al. (1970) found that cortex of rabbit brain contained more polysomal RNA in sleeping than in awake animals. Holmgren and Holmgren (1968) found a 7-fold decrease in the RNA content of neurons of the visceral ganglia ofsnails following a coldinduced hibernation of the animals. Engle and Morrell (1 970) observed the incorporation of uridine in the mirror-focus neurons and in epileptogenic regions of rat brain cortex induced by freezing. The results showed a decrease in the labeled RNA present in the neurons of the mirror focus with no change in the RNA content of the epileptogenic regions as compared to nontreated control cortex. T h e observations were carried out by autoradiographic analysis. Hypophysectomy (Gispen et al. 1970) results in a decrease in the incorporation of uridine into high molecular weight RNA in polysomes obtained from brainstem and liver tissue. A number of studies have been reported on the changes in RNA metabolism during the regeneration of nervous tissue. Oderfield-Nowak and Niemierko (1 969) showed that increased RNA synthesis occurred in rat and cat sciatic nerve after the axons were crushed. The RNA changes were found to be due to an increase in Schwann cell proliferation. A study of neuronal regeneration following axonal transection in mice (Haddad et al., 1969) indicated that the hypoglossal neurons incorporated up to 75% more uridine into RNA 3 days after the transection. Other studies by Bondy and Margolis ( I 970) in chick brain after transection of the optic nerve showed that increased RNA synthesis occurs in the optic lobes up to 17 days after the cut. The RNA changes induced by nonlearning situations were found to give RNA molecules with the same type of base composition as are generally found for the ribosomal RNA (Hyden, 1967). Similarly, potassium chloride-induced convulsions in goldfish brain and general stress situations gave no RNA base composition changes (Shashoua, 1971). Moreover, analysis of the newly synthesized RNA by sedimentation studies in sucrose density gradients gave patterns which showed no increase in specific molecules for any of the regions of the gradient (Shashoua, 1 9 7 3 ~ ) .These results indicate that various behavioral and functionally induced alterations of RNA metabolism, which essentially involve no learning, can cause an increase in RNA synthesis without producing any shifts in the pattern of RNA synthesis or increases in specific RNA molecules. Thus, general stimulation can essentially shift the metabolic system to a higher turnover rate.

C. RNA CHANGES AND LEARNING A number of investigations have suggested a relationship between brain RNA and protein metabolism and the process of the acquisition and storage

RNA METABOLISM IN THE BRAIN

21 1

of new patterns of behavior (see reviews by Hyden, 1967; Glassman, 1969). The findings include observations of (1) an increase in RNA synthesis during the acquisition of a new behaviour (Zemp et al., 1966); (2) the formation of RNA molecules with altered base compositions during training (Hydtn and EgyhPzy, 1962; Shashoua, 1968, 1970); and (3) the requirement for brain protein synthesis during the consolidational step of memory formation (Flexner, et al., 1962; Barondes and Cohen, 1967; and Agranoff et al., 1967a). Table XIX lists the principal types of experimental results obtained for the various training situations and animals employed. TABLE XIX RNA CHANGES DURING TRAINING Training experiment

Animal

Label

Reversal of handedness Rat (in feeding)

None

Y-maze thirst quenching Shock-avoidance jump box Float training

Rat

Uridine

Mouse

Uridine

Goldfish

Float training

Goldfish

Orotic acid Uridine

Imprinting to flashing light

Chick

Uridine

RNA change

Reference

Base composition shift toward mRNA in specific cells Increase in hippocampus Increase in diencephallon Base composition shift toward mRNA Increase in specific RNA molecules Increased synthesis

HydCn and Egyhhzy (1962) Bowman and Kottler (1970) Zemp et al. (1966) Shashoua (1968, 1970a, 1970b). Shashoua (1970b, 1974b) Horn et al. (1971)

The studies in rat brain (HydCn and Egyhizy, 1962) showed that a change in the pattern of RNA synthesis occurred in specific neurons when the right-handed animals were trained to eat with the left hand and vice versa. The RNA from the trained animal brains had a base composition with a higher U/C ratio, indicating that more messenger RNA and nuclear heterodisperse RNA was being formed. These RNA changes occurred during the process of training and could not be found in cortical regions not involved in handedness functions. In another rat experiment, Bowman and Kottler (1970) studied the behaviour of thirsty rats in a Y-maze training situation. The animals were first trained to go to one arm of the Y-maze in order to obtain water and then retrained to go to the opposite arm. This eliminated many of the aspects of novelty and dominant handedness of the animals. During reversal learning the uridine label was introduced intravenously into the animal, and a large increase in the label incorporated into RNA was found for the hippocampus compared to other brain regions. When the label was delivered intracerebrally, however, there was no increased RNA

212

VICTOR E. IHBSHOUA

synthesis. Of the number of possible explanations for these changes, the most interesting and attractive is one suggesting that a n increase in the blood flow into a specific brain region can occur to deliver more label to the hippocampus. The intracranial method should thus be insensitive to the blood-flow changes since direct uptake from the cerebrospinal fluid may occur to give uniform labeling. In a series of experiments Zemp at al. (1968, 1969) found an increased uridine incorporation into RNA in mice brains during a shock-avoidance situation. The animals were trained to avoid a shock following a lightconditioning stimulus by jumping on to a safe shelf in a jump box apparatus. The increased RNA was quite large, ranging from 20’7, to 80%, and occurred very rapidly during the initial phases of the training. This increased RNA synthesis was related to the training component of the behavioral task and was not produced in control animals that received the same number of shocks. Other organs, such as liver, showed no increase in RNA synthesis, indicating that the effects observed were specific to the brain. The RNA synthesis was measured relative to the UMP in the metabolic pool in a double-labeling experiment. In more recent findings, however, Entingh et al. (1972) noted that the UMP of the metabolic pool also changes during the learning, to bias the labeling pattern of the RNA in favour of increased label incorporation. Thus a large amplification of the RNA changes occurs to give more readily detectable data. The actual RNA increase, however, is probably smaller than the initial 20%-8097, inckeases observed. In other studies (Adair et al., 1968a,b) used double-labeling methods to demonstrate a general increase in the RNA associated with the polysomes in the shockavoidance experiment. No specific peaks were observed in the labeling of the RNA in the polysome pattern, suggesting that no specific mRNA is formed. In additional studies, autoradiographic methods were used to demonstrate that the RNA changes produced in the shock avoidance experiments were localized principally in the diencephallon of rat brain. In a series of experiments with goldfish as the experimental animal, a change in the pattern of RNA synthesis was found in the brains of the trained animals (Shashoua, 1968). The behavioral experiment was chosen to present a maximum challenge to the nervous system. The goldfish were trained to swim with a float, equal to their buoyancy, sutured to their ventral side at a position 1-2 mm caudal to their first pair of lateral fins. The animals are initially in an upside-down position (see Fig. 6). I n a 4-hour training period, they achieve the skill of swimming upright in the presence of the float. The task is difficult to master, requiring changes in both motor and perceptual skills. Three types of biochemical experiments were used to detect changes in the pattern of brain RNA synthesis. I n the early studies (Shashoua, 1968), labeled orotic acid was injected intracerebrally at the

RNA METABOLISM IN THE BRAIN

213

I

4

t

t

- g-Q+

-

I11

FIG. 6. The sequence of learning of goldfish in the float-training task. Stage I, initial phase 0% score; stage 11, intermediate phase (50’7, score) achieved after 1 hour; stage 111, final phase achieved after 4 hours. From Shashoua (1968).

onset of the training to label the UTP and CTP, RNA precursors in the brain. The labeled RNA produced was found to have a base composition with a 20-8007, higher ratio ofthe uridine to cytidine as compared to controls. No such changes were observed in the kidneys nor were they observed during nonlearning situations, such as intense swimming in a whirlpool during convulsion induced by KCl administration, and during stress situations where the animals were challenged to learn an impossible task. The impossible task involved using a float equivalent to over 10 times the animals’ buoyancy and the response was to remain in an upside-down posture throughout the labeling period. The base composition changes suggested that a shift in the pattern of RNA synthesis had occurred t o produce RNA with more mRNA and nuclear heterodisperse RNA, with no increase in rRNA. Measurements of the metabolic pool showed that no gross pool changes occur during the learning. However, recent studies (Baskin et al., 1972) found that the brain CO, content could bias the metabolic pool in favor of increased UTP content during activity. These experiments were carried out under conditions of no aeration, and anoxia and acidosis occurred to disrupt the biochemistry of brain. The animals did not learn under these conditions, and shock alone was able to elicit the metabolic pool and RNA

214

VICTOR E. SHASHOUA

changes. If the experiments were carried out in the presence of aeration, no such effects could be observed (Baskin et al., 1972; Shashoua, 1974b). Although the large RNA changes, depicted by the shifts in the base composition data, cannot be accounted for by gross pool changes in the content of U T P and CTP, there is still the possibility that local metabolic pool effects can occur in specific brain regions to bias the data in favor of higher U incorporation during the learning. Two methods, using double-labeling techniques were devised in later studies (Shashoua, 1974b) to examine this question. The second type of biochemical experiments used double-labeling methods with uridine as the RNA precursor. The experimental animals were labeled with one isotope, (U-3H), and the controls with another, U-14C. The brains of the trained and control animals were homogenized together. The cytoplasmic RNA and nuclear RNA components were separated, purified, and analyzed by sucrose density gradient sedimentation methods. An increase in the synthesis of specific RNA molecules was found a t the 13 S region and at positions with values > 3 2 S of the gradient after a 5-hour training period. The double labeling method and the technique of measuring ratios of incorporated label in the learning versus the control groups for specific RNA molecules have the advantage of being independent of metabolic

1-

Controls

u -" c

I F ' Exptls. U - 3 H

Pooled Brains

I

I Pooled Homogenates 1

I

1400 g I 2 . 5 m i n

Pellet

Supernatant

1

J-l

17,0OOg/10 min

Nucieor RNA

90,000g-70m'n,

Crude Synaptasamal I Pellet

SGernalont

1

U

FIG. 7. Double-labeling and fractionation methods used for the isolation of brain RNA components from nuclear, cytoplasmic, and synaptosome fractions. From Shashoua (1974b).

215

RNA METABOLISM IN THE BRAIN

1 '..I\

1.0 EK4h

0.5

I.o E

c

0.5

0

Q

0 I.o

6 c N

0.5

0 0

4 8 12 Fraction Number

16

FIG.8. The sucrose density gradient patterns for the cytoplasmic (C) synaptosomal (S), and nuclear (N) fractions of goldfish brain as a function of training time in the floattraining experiment. The E/C data show the ratios of the 3H/14C-labeled isotopes of uridine incorporated in the RNA for the trained group (E) and control group (C), after 1 hour and 4 hours. The C group was labeled with U-14C, and the experimental group with U-3H. Note that the control groups C/C give a constant ratio of isotopes throughout the gradient and the maximum change is apparent in the synaptosomal fraction for the 4-hour group. From Shashoua (197413).

pool changes. Thus any bias of the pool, for example, in the learning group, can only result in changes in the labeling of all classes of RNA molecules. This merely shifts the base line for the experimenta1:control ratio and does not effect the detection of peak ratios for specific regions of the gradient. The third type of biochemical experiment is illustrated in Fig. 7. Here a double-labeling method was used to compare the newly synthesized RNA associated with brain synaptosomal fraction in trained and control animals. Analysis of the sedimentation pattern of this RNA showed that specific regions of the sucrose gradient had an increased uridine incorporation of the label (see Fig. 8 and Table XX). Control experiments involving pretrained

216

VICTOR E. SHASHOUA

TABLE XX GOLDFISH BRAINRNA C H A N GDURING ~ TRAINING^

A RNA No.

1s 2s

3c 4c

5c 6C

7c

a to 16 17s

18s 19c 2OC 21c 22c

TypeC

yo at Peak 7, ofTotal

+65 + 50 +45 + 30 +60 + 45 + 35

1.6 2.2 7.0 4.0 6.5 8.0 4.0

-

Training Score Y O

55 70 50 52 58 64 55

22 25 28 80

Data from Shashoua (1974b). S = synaptosomal RNA; C = cytoplasmic RNA. E/C = experimental/control; C/C = control/control; control; W/C = work/control; and S/C = stress/conrrol.

Remarks Learning Learning Learning Learning Learning Learning Learning Controls Nonlearning Nonlearning Nonlearning Pretrained control Work control Stress control

a

P/C = pretrained/

animals, which had learned the task for a 7-day period prior to performing it in the presence of label, showed no RNA changes, Similarly, groups of animals that swam vigorously in a whirlpool showed no RNA changes, indicating that physical exercise is not the factor responsible for the type of RNA changes observed, Groups that received label and had floats but did not master the swimming skill, also showed no RNA changes. T h e criterion for learning in such experiments was that the difference between the initial performance score of a group was more than 20y0for the beginning and the end of the 4 5 hour training period. When the training scores improved by 60-70%, then an increase in specific RNA molecules was found. The results suggest that specific classes of RNA molecules, corresponding to 2-47, of the total incorporated label, are obtained as a result of the training (Shashoua, 197413). I n another group of experiments (see Table XIX), a n increase in the incorporation of labeled uracil into the acid-insoluble fraction from upper part of chick forebrain was demonstrated during the imprinting process to a flashing yellow light (Horn et ul., 1971). A split brain preparation was used for these studies. This was achieved by severing the supraoptic commissure 24 hours before the imprinting process. In the experiment, one eye was covered with a translucent plastic to allow light but not patterned vision

RNA METABOLISM IN THE BRAIN

217

during the training procedure. Thus, the training to the flashing light could be introduced through the uncovered eye into one cerebral hemisphere. The untrained hemisphere served as a control. An increase of 5-107, in the incorporation of the uracil was observed in the upper part of the chick forebrain in the stimulated hemisphere contralateral to the exposed eye. No analyses for the formation of specific classes of RNA molecules was carried out. The biochemical difference appears to be the result of the imprinting process, although some nonspecific effects due to sensory stimulation and increased blood flow into the stimulated side may also be involved. VIII. Biogenic Amine Effects on Brain R N A Metabolism

A. STUDIES WITH ACETYLCHOLINE Gisiger (1971) studied the effects of electrical stimulation and acetylcholine on the pattern of RNA synthesis in the rat superior cervical ganglion. The experiments were conducted in vitro. I n initial studies, stimulation with frequencies of 32 per second for 1 hour gave a decrease of about 28y0 in the specific activity of the label in the RNA. After 3 hours, however, the specific activity of the label in the RNA increased by 257,. When tubocurarine was used to inhibit the postsynaptic acetylcholine (ACh) receptor and prevent the depolarization of the neuronal membranes, no increased RNA synthesis occurred. I n experiments with ACh in the incubation medium, a 30-4070 decrease in the incorporation of uridine into RNA occurred during the first hour, but this increased to a level of about 207, higher than control after 5 hours. Thus, ACh effects on RNA synthesis were parallel to those obtained by preganglionic electrical stimulation. Carbacol gave similar results. I n experiments in the presence of tetrodotoxin, ACh was found to stimulate RNA synthesis in the absence of an action potential. Since tetrodoxin selectively blocks the increase in the sodium conductance of neuronal membrane (Kao, 1966) but does not interfere with the ACh-receptor interaction and the generation of excitatory postsynaptic potentials, the effects of ACh on RNA synthesis must therefore not be dependent on the action potential, but rather must be dependent on events that occur after the transmitter reacts with the postsynaptic membrane. In another experiment, Gisiger demonstrated that lowering the membrane potential by the use of high potassium ion does not change the RNA synthesis pattern. Thus the depolarization of a membrane per se does not effect RNA synthesis. However, when ACh was applied in combination with potassium chloride, the increase in RNA synthesis after a 5-hour incubation period was still observable indicating that externally applied ACh is not influenced by the membrane potential. Thus, some factors produced in the subsynaptic

218

VICTOR E. SHASHOUA

region as a rcsult of ACh-receptor interaction may signal the production of RNA.

B. SEROTONIN LEVELCHANGES In studies of the effects of levels of biogenic amines on behavior and brain metabolism (Shashoua, 1973b,c), the concentration of serotonin was found to be related to the arousal level of goldfish. High brain serotonin was associated with a lethargic and inactive behavior while low levels stimulated the activity and arousal of the goldfish. A decrease in the level of serotonin by lOyo in goldfish brain caused an increase in RNA synthesis. This was demonstrated by base composition studies and in double-labeling experiments with uridine as the RNA precursor. Figure 9 shows the sedimentation pattern of the RNA in a sucrose density gradient. The use ofp-chlorophenylalacice to lower brain serotonin resulted in an increase in the label in the cytoplasmic RNA component at regions of the gradient corresponding to 13 S and greater than 32 S. In control experiments, no such increases were obtained. Studies of the effect of increasing the serotonin content in brain (Essman, 1971) gave no RNA changes. These data suggest that an increase in the demand for a given neurotransmitter can stimulate the synthesis of specific RNA molecules in goldfish brain.

F 0

a 0 ‘0

2

4

6

8

10

12

14

FRACTION NO.

FIG. 9. Siicrose density gradient pattern of goldfish brain cytoplasmic RNA showing the effect of p-chlorophenylalanine (PCPA) on the ratio of incorporation of [f~-~H]uridine in the drug-treated group to the control (C) with [“Cluridine. The control versus control ( C / C ) and the PCPA/PCPA give constant ratios while the PCPA/C group shows peak regions of incorporation. From Shashoua (1974a)

.

RNA METABOLISM IN THE BRAIN

219

ON BRAINRNA METABOLISM C. CYCLICAMP EFFECTS

The level of cyclic AMP in superior cervical ganglia and in brain tissue slices can increase in response to electrical stimulation (McAfee et al., 1971) and to the application of catecholamines (Kakiuchi et al., 1969). In viuo, the intracerebral administration of dibutyryl cyclic AMP (Bu,cAMP) to goldfish results in behaviorally hyperactive animals (Shashoua, 1971). A study of the effect of the drug at the 20-50 mg/kg level on the incorpdration of labeled orotic acid as the precursor of UTP and CTP into the newly synthesized RNA showed the formation of RNA with a U/C ratio 20-50x higher than control (Shashoua, 1971). This was suggestive of an increase in the synthesis of the nonribosomal RNA components. Double-labeling studies with uridine as the RNA precursor confirmed these findings. Initially there was an increase in nuclear RNA, and later some of the RNA was found to migrate into the cytoplasm to become associated with polysornes. Figure 10 shows the polysorne pattern with an increase in the RNA associated with specific polysome regions. The results suggested that cyclic AMP, or some subsequent factors triggered by its increased level, can function as a metabolic demand signal for stimulating synthesis of specific RNA molecules in goldfish brain.

D. NOREPINEPHRINE EFFECTS ON BRAINRNA METABOLISM Studies with norepinephrine effects on the pattern of RNA synthesis in organotypic cultures of mouse cerebellum (Shashoua and Wolf, 1972;Wolf and Shashoua, 1973) showed that norepinephrine can stimulate the RNA changes. The base composition of the newly synthesized RNA had a higher U/C ratio than control indicating that a shift of the pattern of synthesis toward more mRNA and nuclear heterodisperse RNA occurs in the presence of norepinephrine. IX. Drug Effects on R N A Synthesis

A number of studies have reported on the effects of drugs on the pattern of RNA synthesis in brain (see Table XXI). Prives and Quastel (1969) studied drug effects on the pattern of uridine incorporation into cerebral cortex slices from adult rats. The tissues were incubated in Krebs’ Ringer in the presence of 10 m M glucose, in an oxygen atmosphere. Electrical stimulation of the slices was found to decrease the rate of conversion of uridine to UMP and U T P and its incorporation into RNA. Tetrodotoxin blocked this inhibitory effect. Ouabain at the 1 p M level was found to inhibit RNA synthesis by 32y0.In tissue slices potassium chloride was found to increase the RNA synthesis by 54y0 whereas it had no effect of inhibited RNA synthesis in intact ganglia (Gisiger, 1971) and in goldfish brain (Shashoua, 1968). Acetylcholine in the presence of eserine gave an increased RNA

h3 h3 0

2

3lo

TABLE XXI DRUGEFFECTS O N RNA SYNTHESIS

Label

Time (hours)

Rat Rat

Uridine Uridine

3 3

Tetrodotoxin

Rat

Uridine

3

KCI KCI Tranylcypromine

Rat Uridine Goldfish Uridine Rabbit -

4

Drug

Animal

Acetylcholine (ACh) D-Tubocuranine

Ouabain

Rat

Uridine

3

Locus

-i RNA changes

30y0 Increase Inhibits stimulation of R N A synthesis by ACh Superior cervical ganglion No effect on stimulation of R N A by ACh Superior cervical ganglion No effect Total brain No change Vestibular neurons Increase Superior cervical ganglion Superior cervical ganglion

Vestibular glia

Decrease

Cortex slices

Inhibited RNA synthesis by 32y0

Reference Gisiger (1971) Gisiger (1971) Gisiger (1971) Gisiger ( 197 1) Shashoua (1970) H y d h and Eghlzy ( 1968) Hydkn and E g h b y (1968) Prives and Quastel ( 1969)

E8 F

Tetrodotoxin

Rat

Uridine

Cortex slices

KCl

Rat

Uridine

Cortex slices

Blocked inhibiting effect of electrical stimulation At 5-1 5 mM gave 32 yoincrease

KC1

Rat

Uridine

Cortex slices

At 50 mM gave decreased RNA

ACh (1 mM) + KCI (15 mM) Carbamylcholine

Rat

Uridine

Cortex slices

lOOyoincrease

Rat

Uridine

4

Cortex slices

Increased RNA

Goldfish Uridine

4

Total brain

Increased RNA

Prives and Quastel (1969) Prives and Quastel (1969) Prives and Quastel ( 1969) Prives and Quastel ( 1969) Prives and Quastel ( 1969) Shashoua (1974a)

Rat Uridine Goldfish Uridine Chick Formate

4 4

2

Total brain Total brain Fetal brain

Decreased RNA Increased nuclear and mRNA Increased RNA (+ 14Oy0)

Essman (1971) Shashoua (1971) Caldarera et al. (1969)

Rat

2

Total brain

Increased rRNA

Jacob et al. (1969)

p-Chlorophenylalanine Serotonin ButacAMP Spermidine + spermine Hydrocortisone

32P

222

VICTOR E. SHASHOUA

Froction Number

FIG. 10. The polysorne gradient for goldfish brain, showing the effect of dibutyryl adenosine cyclic 3': 5'-monophosphate on the incorporation of uridinc into RNA in a double-labeling experiment. Upper section of the graph depicts the 3H/'4C ratios for the experimental -( ) versus control and control versus control (0-0) groups. Lower graph shows the polysome profile (AQe0)and the labeling pattern for the experimental group (- - - - -) with L'-3H label and control group () with U-I4C label. Drug dose 50mg/kg. From Shashoua (1971).

synthesiy. Carbarnylcholine stimulated the RNA synthesis rate both in the presence and in the absence of eserine. When sodium was removed fi-om the incubation medium, the rate of stimulation of RNA synthesis by ACh was diminished. Large RNA stimulation effects in rabbit brain were observed when tranylcypromine was injected into the animals (Hydin and Egyhky, 1968). However, rm base composition changes occurred. X. A Theoretical Model for Information Storage

A number of hypotheses have been proposed for the process of information storage in the brain. The early work of Flexner et al. (1962) suggested

223

RNA METABOLISM IN THE BRAIN

that there are two critical phases in the formation of a new memory. The first is a “short-term memory” stage, which is not sensitive to inhibitors of protein synthesis and the second is “long-term memory,” which is sensitive to inhibitors of protein synthesis, such as puromycin and cycloheximide. Many studies of the time sequence of delivery of the inhibitors relative to the time of training have been carried out (Barondes and Cohen, 1967; Agranoff et al., 1967a, among others). I n the analyses of these studies, Shashoua (1972) proposed a hypothetical scheme for the coupling of the information storage pracess with brain

1

ENVIRONMENT

I SENSORY

RECEPTORS

I I

CJj

@ TRANSDUCTIONS

ELECTRICAL - TIME

ELECTRICAL- SPACE

TEMPORARY CHEMICAL- SPACE

KLYSOYE

I

MEMBRANES

I

FIG. 11. Diagram of the sequence of transduction processes required for coupling environmental information to brain metabolism symbol for a generalized transducer; hV, light inputs; A) heat inputs; @, chemical inputs; and , mechanical inputs. Each stage in the information processing is considered to have specific time constants and to be a multicomponent event representing the transit status of information. From Shashoua (1972).

+,

f

224

VICTOR E. SHASHOUA

metabolism. I n this scheme (see Fig. 11) environmental information, as detected by the heat, light, chemical, and the mechanical sensory receptors of an organism, is converted by a series of four transduction steps into a longterm “metabolic demand signal,” which is specific to the state of information recording in specific neural circuits. This “demand signal” is postulated to act as a “trigger signal” to elicit through three additional transduction steps the synthesis of proteins for the modification of membranes in the nervous system. Each of the transduction steps outlined is a multicomponent and a multistep event, representing a phase in the continuous processing of the input into a suitable biochemical form for storage. T h e basic assumption of the model is that the brain operates as a “novelty detector” with a capacity for extracting the new from the old features of a given input. Operationally, this may be accomplished through a series of steps that act as filters to allow only the new aspects of a given input to be transmitted to the next processing level. Thus, distinct biochemical changes leading to cellular modifications occur only in the specific cells committed to new information; all other cells in the path of the input may increase their metabolic rate only in a manner characteristic of an increased “use” function. This model for information processing has been derived from the following considerations. In the first stage, information from the sensory receptors is transduced into a generalized type of output that is in an “electrical time” mode, i.e., the digital electrical spike output of neurons. The information storage capacity in this mode is quite limited because the same receptor system that produces a given quantum of information must be used over again in generating the next segment of a message. Thus, this type of output must quickly be converted into some other form for use in additional processing towards the ultimate goal of producing information storage and retrieval. On the two possibilities available, i.e., a transduction of the (1) electrical or of (2) the time component of the output, the most likely transduction process to occur is the conversion of the time-aspect into a space-aspect to give an “electrical-space” mode of information encoding. This is supported by the findings in which digital spike train inputs are converted to analog signals (Grundfest, 1969; Werner and Mountcastle, 1965). Thus, at this stage, the information is considered to be a distribution of “space charges” in a three-dimensional network over many newal elements in different brain areas. There are a number of experiments that support such a notion. I n particular, the studies of the effect of electroconvulsive shock (ECS) reported by Chorover and Schiller (1965) indicate that, in one-trial passive avoidance learning, ECS can induce retrograde amnesia if it is administered within 10 seconds (see Table XXII) after training. Additional experiments suggest that this susceptibility of the memory trace

TABLE XXII ESTIMATED TIME CONSTANTS FOR THE TRANSDUCTION PROCESSES Transduction process" Simulated by Inhibited by 7 i f a Range

References

EN to E-T

2 ET to ES

ES to CS

cs to LC

-

-

-

CAMP -

1

Ouabain (?) ECS

3

Anticholesterases 10-70 Sec Minutes to hours Chorover and Deutsch (1967, Schiller (1965) 1971)

Quartermain (1965) McGaugh (1966)

4

Up to 2-3 hours Shashoua (1971)

5 LC to RNA

Actinomycin D 15 Minutes up to hours Hyd+ and Eghhzy (1962) Glassman (1969) Agranoff et al. (1967b) Shashoua (1970a)

6 RNA to protein I

Puromycin, cycloheximide 3-10 Hours

7 Protein to membranes

Peptidyl puromycin Up to 30 days

w

T 0

Flexner et nl. (1962) Barondes and Cohen (1967) Agranoff et al. (1967a) HydCn and Lange (1971)

Roberts et al. (1970)

Shashoua (1968, 1974b) a EN = environment; E-T = electrical-time components; ES = electrical-space mode; CS = chemical-space mode; LC = long-term chemical signal. b ECS = electroconvulsiveshock.

c

E

2 1

B

!i z

226

VICTOR E. SHASHOUA

to disruption by ECS can last as long as 30-70 seconds (Quartermain et al., 1965; McGaugh, 1966). The third transduction process to occur in this model is the conversion of the electrical-space into a “chemical-space” signal. By this means all the electrical elements of the input are removed to generate a chemical signal at the same loci where the electrical changes were present. These temporary chemical changes may be at the synaptic sites, where some sort of chemical coupling between pre- and postsynaptic sites occurs. There are many obvious candidates for such chemical changes including concentration changes of transmitter substances, or the production of small peptidc molecules (Ungar, 1970). Temporary ionic changes would no doubt also be involved in the coupling. The time constant (see Table XXII) for this “informational-mode” may vary depending upon the principal neurotransmitter used for the given neural circuit in which the temporary change resides. Thus, a very fast time constant may be characteristic of neural circuits in which ACh is the transmitter, since the deactivation process by acetylcholinesterase is extremely rapid. The studies of Deutsch (1971) bear on this proposed mechanism. Their studies indicate that memory retention is inhibited when anticholinesterases are administered intracerebrally within 30 minutes after training, but therc arc no effects if such drugs are given 3 days after training. These Experiments suggest a primary time constant for the chemical-space signal of about 30 minutes for neural circuits involving -4Ch. Thus, a variety of time constants may define the life-times of the primary processes involved in encoding the chemical-space mode, and these time constants may depend on which indoleamines, catecholamines, or peptide hormone? are used in the particular neural circuits involved in specifying the new input. The fourth transduction process is postulated as a means by which events occurring a t the cell membrane are communicated to the cell nucleus. Thus, the “temporary chemical space mode” is converted into a “long-term chemical” signal. At this stage the specificity of the information is essentially lost and the function of the long-term chemical is simply to transmit a biochemical demand change to the cell nucleus for specific membrane proteins. Adenosine cyclic 3’, 5’-monophosphate (CAMP) may have such a function in the nervous system (Shashoua, 1971). This function for CAMPis supported by a variety of findings which indicate that electrical stimulation of superior cervical ganglia (McAfee, et al., 1971) or of brain tissue slices (Kakiuchi et al., 1969) can give rise to increased CAMP levels in nervous tissue. In addition, brain tissue slices can be stimulated to synthesize CAMP by catecholamines, particularly norepinephrine. As shown in Fig. 11, the function of the long-term chemical is to signal the cell nucleus to synthesize

RNA METABOLISM IN THE BRAIN

22 7

RNA which is to be used-in subsequent steps for the synthesis of proteins needed to modify cell membranes in the last step of the information processing. There are a number of molecules that could be considered for use as the “long-term metabolic demand signal.” Each molecule in this category may be specific to a class of neural circuits. Thus CAMPmay be specific to norepinephrine and dopamine circuits (Shashoua, 1971), and very likely to serotonin circuits as well. Recent studies (Kuo et al., 1972) suggest that guanosine cyclic 3’, 5’-monophosphate may be involved in ACh circuits. There may well be other types of metabolic demand signals, which need not be nucleotides (Ungar, 1970), for use in this type of function. From the experiments with CAMP, the time constant for this stage appears to be about 2-3 hours, since RNA synthesis shows a maximum change at about 3 hours after intracerebral administration of the drug and there are no effects after 10 hours. The transduction step from the long-term chemical signal to an RNA has been reviewed by Glassman (1969). At this stage the integrity of the information is no longer preserved. The membrane structures, which in stage 3 are involved in generating the chemical-space signal, are essentially withdrawn from receiving further inputs and function as a holding pattern until the metabolic systems can produce proteins to fix these patterns permanently. Thus, stage 3 may have one time constant by which it can signal the nucleus via a long-term chemical signal and another, longer, time contant during which the holding pattern awaits modification by proteins. Such a notion is supported by the findings of Deutsch (1971) in which anticholinesterase have both short- and long-term effects. The sixth stage is one in which mRNA is used for the synthesis of proteins. The time constant for this stage has been specified in a variety of experiments (Barondes and Cohen, 1967; Agranoff et al., 1967b) through the use of protein synthesis inhibitors to prevent long-term memory storage. Again, a number of time constants can be deduced from experimental data ranging from as little as 3 hours to about 10 hours (see Table XXII). This may be dependent on the experimental animal used and the difficulty of the task to be remembered. The last stage in the information processing is the assembly of proteins into membranous elements at the “chemical-space sites” within the nervous system. The types of changes that may occur include a modification of the postsynaptic membranes and the production of more synaptic vesicles in a given neural circuit; it may also include the production of the new dendritic or axonal outgrowth and an increase in myelination of specific axons. In a recent paper by Roberts et al. (1970) it was shown that when puromycin is administered 24 hours after a training session the peptidyl derivative formed by the disruption of protein synthesisremains within the nervous tissue causing

228

VICTOR E. SHASHOUA

amnesia for periods of as long as 30 days. Intracerebral saline injections during this period, however, can restore the memory, probably by releasing the puromycin peptides from their membrane-bound sites. Roberts et al. believe that these puromycin peptides have binding characteristics similar to norepinephrine. If, in fact, this occurs, then the puromycin peptides might interfere with the membrane assembly processes involved in new vesicle formation. This type of binding suggests that the time constant for the final transduction process can be quite long. In this proposed mechanism, information specificity is provided by the neural circuitory while biochemical events define the means for their modification. Essentially, the mcssage writes itself and biochemical changes constitute a “repair” mechanism by which classes of proteins and their modification are deposited a t the same membrane sites, which are specified in the circuits depicting the new information. Thus, message-specific proteins are not required. Moreover, once a structural change in a neural circuit has occurred, there is no necessity for long lifetimes for the proteins used. This is consistent with the relatively short half-life of 14 days reported by Lajtha (1964) for over 987’. of brain proteins. T h e proposed mechanism takes into account the dynamic nature of brain protein and RNA metabolism Thus, information recording in a biochemical sense becomes a “microevent” in brain development. After a membrane change has occurred, its components become part of the overall structure of the brain and enter into ongoing dynamics of brain metabolism, regenerating at the same general rate as other brain structures. In this way, the specialized informationrecording function of nervous tissue can utilize processes and substrates, which may be common to all cells but which are maximally developed in brain. REFERENCES Adair, L. B., Wilson, J. E., Zemp, J. W., and Glassman, E. (1968a). Proc. Nut. Acad. Sci. US. 61, 606. Adair, L. B., Wilson, J. E., and Glassman, E. (1968b). Proc. Nut. h a d . Sci. US. 61, 917. US.61, 606. Agranoff, B. W., Davis, R . E., and Brink, J. J. (1967a). Proc. Nut. Acad. Sci. US. 154, 788. Agranoff, B. W., Davis, R. E., Casola, L., and Lim, R. (1967b). Science 158, 1600. Amaldi, P., and Ruska, G. (1970). J . Neurochem. 17, 767. Appel, S. H. (1966). Recent Aduan. Biol. Psychiatry 9, 347. Appel, S. H. (1967). Nulure (London) 215, 1253. Appel, S. H., Davis, W., and Scott, S. (1967). Science 157, 836. Autilio, L. A., Appel, S. H., Pettis, P., and Gambetti, P. L. (1968). Biochemistry 7, 2615. Balazs, R., and Cocks, W. A. (1967). J . Neurochem. 14, 1035. Barondes, S..H., and Cohen, H. D. (1967). Brain Res. 4, 44. Barondes, S. H., and Jarvik, M. E. (1964). J . Neurochem. 11, 187. Baskin, F., Masian, F. R., and Agranoff, B. W. (1972). Brain Res. 39, 151. Bateson, P. P. G., Horn, G., and Rose, S. P. R . (1972).Brain Res. 39, 449.

RNA METABOLISM IN THE BRAIN

229

Berry, R. W. (1969). Science 166, 1021. Berry, R. W., and Cohen, M. J. (1972). J. Neurobiol. 3, 209. Bieth, R., and Mandel, P. (1953). Expcrientia 8, 185. Bok, D. (1970). Invest. Ophthalmol. 9, 516. Bondy, S. (1966). J. Neurochem. 13, 955. Bondy, S. (1971). Exp. Brain Res. 13, 135. Bondy, S. C., and Madsen, C . J. (1971). J. Neruobiol. 2, 279. Bondy, S. C., and Margolis, F. L. (1970). Exp. Neurol. 27, 344. Bondy, S. C., and Roberts, S. (1967). Biochem. J. 105, 1111. Bondy, S. C., and Roberts, S. (1968). Biochem. J. 109, 533. Bondy, S. C., and Roberts, S. (1969). Biochem. J. 115, 341. Bowman, R. E., and Kottler, P. D. (1970). In “Biochemistry of Brain and Behavior” (R. E. Bowman and P. Datta, eds.), p. 212. Academic Press, New York. Bray, J. J., and Austin, L. (1968). J . Neurochem. 15, 731. Burdmann, J. A. (1969). Brain Res. 15, 515. Cain, D. F. (1967). J. Neurochem, 14, 1175. Caldarera, C. M., Moruzzi, M. S., Rossoni, C., and Barbiroli, B. (1969). J. Neurochcm. 16, 309. Casola, L., Davis, G. A., and Davis, R. E. (1969). J. Neurochem. 16, 1037. Chorover, S. L., and Schiller, P. H. (1965). J. Comp. Physiol. Psyoitol. 59, 43. Daneholt, B., and Brattgard, S. D. (1966). J. Neurochem. 13, 913. Dawson, D. M. (1967). J. Neurochem. 14, 939. Deutsch, J. A. (1971). Science 174, 788. Dellweg, H., Gerner, R., and Wacker, A. (1968). J. Neurochem. 15, 1109. Dravid, A. R., Pete, N., and Mandel, P. (1971). J. Neurochem. 18, 299. Dutton, G. R., Campagnoni, A. T., Mahler, H. R., and Moore, W. J. (1969). J. Neurochem. 16, 989. Edmonds, M., and Abrams, R. (1963). J. Biol. Chem. 238, 1186. Edstrom, A. (1964). J. Neurochem. 11, 309. Edstrom, A., Edstrom, J. E., and Hokfelt, T. (1969). J. Neurochem. 16, 53. Edstrom, J. E. (1956). J. Neurochcm. 1, 159. Edstrom, J. E. (1964). Methods Cell Physiol. 1, 417-447. Edstrom, J. E., and Eichner, D. (1957). Z . Mikrosk. Ana. Forsch. 63, 413. Edstrom, J. E., and Pigon, A. (1958). J. Neurochem. 3, 95. Engel, J., Jr., and Morrell, F. (1970). Exp. Neurol. 26, 221. Entingh, D., Entingh, T., Glassman, E., and Wilson, J. E. (1972). 2nd Ann. Meet., SOC. Neurosci. Abstract No. 38.2. Essman, W. B. (1971). In “Biology of Memory” (G. Adams, ed.), p. 213. Plenum, New York. Flangas, A. L., and Bowman, R. E. (1970). J. Ncurochem. 17, 1237. Flexner, J. B., Flexner, L. B., Stellar, E., De La Haba, G., and Roberts, R. B. (1962). J . Neurochem. 9, 595. Gerber, G., and Altman, K. J. (9160). J. Biol. Chcm. 235, 2682. Gillespie, D., and Spiegelman, S. (1965). J. Mol. Biol. 12, 829. Gisiger, V. (1971). Bruin Res. 33, 139. Gispen, W. H., de Wied, D., Schutman, P., and Jansy, H. S . (1970). J. Neurochem. 17,751. Glassman, E. (1969). Annu. Rev. Biochem. 38, 605. Gramp, W., and Edstrom, A. (1963). J. Ncurochem. 10, 725. Grundfest, H. (1969). In “Structure and Function of Nervous Tissue” (G. H. Bourne, ed.), p. 463, Academic Press, New York.

230

VICTOR E. SHASHOUA

Gurgo, C., Rutigliano, B., Pagliuca, N., and Giuditta, A. (1970). Biochem. Biophys. Acta 198. 31. Guroff, G . , Hogans, A. F., and C‘denfriend, S. (1968). J. Neurochem. 15, 489. Haddad, A.. Iucif, S.,and Cruz, A. R. (1969). J . Neurochem. 16, 865. Holmgren, R. l’.,and Holmgren, B. (1968). Brain Res. 8, 220. Horn, G., Horn? A. L. D., Batrson, P. P. G., and Rose, S. P. R. (1971). Nature (London) 229, 131. Hurwitz, J . Ii.. Rressler, .4., and Kaye, A. (1959). Riochcm. Biophys. Res. Commun. 1, 3. Hyden, H.(19.55). In “Keurochemistry” (K. A. C. Elliott, I, H. Page, and J. H. Quastel, eds.), p. 204. Thomas, Springfield, Illinois. Hydtn, H. (1959a). Proc. Int. Congr. Biochem., 4th, 1958 Symp. 111, p. 88. Hydcn, H. (1959b). Nature (London) 177, 433. Hydtn, H. (1960). In “The Cell” (J. Brachet and A. E. Mirsky, eds.), Vol. 4, p. 215. Academic Press, New York. HydPn, H., and Egyhkzy, E. (1962). Proc. A‘at. Acad. Sci. 48, 1366. HydPn, H.,and Egyhizy, E. (1968). Neurology 18, 732. Hydin, H., and Lange, P. W. (1971). In “Handbook ofh’eurochemistry” (A. Lajtha, ed.), Vol. 111, p. 221. Plenum, New York. Hyden, H. V. (1943). ACfQPhysiol. Scand. 6, Suppl. 17, 1. Hydrn, H. V. (1964). Recent Advan. Biol. Physiol. 6 , 31. Hyden, H . V. (1967). In “The Neuroscirnct9-(G. C. Quarton, ‘r. Mclnechuk, and F. 0. Schmitt. eds.), p. 248, Rockefeller Univ. Press, New York. Ingoglia, N. A.. Grafstein, B., McEwen, B. S.,and McQuarrie, I. G. (1973). J . Neueurochem. 20. 1605. Jacob, S. J., Sajdel, E. M., and Munro, H. N. (1969). Eur. J . Biochem. 7 , 449. Jakoubek, B.$ and Edstrijm, J. E. (196.5). J . Neurochsm. 12, 845. Jakoubek, B., and Semiginovsky, B. (1970). Int. Rev. Neurobiol. 13, 255. Jarvik, M. E. (1972):Annu. Rev. Psychol. 23, 457. Johnson, T.C.(1967). J . Neurochem. 14, 1075. Johnson, T. C. (1968). J . Neurochem. 15, 1189. Kakiuchi, S., Rall, 1’. W., and McIlwain, H. (1969). 3. Neurochem. 16, 485. Kao, C:. Y. (1966). Pharmacol. Rev. 18, 997. Kernell, D., and Peterson, R. P. (1970). J. Neurochem. 17, 1087. Khan, A. A., and Wilson, J. E. (1965). J. Neurochem. 12, 81. Koenig, E. (1965). J . .Veeurochem. 12, 357. Koenig, E. (1967). J. Neurochem. 14, 437. Kuo, J., Lee, T., Reves, P. L., U’alton, K. C., Donnelly, ‘I‘. E., and Greengard, P. (1972). J . Biol. Cham. 247, 16. Lajtha, .4.(1964). b t . Reu. Neurobiol. 6, 1-98. Lasek. R. J. (1970). J . Neurochem. 17, 103. Liu, C. N..Bailey, H. L., and Windle, W.F. (1960). J . Comp. Neurol. 92, 169. Loeb, J. N.,Howell, R. R., and Tomkins, G. M. (1965). Science 149, 1093. Lsvtrup-Rein. H. (1970). J. Neurochem. 17, 853. Lovtrup-Rein. H., and Grahn, B. (1970). J. Neurochem. 17, 845. Lovtrup-Rein, H., and McEwen, B. S. (1966). J . Cell Biol. 30, 405. McAfee, I). A., Schorderet, M., and Greengard, P. (1971). Science 171, 1156. McGaugh, J. L. (1966). Science 153, 1351. Mandel, P.,Jacob, hl., and Mandel, L. (1950). Bull. SOC.Chim. Biol. 32, 80. Mandel, P.,Dravid, A. R., and Pete, N. (1967). J. Neurochem. 14, 301. Merritt,.J. H., and Sulkowski, T. S. (1970). J . Neurochem. 17, 1327.

RNA METABOLISM IN THE BRAIN

231

Miani, N., Di Girolamo, A., and Di Girolamo, M. (1966). J . Neurochem. 13, 755. Ochs, S. (1972). Science 176, 252. Oderfield-Nowak, B., and Niemierko, S. (1969). J . Neurochem. 16, 235 Perry, R. P. (1967). Prog. Nucl. Acid Res. Mol. Biol. 6, 220. Peterson, J. A., Bray, J. J., and Austin, L. (1968). J . Neurochem. 15, 741. Peterson, R. P. (1970). J . Neurochem. 17, 325. Peterson, R. P., and Kernell, D. (1970). J . Neurochem. 17, 1075. Prives, C., and Quastel, J. H. (1969). Nature (London) 221, 1053. Quartermain, D., Paolina, R. M., and Miller, N. E. (1965). Science 149, 1116. Rambach, W. A., Moomaw, D. R., Alt, H. L., and Cooper, J. A. D. (1952). Prod. SOC. Exp. Biol. Med. 79, 59. Ringborg, U. (1966). Bruin lies. 2, 296. Roberts, R. B., Flexner, J. B., and Flexner, L. B. (1970). Proc. Nut. Acad. Sci. U.S.66, 310. Rose, S. P. R. (1970). In “Short-term Changes in Neural Activity and Behavior. (G. Horn and R. A. Hinds, eds.), p. 218. Cambridge Univ. Press, London and New York. Schaer, J. C., Grieder, A., Heiniger, H. J., and Schindler, R. (1969). Exp. Cell Res. 56,449. Schneider, D., and Roberts, S . (1968). J. Neurochem. 15, 1469. Schneider, W. C. (1946). J . Biol. Chem. 164, 747. Schneider, W. C., and Klug, H. L. (1946). Cancer Res. 6, 691. Shashoua, V. E. (1968). Nature (London) 217, 238. Shashoua, V. E. (1970a). Proc. Nut. Acad. Sci. US.65, 160. Shashoua, V. E. (1970b). J . Cell. Biol. 47, 188a. Shashoua, V. E. (1971). Proc. Nut. Acad. Sci. US.68, 2835. Shashoua, V. E. (1972). Znt. J . Neurosci. 3, 299. Shashoua, V. E. (1973a). Exp. Brain Res. 17, 139. Shashoua, V. E. (1973b). Science 181, 572. Shashoua, V. E. (1974a). Brain Res. (in press). Shashoua, V. E. (197413). Brain Res. (in press). Shashoua, V. E., and Wolf, M. K. (1971). J . Neurochem. 18, 1149. Shashoua, V. E., and Wolf, M. K. (1972). In Vitro 7, 250. Sttvenin, J., Mandel, P., and Jacob, M. (1969). Proc. Nut. Acad. Sci. U S . 62, 490. Sung, S. C. (1969). Can. J. Biochem. 47, 47. Tencheva, Z. S., and Hadjiolov, A. A. (1969). J . Neurochem. 16, 769. Ungar, G., ed. (1970). “Molecular Mechanisms In Memory and Learning”. Plenum, New York. Vesco, C., and Giuditta, A. (1967). Biochim. Biophys. Acta 142, 385. Vitale-Neugebauer, A., Giuditta, A., Vitale, B., and Giaquinto, S. (1970). J . Neurochem. 17, 1263. Volpe, P., and Giuditta, A. (1967). Nature (London) 126, 154. Werner, G., and Mountcastle, V. B. (1965). J. Neuro~h~siol. 28, 359. Whittaker, V. P. (1959). Biochem. J . 72, 694. Whittaker, V. P., Michaelson, I. A., and Kirkland, R. J. A. (1964). Biochem. J . 90, 293. Wolf, M. K., and Shashoua, V. E. (1973). Trans. Amer. SOC. Neurochem. 4, 59. Yamagami, G,, and Mori, K. (1970). J . hreurochem. 17, 721. Zemp, J. W., Wilson, J. E., Schlesinger, K., Boggan, W. D., and Glassman, E. (1966). Proc. Nat. Acad. Sci. US.55, 1423. Zomzely, C. E., Roberts, S., Gruber, C. P., and Brown, D. M. (1968). J . Biol. Chem. 243, 5396. Zomzely, C. E., Roberts, S., and Peache, S. (1970). Proc. Nut. Acad. Sci. U.S. 67, 644.

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A COMPARISON OF CORTICAL FUNCTIONS IN MAN AND THE OTHER PRIMATES By R. E. Passingham and G. Ettlinger The Department of Experimental Psychology, South Parks Road, Oxford, and The Institute of Psychiatry, D e Crespigny Park, London, England

I. Introduction . 11. Anatomy . A. Methods . B. Sensory Functions . C. Motor Functions . D. Learning . E. Memory . F. Lateralization of Function . G. Cross-Modal Abilities . H. Language . I. Conclusion . 111. Behavior . A. Sensory Functions . B. Motor Functions . C. Learning . D. Memory . E. Lateralization of Hemispheric Function and Exchange of Information between the Hemispheres. . F. The Exchange of Information between Sense-Modalities . G. Language . H. Conclusion . IV. Discussion . . References .

233 237 237 240 241 243 25 1 252 253 254 257 260 260 262 264 2 73 277 282 284 287 290 292

1. Introduction

The nonhuman primates may be studied for one of several reasons. They are useful as substitutes for people in medical and psychological research, when it would be unethical or impractical to carry out the experiments on man. They are also of value to anthropologists for the study of the evolution of man’s ancestors. Finally, they are of interest to zoologists, since primates form a large and successful order of mammals. Nonhuman primates are much used by medical scientists, such as neurophysiologists and neuropsychologists. For such work, mammalian species are chosen which are similar to man in the respect being studied-for 233

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example, the laboratory rat for work on the hypothalamus and the rhesus monkey (Macaca mulatta) for work on the neocortex. Comparisons between species are made only so as to select the animal best suited for the particular research. Thus Drewe et al. (1970) compared the behavior of the rhesus monkey and the chimpanzee (Pan troglodytes) and suggested that the chimpanzee might be a better model for neuropsychological research than the more commonly used rhesus monkey. Criticisms that nonhuman primates are used in this type of research as “dummy humans” (Morris, 1967), and that the natural history of the animals is ignored (Warren, 1973), are irrelevant, since the purpose of such research is exactly to use them to stand in for people. The interests of comparative psychologists and physical anthropologists are different. They study the modern nonhuman primates because they provide indirect evidence of man’s ancestors. They may be arranged in a pseudo-evolutionary series such that the closer a group is to man in the series, the more recently it shared a common ancestor with him (le Gros Clark, 1971) (see Fig. 1). However, such a series may be misleading since modern primates cannot be treated as “living fossils.” Since their divergence from man’s ancestral line, they have radiated into different niches and changed both in anatomy and behavior. Furthermore, different characters have changed to different degrees in the same species, and it will not always be easy to tell which are inherited from the ancestor in common with man, and which have been independently acquired since diverging from that ancestor (le Gros Clark, 1971; Martin, 196813). The only direct evidence comes from the fossil record (see Fig. l ) , and this may be used to determine to what extent characters in living primates are characters of common inheritance. For example, as Martin (1968b, 1973) has pointed out, it cannot be assumed that the brain size of modern prosimians is the same as that of ancestral prosimians; but this may be checked by comparing them with the cranial capacities of fossil prosimians (Radinsky, 1970). The fossil record provides an independent check for many aspects of hard anatomy and for some aspects of behavior, such as locomotion. But it is of little help for soft anatomy, FIG. 1. Evolution of man’s ancestors. Line ABCDEF = series of ancestors of man. Time on left ordinate is in millions of years. Bars indicate range in time offossil primates, the groups being placed to the right of the line leading to the taxon to which they belong. Only those fossil groups are included which are of value in reconstructing the line ABCDEF. The dates and classification are based on Simons (1969, 1972) and Romer (1966). The dates of divergence are very inexact. Pleist., Pleistocene; Z, Zalamdalestes (Zalamdalestidae) ; L, Leptictidae; Par, Paromomyinae; C, Carpolestidae; PI, Plesiadapidae; A, Anaptomorphinae; Ad, Adapidae; M, Microchoerinae (Necrolemurinae) ; Om, Omomyinae; B, Branisella; Ce, Cebidae; Pa, Parapithecinae; Cer, Cercopithecinae; Co, Colobinae; Plio, Pliopithecinae; 0, Oligopithecus ; Ae, Aeggfitofiithecus;D, Dryopithecinae; R, Ramapithecur ; A, Auctralopithecus; H, Homo.

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such as the fine structure of the brain, although in lower, but not in higher, primates something may be deduced about cortical areas from impressions of the sulci on the cranial endocast (Radinsky, 1972). Similarly, the fossil record gives no information about such aspects of behavior as the capacity for learning. I n such cases the only available evidence comes from the study of a modern series. Wherever possible, however, such a modern series should include many species from each major taxon. No single species can be taken as fully LL representative” of other species within a taxon, since there is considerable variation in anatomy and behavior within the major taxa. Furthermore, although particular species, such as the prosimian mouse lemur (Microcebus mrinus), may be more “primitive” in certain respects than other species in their grouping, they may differ in other ways from the ancestor they share in common with man. However, where research is carried out in such areas as comparative neuropsychology, it may be completely impractical to study a series of more than a very few species (Masterton et al., 1969; Masterton and Skeen, 1972). Both medical scientists and anthropologists are primarily interested in man, whereas zoologists are primarily interested in other animals. Zoologists have therefore criticized the anthropologists for being anthropocentric in their study of the nonhuman primates, and for suggesting “trends” within primates which are not trends for the primate order as a whole. But this criticism comes from a misunderstanding of the primate series as suggested by le Gros Clark (1971). This series is intended to be indirect evidence not for primate radiation, but for man’s ancestry. In le Gros Clark’s (1971) own words the aim is to reconstruct “The Antecedents of Man”; the “trends” are not trends of primate evolution but for man’s ancestors. For this purpose, the radiation of primates within each taxon of the series is error variance. For comparative zoologists, however, it is the primary source of interest. For example Charles-Dominique and Martin (1970) have used evidence from both living and fossil prosimians to reconstruct the evolution and adaptive radiation of lemurs and lorises. The aim of this paper is that of comparative psychology, to reconstruct the relations between the brain and behavior in man and his ancestors. The hope is that it will one day be possible to relate changes in the behavior of man and his ancestors to changes in the structure of their brains. The discussion in this paper is confined to cortex, mainly to neocortex, partly because neocortex is the structure which has changed most in primate evolution (Stephan and Andy, 1969), and partly because man’s special attributes might be thought to depend on it. There are two ways in which the comparative method can be used to establish the relations between the brain and behavior; by correlating the differences in the brain of several species

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with differences in their behavior; and by comparing the effects on the behavior of several species of experimentally altering their brains. The first method is discussed in Skction 11, and the second in Section 111. II. Anatomy

’

A. METHODS 1. Behavior It is often assumed that the differing abilities, both within the nonhuman primates and between the nonhuman primates and man, must be accounted for by differences in the structure of the neocortex. However, such claims require that there be adequate knowledge of the differences in ability within the primate order. Such differences might either be ones of efficiency in learning or of the capacity for particular types of learning. Within the nonhuman primates information on differing abilities comes mainly from attempts to assess them in the laboratory (Warren, 1965). Warren (1973) has pointed out the difficulties involved in such attempts, and suggested that the tests commonly used, such as learning sets, are not valid or reliable. Their validity is questioned because it can be shown that some of the variance is accounted for by specific factors, such as sensory and motor capacities, and by species-specific abilities. However, while such factors might make comparisons suspect between species in widely different orders, they may account for less of the variance in comparisons of species within an order. The reliability of the tests is questioned because the results are affected by the type of apparatus used, and also because there are large differences in the performance of animals within a species. Warren (1973) therefore concludes that such tests cannot be used to rank species in ability. For example, he quotes Maier and Schneirla’s (1964) comparison of the performance of animals on the delayed response task, in which no clear phylogenetic trend can be seen, and also points to the overlap of animals such as cats with primates on this and other tasks. However, the comparisons made by Maier and Schneirla (1 964) are invalid since they are made across completely different conditions of testing, such as number of trials a day, intertrial interval, and so on. Given the evidence for the role of interference in delayed response tasks (Jarrard and Moise, 1971), it is not meaningful to compare animals tested for one or two trials a day with animals tested for forty. Furthermore, there is no phylogenetic scale that ranks primates above carnivores, since they diverged at the same time and do not form a pseudoevolutionary series such as can be constructed within the primates. They can only be ranked on a scale of brain or neocortical development, and the size of their neocortex gives no reason to think that cats should perform worse than prosimians. Similarly overlap in abilities would be expected between prosimians and New World Monkeys and between New World and Old

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World monkeys, since there is overlap for their indices of encephalization (Stephan, 1972). I n fact it is not possible to reject the hypothesis that the primates may be ranked in ability on laboratory tasks, because it has never been tested. S o one has ever carried out a study af learning set, reversal, or delayed response oil a large number of primates under equivalent conditions. Thc best study is that of Harlow el d.(1932; Marlow and Harlow, 1932) on delayed response, and in that study a fairly orderly patterns of results was found. Only when a large-scale study has been carried out will it be possible to correlate the order of abilities with the development of the neocortex as assessed by Stcphan and Andy (1969). Most of Warren’s (1973) objections suggest only that the correlation would not be perfect, but the correlation might nonetheless be fairly high. Man is much better than other primates at such laboratory tasks, but his possession of language complicates the comparisons since it is difficult to provide a task on which man could not use verbal coding. For example, language may make the delayed rcsponsc task very much easier. Furthcrmore it is difficult to decide whether this task is like the human short-term memory tasks or not, since visual short-term memory is little studied in man. Most studies use digits or words, for which errors often result from acoustic conftisions (Broadbent, 1971); and most studies of rehearsal investigate verbal rehearsal. It might be possible to establish whether other primates have short-term memory, and whether delayed response and matching tasks test it, by determining whether retrieval is affected by a distracting task, or only by interfering stimuli which are similar to those being remembered. In human short-term memory the effect is probably one of distraction, rather than of interference as in long-term memory (Broadbcnt, 1971). The issue is not yet settled for nonhuman primates by the studies of Jarrard and Moise (1971) and Jarvik el ~ l (1969), . since they have not compared memories for the long or short term. , Man’s abilities can be compared with those of other primates not only on laboratory tasks, but also on those abilities observed in the field. Several qualitative diKerences claimed up till only a few years ago have been disproved by recent ficld studies. Man ha’s been defined as the “Toolmaker” (Oakley, 1967); and yet it is now clear that not only do many primates occasionally use tools, but cebus monkeys (Cebus) and chimpanzees also make them (in Van Lawick Goodall, 1970). Kroustov (1970) carried out a study from which he concluded that chimpanzees, unlike man, cannot use tools to make tools. But his attempt to persuade a chimpanzee to use a stone tool to shape a piece of wood was absurd, since he never gave the animals experience with the stone tool first, so that they could learn its properties. Furthermore, Wright has succeeded in teaching an orang-utan (Pongo pygmaeus) to make a stone tool using a hammerstone (in Pfeiffer, 1972). Man has also been

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defined as being the only primate who possesses culture (White, 1959), but it is now known that traditions of behavior are transmitted in other primates (Kummer, 1971). One remaining distinction is that only man has language. However, although other primates are not specialized for auditory communication, chimpanzees seem to have certain abilities which are preadaptive for language. I t has been shown that they can be trained to intentionally communicate (Gardner and Gardner, 1971) and that they can appreciate symbols (Premack, 1971b). The distinctive human ability of language evolved in our ancestors, and the study of other primates may still help us to understand that evolution. 2. Brain Similarly the evolution of the human brain can be adequately described only when it is seen in the perspective of trends within the other primates. Comparisons of man’s brain and those of nonhuman primates might indicate that there is an area or structure which is unique to man’s brain, or that an area is larger in man’s brain than it is in that of the nonhuman primates. The second finding would not be possible to interpret as it stood, since it is known that the weight of the brain is related to the size of the body, and that the size of an area is related to the size of the brain. These relationships would have to be taken into account by the use of brain: body or area: brain ratios. However, it is not valid simply to compare the brain:body ratio across differing weights, since the ratio itself decreases with increasing body weight, whether the body weight is changing in ontogeny or phylogeny (Schultz, 1941). It is for this reason that the comparison by Cobb (1965) and others of the brain :body ratio of 1 :12 for the squirrel monkey (Saimiri sciureus) and that of 1 :45 for man is invalid and cannot be used to throw doubt on the usefulness of the brain:body ratio. It is necessary to determine first the relationship between brain and body with increasing body size, and between a brain area and the whole brain with increasing brain size, and then to ask whether an obtained value fits the one predicted by these relations. This technique was used by Stephan and Andy (1969; Stephan, 1972) to study changes in the brain within the nonhuman primates, and by Passingham (1 973) to study differences between the brains of man and of other primates. The brain: body ratio is useful because brain weight and body size can be shown to be related (von Bonin, 1937). In man the brain can be shown to be more closely related to height than weight (Pakkenberg and Voight, 1964). These relationships presumably result from’thefact that a percentage of the body weight and height is made up by muscle, and the size of the brain is to some extent determined by the number of efferent nerves to muscles. But the relationship would be looser the more the fat, since there is no reason why the brain should be related to body fat. It appears that a t very large a

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and very small body weights, such as for the gorilla (Gorilla gorilla) and the talapoin (Cercopithecus tulapoin) , the ratio may give paradoxical results, suggesting that the gorilla’s brain is much less and the talapoin’s much better developed than we would suspect to be the case (Stephan and Andy, 1969). It may be that the parabolic relationship as suggested by Count (1947) would be a better fit for extreme values of body size than a linear relationship. Not only does the brain:body ratio break down at low and large body weights, but it may well also be invalid as a means of comparing land and sea mammals. Stephan (1972) finds that seals (Pinnipediu) and toothed whales (Odontoceti) appear to have a brain which is at least as well developed as the highest nonhuman primates. It is quite possible that they do, since seals, like chimpanzees and cebus monkeys, are good circus performers, and dolphins, closely related to whales, are similarly good at circus tricks. All these have high encephalization indices in the data of Stephan (1972), although the dog, which is also a good circus performer, may have a lower index of encephalization (von Bonin, 1937). On the other hand, the ratio may be quite meaningless when used to compare a mammal whose body structure is adapted to locomotion on land with one that lives in the sea. It would be better to relate the brain not to the body weight or height, but to the total number of sensory and motor nerves reaching and leaving the brain. Such a relation would give information on the amount of central processing over and above that needed for the receiving of information and execution of commands. Krompecher and Lipak (1966) have suggested comparison with the spinal cord, which carries many, but not all, of these nerves. Radinsky (1967, 1970) has argued that, where only the skull and not the brain or spinal chord are available, comparison might be made with the width of the foramen magnum. This has the advantage that it may also be used for fossil skulls.

FUNGTIONS B. SENSORY The most striking change in the evolution of the sensory systems from insectivores to primates, and from prosimians to simians, is the decrease in the use of the sense of smell and increase in the importance of vision. The snout of fossil insectivores of the Cretaceous is longer than that of fossil prosimians (Romer, 1966), and the snout of fossil prosimians longer than that of fossil catarrhines (Simons, 1962). This may reflect both the decrease in the size of the olfactory turbinal system and the decreasing use of the mouth to obtain and hold food. Radinsky (1970) has commented that in Smdodectes, a fossil prosimian of the middle Eocene, the olfactory bulbs are reduced, although they are relatively larger in Tetonius. Comparison of a modern series by Stephan and Andy (1970) shows that the olfactory bulbs of the prosimian Cheirogaleinae and Galagidae tend to be within the range

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24 1

of insectivores, and that those of the prosimian Lemurini and Lorisidae are smaller. There is a further decrease in the simiae, the olfactory bulbs of man being little reduced compared with the great apes. As yet, Stephan and Andy (1970) are only able to provide qualitative evidence that there has been a corresponding decrease in the olfactory structures in the paleocortex and amygdaloid nuclei. The decrease in the importance of olfaction is paralleled by the increase in the use of vision, particularly of stereoscopic vision. Simons (1962) has summarized the evidence from the study of the size and orientation of the orbits in fossil prosimians. The convergence of the orbits correlates in modern primates with the number of uncrossed fibers in the optic tract. The common tree shrew (Tupaia glis), an insectivore (Martin, 1968a), has laterally placed eyes and under 10% of its optic fibers are uncrossed, compared with prosimians, such as the galago (Galago crassicaudatus) and the slow loris (Nycticebus cougang), which have greater convergence of the orbits and 4040% of uncrossed fibers (Cartmill, 1970; Giolle and Tigges, 1970). Optic fibers pass to many centers, of which the two most studied are the superior colliculi (optic tectum in nonmammalian vertebrates) and the striate cortex. The only comparative evidence on the superior colliculi in primates comes from Tilney (1927), and it is inadequate, since he compared the size of the superior colliculi with the cross section of the brain at that level, without independently determining whether that cross section itself changed. Stephan (1972) has data only for the mesencephalon as a whole. Striate cortex is truly striate in primates, but it is similar in a t least two other semiarboreal mammals-an insectivore, the tree shrew (Snyder and Diamond, 1968), and a rodent, the gray squirrel (Sciurus carolinensis) (Kaas et al., 1972). It increases in the prosimians, but within simians there is little trend (Stephan, 1969); and man’s striate cortex is of the size predicted for a primate of his body weight (Passingham, 1973). The striate cortex forms proportionately less of the total neocortex in higher than in lower primates (Stephan, 1969). Figure 2 shows that the striate cortex of man is 2.4 times as small in proportion ot the total neocortex as would be predicted for a primate with his size of neocortex. This implies that other areas of neocortex have grown more than would be predicted. C. MOTORFUNCTIONS The basic primate adaptation is the development of a prehensile hand for clinging to small branches (Cartmill, 1972). The ancestral insectivores probably explored and manipulated their environment in a similar way to modern insectivores, exploring with their noses and whiskers, and manipulating with their mouths. Instead of the clawed hands and feet, as in tree shrews, primates developed hands with nails and tactile pads for exploration and

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1.01 2.0

1

1

3.0 4.0 5.0 Log Neocortex (mm3)

6.0

FIG.2. Krgression line for striate rortex on neocortex. Log striate cortex (nm3) = -0.13 + 0.78 x log neocnrtrx (mm3). H = Homo. Data from H. Stephan (personal communication) and Stephan et al. (1970).

manipulation. Thc representation of the mouth, hands, and feet in the neocortical sensory and motor areas must have changed, although fossil endocasts can provide only a little evidence of such changes (Radinsky, 1972). If the representation is compared in modern mammals, it can be seen that in both the rat and the cat the mouth area has a larger representation and that the representation of each digit is smaller than in the rhesus monkey (Woolsey, 1938). Woolsey (1958) has also shown that the representations of the hand and foot arca in the somatosensory and motor cortex have increased disproportionately within the primates, causing distortion of the map. The large representation of the hand in the somatosensory and motor cortex has led Washburn (1959) to suggest that it might be correlated with his skill with tools. However, Passingham (1973) has argued that the maps of rhesus monkey and of man compared by Washburn (1959) are not strictly comparable. Furthermore, crude measurements suggest that if anything there is a decrease in the representation of the foot rather than an increase in the proportion of the total map accounted for by the hand area. Such a change might be correlated with the decrease in the mobility and individual use of the toes in man compared with other primates. Even if the hand area was larger in man than in rhesus monkey, no conclusion about differences between man and other primates could be reached until a comparable map is published for chimpanzees. Noback and Moskowitz (1963) have put forward another suggestion to account for man’s manual skills. They quoted the work of Kuypers (1958a,b) on the projection of fibers from caudal motor cortex in rhesus monkey, chimpanzee, and man. Kuypers (1958a,b) found that in the chimpanzee

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many more fibers terminated directly on the motor neurons of the pons and lower brainstem than did so in the rhesus monkey, in which most fibers were found to terminate in the lateral tegmentum. However, the difference between the number of such fibers in chimpanzee and man was found to be slight, and such a small anatomical difference could clearly not account for any large behavioral differences.

D. LEARNING The primates are characterized less by anatomical and behavioral specializations, than by a generalized anatomy and adaptability of behavior (le Gros Clark, 1971). This may be seen not only in their ability to tolerate different environments, to accept different foods, and to move in different ways, but also in their capacity for both individual and social learning (Jolly, 1972). This capacity might be correlated with the size of the brain, of the neocortex, of association cortex, or with the organization of cortex. 1. Brain Size

The brain size of fossil primates may be roughly estimated from the cranial capacities of their skull, and related to the size of the hindbrain as estimated from the size of the foramen magnum (Radinsky, 1967). Using this ratio Radinsky (1970) has shown the similarity in brain size of some fossil prosimians to modern prosimians. For a living series, both brain and body can be weighed. Comparisons of primates with other mammals suggest that the indices of “encephalization” are greater for simians than prosimians, and for primates than for other groups, although there may be overlap between groups (von Bonin, 1937; Stephan, 1972). It appears that the greatest selection pressure for brain weight has been within the primates. This pressure must have been great for man, because his brain is 3.1 times as heavy as would be predicted for a primate of his body weight and 3.0 times the size predicted for a simian (see Fig. 3).

2. Neocortex The neocortex is large in proportion to the total brain in primates. Harman (1957) claimed that the proportion was 300/, in rodents, 40-46% in carnivores, and 4 6 4 8 % in primates. The volume of neocortex also increases within the primates, such that in chimpanzees it is roughly 60 times as large as would be predicted for a hypothetical insectivore of the same body weight (Stephan and Andy, 1969). Furthermore in man it is roughly 3 times as large as would be expected for a primate matched for body size (Passingham 1973). Not only does the volume of the neocortex increase within the primates, but so also does the ratio of neocortex: brain (volume) : in prosimians it is 50.5%, in monkeys 66.8%, in apes 74.4%, and in man 80.4% (based

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3.0L 1.5

2.0

2.5

3.0

AND G . ElTLINGER

3.5

,

4.0

I

4.5

5.0

I

Log Body Weight (Grams)

FIG.3. Regression lines for brain on body weight. Lower line is for nonhuman primates, upper line for simians. Dots are for prosimians, and circled dots for simians. For the nonhuman primates log brain weight (mg) = 1.96 0.76 x log body weight (grams). For simians log brain weight (mg) = 2.31 + 0.69 x log body weight (grams). H = Homo. Data from Stephan ct al. (1970).

+

on data from Stephan et al., 1970). However, although man has proportionately more neocortex than other primates, he does not have more neocortex than would be predicted for a primate of his brain size (see Fig. 4).

3. Association Cortex Evidence from lesion studies (see Section 111) suggests that the areas of neocortex most critical for learning are the “association areas.” These were defined by Flechsig (1901) as being the “terminal zones” in myelinization during ontogeny, that is, the last area to myelinate. They were called “association areas” because they were thought to associate “ideas.” They are now defined, however, in one of three other ways; by cytoarchitecture, by studies using electrical recording and stimulation, or by thalamic connections. On cytoarchitectural grounds, areas are said to be ‘Lassociationareas” if they are neither koniocortex nor agranular cortex (Bailey and von Bonin, 1951). By the criteria used in evoked potential and stimulation studies,

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log Brain Weight ( m g )

FIG. 4. Regression line for neocortex on brain weight. Log neocortex (mm3) = + 1.10 x log brain weight (mg). H = Homo. Data from Stephan et al. (1970).

-0.71

association cortex is that neocortex which is not part of a sensory or motor map (Rose and Woolsey, 1949). Finally, association or “secondary” areas may be defined as being those areas which are connected to a thalamic nucleus which has no extrinsic connections (Rose and Woolsey, 1949). But as Chow and Hutt (1953) have demonstrated, differing areas are defined by these three methods. For example, the area PC in the parietal cortex of the rhesus monkey is not koniocortex, yet it receives connections from an extrinsic nucleus of the thalamus, the nucleus ventralis posterior (Walker, 1938a; von Bonin and Bailey, 1947). Similarly some of the evoked potential maps derive their input not from thalamic extrinsic nuclei but from corticocortical connections (Cowey, 1965). Cortex which is not granular in the rat receives projections from the dorsomedial nucleus, which in the rhesus monkey projFcts Ito granular frontal cortex (Leonard, 1969), so that comparisons on the basis of cytoarchitecture would conclude that the rat had no frontal association cortex, and yet it is clear from thalamic connections that it does have frontal association cortex. There is, however, a problem

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in the use of the distinction between intrinsic and extrinsic nuclei suggested by Rose and Woolsey (1949), that nuclei not known at that time to have extrinsic connections are now known to do so. For example, the pulvinar has projections from the superior colliculi in the tree shrew (Snyder and Diamond, 1968), squirrel monkey (Mathers, 1971), and rhesus monkey (Mishkin, 1972), and further research may show that it receives projections from other structures. It is not clear, therefore, how comparisons of association cortex in different primates can be made. The problem is aggravated by the fact that the term “association cortex” implies a function; that association cortex is cortex which “asso~iates,”or more generally subserves cognitive rather than sensory or motor functions. Association areas should therefore be defined on the basis of studies of function. However, ablation studies are not yet able to provide information on the exact boundaries of areas with particular functions. It seems most practical to compare the extent Qf areas with projections from particular thalamic nuclei, and to determine by ablation studies what function these areas have. I t will then be unnecessary to make a distinction between extrinsic and intrinsic thalamic nuclei, and comparisons may be made of areas that have the same function. While the hedgehog, for example, appears to have little association cortex as defined by the old criteria (Diamond and Hall, 1969; Kaas et al., 1970), it does have areas tp which the lateral posterior nucleus and the dorsomedial nucleus project, and it may be that by the new criterion these are homologous with the areas to which these same nuclei project in primates. There is little quantitative information on the relative sizes of the different thalamic nuclei in primates. Stephan (1 969) has published evidence for the lateral geniculate in a large series of primates, and Masterton and Skeen ( 1 972) have measured the dorsomedial nucleus in three hedgehogs (two Hemzechinus auritus, one Paraechinus hindei), two tree shrews ( Tujaia glis) and two bushbabies (Galago senegalensis). But Masterton and Skeen (1972) failed to make clear how their relative values for volume of the dorsomedial nucleus were calculated. Hopf (1965) has presented data for all the thalamic nuclei in four primates including man, Table I gives the values obtained by Hopf (1965) for the pulvinar and dorsomedial nucleus in the ring-tailed lemur (Lemur catta), crab-eating macaque (Macaca fusciculuris), orang-utan, and man. The pulvinar projects in rhesus monkey to prestriate and inferotemporal association cortex (Chow, 1950), and the dorsomedial nucleus to frontal association cortex (Walker, 1938a). The percentages of the thalamus accounted for by each of these two nuclei increase as the thalamus increases in size. There is no indication from the figures in Table I that the percentages for these nuclei are greater in man than would be expected in view of the increase in the thalamus as a whole.

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TABLE I VOLUMES OF THALAMUS (GRAYSUBSTANCE), PULVINAR, AND DORSOMEIXAL NUCLEUS IN FOURPRIMATES

Species Homo Pongo pygmaeus Macaca farcicularis Lemur catta

Thalambs (gray substance) (cm3)

Pulvinar (cm3)

Dorsomedial nuc1eus (cm3)

14.71 8.05 1.49 0.72

4.03 (27.4) 1.95 (24.2) 0.31 (20.8) 0.09 (11.8)

2.03 (13.8) 0.76 (9.4) 0.13 (8.4) 0.06 (8.2)

a Figures in parentheses are the values for the volume of that nucleus/volume of thalamus x 100. Data from Hopf (1965).

Unfortunately, data on projections from thalamus to neocortex are not available for a series of primates. The only possible information on which comparisons could be made come from cytoarchitecture, and there is only one series for which the same methods and criteria were used, that studied by von Bonin and Bailey (1961). Comparable cytoarchitectural maps are provided for two insectivores, the elephant shrew (Elephantulus myurus) and the tree shrew (Tupaia glis), and for six primates, the bushbaby (Galago demidovii), the tarsier ( Tarsius spectrum), the marmoset (Callithrixjacchus), the rhesus monkey (Macaca mulatta), the chimpanzee (Puntroglodytes) and modern man (Homo sapiens sapiens). However, quantitative data are available only for the tarsier, the marmoset, the guenon (Cercopithecus), the chimpanzee, and man (Shariff, 1953), and the data for the marmoset have been questioned (Haug, -1956). Shariff (1953) reported for these five primates the volumes (mm3) of koniocortex, agranular cortex, and eulaminate cortex, but it is not clear how he classified cortex which would later have been termed transitional (Bailey and von Bonin, 1951). It was found that the percentages of volume of eulaminate cortex:volume of total neocortex were for the tarsier 57.2%, marmoset 66.6%, guenon 69.7%, chimpanzee 82.9%, and man 84.9%. The percentage therefore increases with increasing brain size, but Passingham (1973) has shown that the percentage for man, although greater than that for other primates, is no different from the percentage predicted for a brain ,of his size. Holloway (1968) argued that man had more eulaminate cortex: koniocortex, using the A: S ratio (association: sensory). But he did not carry out a regression analysis to see whether the percentage for man differed from that predicted. Furthermore, the ratio has little justification ; the relevant ratio is that of association cortex to both sensory and motor cortex. It is possible that, while man has no more association cortex than expected for a primate of his brain size, some subareas of his association

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cortex might be larger than expected, although others would necessarily be smaller. This question has sometimes been discussed on the basis of studies of the gross proportions of the lobes, carried out either on fossil endocasts or on modern brains. But measurements on the lateral surface take no account of cortex buried in fissures. Furthermore, the measures usually taken include both primary and association cortex, and cannot therefore be used to make statements about association cortex. Radinsky (1970) has argued that the frontal lobes of the fossil prosimians Smilodect.es, Tetonius, and Necrolemur are small, but his measure is one of both motor and association cortex, and it ignores any frontal association cortex that might be, as in the rat, on the medial surface. Measures of the temporal lobe, however (Radinsky, 1970), might give a better representation of association cortex since in the tree shrew (Diamond et al., 1970) and in primates, such as thq rhesus monkey (Walker, 1938a), the projections of the medial geniculate nucleus terminate in only a small area. It should be possible to study the downward extension of the temporal lobe in fossil primates, as in the tree shrew and squirrel (Kaas et al., 1972), and perhaps to correlate it with the importance of vision in these fossil primates. Von Bonin (1963) has reviewed the attempts to determine the size of association and other areas from the endocasts of fossil hominids. Holloway (1972b) has recently published a study of a natural endocast of a robust australopithecine (SK 1585), and has suggested that there is an increase in parietal and temporal cortex compared with the brain of pongids. This assessment is based on the estimated position of the lunate sulcus. But the lunate sulcus cannot be identified on this endocast (Holloway, 1972b), and its identification on the endocast of another Australopithecine, the Taung skull, is controversial (von Bonin, 1963; Holloway, 1972a). Von Bonin (1963) took gross encephalometric measures on a series of endocasts of fossil hominids. The technique was similar to that used with modern brains as described by von Bonin (1941). Table I1 shows his results for those measures for which data on both fossil endocasts and modern brains are available. These values may be compared with those for the rhesus TABLE I1 ENCEPHALOMETRIC INDICES FOR FOSSIL HOMINIDS a Species

N

Frontal length

Parietal length

Occipital length

dushalopithecus Homo erectus Homo sapiens ncandwthalensis Homo sapiem s a p h

2 5

61.4 (60.3-62.5) 58.4 (52.0-63.0)

21.3 (20.0-22.6) 25.6 (17.5-30.0)

17.3 (17.1-19.5) 16.1 (13.6-19.5)

6 5

54.8 (50.4-60.0) 59.7 (53.1-59.9)

28.2 (21.7-36.9) 27.7 (21.8-30.4)

17.5 (11.5-21.9) 15.4 (11.4-21.8)

Data from von Bonin (1963). The indices for frontal, parietal, and occipital length are defined in Passingham (1973).

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249

monkey, chimpanzee, and modern man, to be found in Passingham (1973). Given the variation within the species and subspecies, most of the differences between them appear insignificant on the measures of frontal, parietal, or occipital length. Furthermore, the more significant differences, for example between the parietal lobe in Neanderthal man and modern man, are probably due to the problems of identifying points on endocasts. These results are not surprising since measurements on brains show that there are no differences on these indices between modern man and chimpanzee. Furthermore, man does not differ significantly from chimpanzee on the measures of temporal length and frontal, parietal, and temporal breadth (von Bonin, 1941 ; Passingham, 1973). On the other hand, the chimpanzee does differ on some of the measures from the rhesus monkey. These results show only that the brains of fossil hominids and modern man appear to be similar in the proportions of the lobes to the chimpanzee. The available data on which comparisons can be made for subareas of association cortex is that provided by studies of cytoarchitecture. The frontal lobes have for long been thought to subserve activities and qualities that distinguish man from other primates (for review, see Gross and Weiskrantz, 1964). However, only Brodmann (1912) has ever measured the frontal association cortex in a series of primates including man. Passingham (1973) has shown that man has a prefrontal area which, on Brodmann’s data, is roughly two times as large as that predicted for a primate of his neocortical area. However, there are two reasons for doubting that this conclusion is well founded. First, von Rose (1935) gave proportions of prefrontal cortex for man which were much lower than those given by Brodmann (1912), while giving proportions of the whole frontal lobe which were similar. This suggests that they disagreed radically on the boundaries between prefrontal and frontal cortex. Second, as has already been noted, cytoarchitecture may be a poor guide to prefrontal cortex as judged from thalamic connections, particularly in lower mammals. Until there are further studies, it is only safe to conclude that man has relatively more frontal association cortex than other primates, but that it is not known whether he has more than would be expected for a primate with his size of neocortex. Because the results for the frontal association cortex have been disappointing, some have pinned their hopes on the temporal or parietal association cortex. The dorsoventral extent of the brain is relatively greater in man than in the chimpanzee (von Bonin, 1941), and Weidenreich (1948) has shown that in fossil hominids the increase in height is most marked at the level of the parietal lobe, However, as has been pointed out before, such measures are not measures of association cortex. Von Bonin (1963, p. 51) stated that the parietal association field is “quite small in monkeys, gets bigger in anthropoids, and is very large in man. These facts are, of course,

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well known.” His evidence presumably came from a qualitative analysis of the parietal areas PE, PG, and PF in different primates (von Bonin and Bailey, 1961). But no quantitative data are provided. There is no doubt that man has more such cortex, just as he has more prefrontal cortex and more total association cortex, but without quantitative data we cannot say whether he has more than would be expected for a primate with his size of neocortex. Indeed, Shariffs (1953) results for total association cortex, if they are ;o be relied on, suggest that he does not. Nor are quantitative data available for temporal association cortex. Only for prestriate cortex is there any information, and while the ratio of prestriate to occipital cortex is higher in man, he has exactly that proportion which would be predicted from the trends in other primates (Solnitzky and Harman, 1946; Passingham, 1973). It is not yet possible to make comparisons of association cortex between primates as defined by thalamic connections. There is information on the rough areas in which thalamic projections terminate for the tree shrew (Diamond et al., 1970) and three primates, the rhesus monkey (Walker, 1938a). chimpanzee (Walker, 1938b), and man (Walker, 1966). However, it is not possible on the basis of published data to estimate the size of the various areas to which they project. The only suggestion in the literature that there might be differences in the projections in different primates comes from Rose and Woolsey (1949)’ who argued that “true” association cortex is neocortex without afferents from the thalamus and dependent on corticocortical connections. Walker (1938a,b) reported that the temporal association cortex of the rhesus monkey and of the chimpanzee had no thalamic projections, but Chow (1950) showed that he was wrong for the rhesus monkey, since there were projections from the pulvinar to inferotemporal cortex, although only slight thalamic projections to superior temporal association cortex. The view that the temporal lobe of man did not have similar projections was shown to be incorrect by Simpson (1952) and Walker (1966). While there may be a remaining area in man’s superior temporal association cortex with a few thalamic connections, there is no reason to think that it differs from the similar area in the rhesus monkey. 4. Cell Density

Since no marked differences have been found in the gross structure of the human neocortex as compared with that in other primates, it is natural to try to find the basis for man’s abilities in comparisons of the finer structure of primate brains. However, the only published evidence permitting such comparisons is that of Shariff (1953) on cell size and density. In mammalian brains (Tower, 1954) re11 density decreases with increasing brain weight. Similarly in primates, as Shariffs (1953) data show, cell density

CORTICAL FUNCTIONS IN PRIMATES

25 1

decreases as neocortical volume increases. However, while man has a lower cell density than other primates it is no lower than would be expected for his neocortical volume (Passingham, 1973). Holloway (1968) has reviewed the evidence suggesting that the lower the cell density, the greater the proliferation of cell processes, and thus the greater the chances for interaction between cells. But evidence on dendrite branching is available for only one primate, man (Sholl, 1956), so that there are as yet no comparative data on a primate series with which to test this possibility. Furthermore, since it has already been shown that the changes in cell density are associated with changes in neocortical size, this might be true also of the proliferation of cell processes.

E. MEMORY A more specific suggestion than the one that there are differences in the capacity to learn within primates is that there are differences in the ability to remember. Krantz (1968) suggested that “persistence hunting,” as in Homo erectus and Homo sapiens, selects for a bigger brain and better memory. He cites evidence from Rensch (1956) that memory is related to absolute brain size in vertebrates. Unfortunately, however, no one has carried out a study to see whether there is any such relation between memory and either absolute or relative brain size in primates. Furthermore, while tests have often been carried out of the ability of primates to remember over a few seconds or minutes, there are few studies of their ability to remember over weeks or years (Strong, 1959; Davis, 1971). Nor is there yet any good evidence as to whether there is a distinction in nonhuman primates between short- and long-term memory. Studies on human patients have suggested that the hippocampus (archicortex) is involved, together with other limbic structures, in the storage or retrieval of long-term memories (Scoville and Milner, 1957; Warrington and Weiskrantz, 1970). Comparisons have sometimes been made between the count of 500,000 fibers in the fornix for the rhesus monkey (Daitz and Powell, 1954), the main efferent fiber system from the hippocampus, and the count of 1,200,000 for man (Powell et ad., 1957). Such comparisons of absolute fiber counts cannot be meaningfully made without relating them to the brain size. Furthermore, a more relevant comparison would be one between man and the chimpanzee. Stephan and Andy (1970) have reported comparative data for hippocampus in primates, and they found that it increases relative to body weight from insectivores to prosimians and then shows no clear trend. Figure 5 compares the value predicted for man from the trends within other primates, and the value obtained. When related to body weight the hippocampus is 2.1 times as large as expected. On the other hand, it will be seen from Fig. 6 that the relation between

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R. E. PASSINGHAM A N D G . ETTLINGER

ld1.0

1.5

2.0

1

2.5 3.0 3.5 4.0 Log Body Weight (grams)

4.5

5.0

I

5.5

FIG.5. Regression line for hippocampus on body weight. Log hippocampus (mm3) = 1.12

f 0.53 x

log body weight (gtam). H = Homo. Data from Stephan et al. (1970).

hippocampus and brain size which is found for other primates also fits man: in other words, the increase in hippocampus is accounted for by the change in total brain size. ??.LATERALJZATIUN OF FUrJCTioN In the neocortex of man many functions are associated more closely with one hemisphere than with the other. Of these functions, the most strongly lateralized is language, which is represented in the left hemisphere of almost all right-handers (Milner ct al., 1964). Several attempts have therefore been made to discover whether there are anatomical differences between the left and right hemisphere. Von Bonin (1962) reviewed these and concluded that the only difference was that there was a tendency for the left sylvian fissure to be longer than the right. Geschwind and Levitsky (1968) suggested that this difference is probably related to the difference they found between the left and right planum temporale, an area in the supratemporal plane near Heschl's gyrus. This was found to be larger on the left in 57'7, of cases, and on the right in 11 %. The fact that the right planum temporale tends to be smaller might be due to the greater development of nontemporal areas in that hemisphere, but no measures of volume or area of neocortex are given

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CORTICAL FUNCTIONS IN PRIMATES

2.51 3.0

I

3.5

4.0 4.5 5.0 5.5 Log Brain Weight (me)

6.0

6.5

%

FIG. 6. Regression line for hippocampus on brain weight. Log hippocampus (mm9 = -0.05 + 0.65 x log brain weight (mg). H = Homo. Data from Stephan et al. (1970).

for the hemispheres as a whole. Geschwind (1972) also quoted evidence from Wada that this difference is found in the brains of newborns. It is tempting to conclude that this anatomical asymmetry in human brains is related to language, but such a conclusion would be premature until studies have been carried out on other primates to establish whether they also have a n asymmetry in the supratemporal plane.

ABILITIES G, CROSS-MODAL Althougli nonhuman primates do not naturally have language, they might have cognitive abilities which are preadaptive for language. Geschwind (1965) has suggested that cross-modal abilities might be necessary for language, and that the angular and supramarginal gyri of the parietotemporal cortex might subserve cross-modal abilities, Comparisons of man with other primates in cross-modal abilities are discussed in Section 111; here it is necessary only to compare the brains. Geschwind (1971) stated that the angular gyrus is better developed in man than in the chimpanzee, and even more so compared with the rhesus monkey. But the angular gyrus includes the cytoarchitectural areas PF and PG in the parietal lobe, and, as already stated, there is no quantitative evidence which would allow comparisons of this area in the rhesus monkey, chimpanzee, and man. Furthermore, von Bonin and Bailey (1947) have quite clearly identified a large area in the parietal lobe of the rhesus monkey which is homologous with the angular gyrus in man. Finally, while it is assumed that there must be

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connections to this area from all modalities in man, it has been shown to be the case for the rhesus monkey (Jones and Powell, 1970; Pandya and Kuypers, 1969). There is therefore, as yet, no clear evidence that there are major differences between the angular gyrus in man and in other primates. H. LANGUAGE 1. Language Areas

It was argued in Section A, 1 that the only major difference between the behavior of man and that of primates lay in man’s possession of language. Both stimulation and lesion studies have localized the areas in the dominant hemispheres which are most critical for language to Wernicke’s area for speech comprehension, and Broca’s area for speech production ( Penfield and Roberts, 1959). The posterior area includes the angular and supramarginal gyri and posterior temporal cortex. The anterior area is in the third frontal convolution. Konorksi (1967) has stated that the arcuate fasciculus, connecting the posterior part of the temporal cortex with the frontal cortex, does not exist in chimpanzees. His evidence is based on a study by Bailey et al. (1943) on the chimpanzee using strychnine neuronography. However, such connections were reported for the rhesus monkey by Bailey et al. (1950) using the same technique, and have been found in the same animal using anterograde degeneration techniques (Jones and Powell, 1970; Pandya and Kuypers, 1969). Von Bonin (1952) also reported for the chimpanzee that there were projections from superior temporal cortex to the area of frontal cortex termed FCBm. The designation FCBm was given by von Economo (1929) to the area in the third frontal convolution of man’s brain called Broca’s area. The main characteristic of this area’s cytoarchitecture is that there are large pyramidal cells in the third and fifth layer, Von Bonin (1952) stated that a homologous area could be seen in the brains of the bushbaby, marmoset, rhesus monkey, and chimpanzee, although von Bonin and Bailey (1961) suggested that this area might be more difficult to recognize in these primates than had a t first been suggested. In man it has also been shown that electrical stimulation of this area inhibits ongoing speech (Penfield and Roberts, 1959). Various points in the brains of the rhesus monkey (Robinson, 1967, 1972) and the squirrel monkey (Jurgens and Ploog, 1970) have been stimulated electrically in an effort to evoke vocalizations, but no one has reported whether stimulation of FCBm inhibited ongoing vocalizations. On the other hand Myers (1972) has stated that bilateral ablations of this area in the rhesus monkey do not impair spontaneous vocalizations. Whether there are homologous areas in other primates for Broca’s area in man cannot as yet be settled.

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2. Brain Size Since the clearest behavioral difference between man and other primates is that of language, and the clearest anatomical difference is that of brain size, it is natural to ask whether language is related to brain size. Lenneberg (1964) firmly denied that it is, and based his arguments on the fact that nanocephalic dwarfs with brains of low absolute size are able to understand and produce language. However, the value for cranial capacity given by Lenneberg (1964) for a dwarf is the value of a hypothetical dwarf, and it is obtained by plotting the cranial capacities and body weights of the dwarfs in Seckel (1960), and then predicting the most likely cranial capacity of a dwarf weighing 13.5 kg. Since at that time nothing was known of the growth rates of the brains of these dwarfs this procedure may be invalid because it assumes a particular growth rate. Furthermore, no adequate histological study has ever been carried out on these brains to determine whether their cell sizes are abnormal or whether the number of cells is larger than would be expected for brains of that size. Only gross examinations have been carried out (Seckel, 1960). The brain weight of nanocephalic dwarfs is available for only two cases, and for only one of these, case 6, was an adult brain weight obtained (Seckel, 1960). The measures for the other dwarfs are of cranial capacity estimated from external measurements of the skull, and for only one dwarf, case 9, was a n adult estimate made. However, although case 6 had a brain which was much larger in relation to his body weight (at age 22 years 11 months, brain weight 665 g, body weight 7.2 kg) than that of case 9 (at age 22 years, brain 51 7 g, body weight 18 kg) he had a vocabulary of only 12 words. The only case, therefore, that can usefully be compared with great apes is case 9. Figure 7 shows the regression for cranial capacity on body weight for 45 great apes (19 orang-utans, 23 chimpanzees, and 3 gorillas) for which data were provided by A. H. Schultz (personal communication). The cranial capacity: body weight for case 9 is plotted on this figure, and Table I11 shows that the cranial capacity is 1.7 times larger than would be predicted for a great ape of that body weight, and this difference is significant. Also plotted on the figure are the estimated cranial capacities and body weights for some fossil hominids. The cranial capacities are taken from Tobias (1971) and Holloway (1970, 1972a), and the estimated body weights from Tobias (1971) and Lovejoy and Heiple (1970). Ranges are given for body weight because of the difficulty of giving exact estimates on the basis of postcranial bones. These calculations, comparing the fossil hominids with great apes, differ from those of Jerison (1955, 1963) and Tobias (1971) in making no further attempt to convert cranial capacities into estimated number of cells. I t can be seen from Table I11 that the values for Australopithecus africanus are similar to that of case 9, although they would be much

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R. E. PASSINGHAM AND 0. ETTLINGER

HS 3.1

1.1 1.2 1.3 1.1 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 LOQ Body Weight (Kg)

FIG. 7. Regression line for cranial capacity on body weight for the pongids. Log cranial capacity = 2.17 0.24 x log body weight (kg). Data for pongids from A. H. Schultz (personal communication). D = Dwarf, AA = Australopithecus africanus, AH = Australopithecus habilis, J = Homo erectus erectus (Java), P = Homo erectus pekinensis (Peking), HS = Homo sapicnr sapicnr. Continuous fine for AA is for cranial capacity as estimated by Tobias (1971), and dotted line is for cranial capacity as estimated by Holloway (1972a).

+

lower than values estimated for case 6. The values for other hominids are all higher. It is certainly not possible to determine whether any hominid possessed language by comparing it with the nanocephalic dwarf, case 9. However, attempts have been made to decide this in other ways. Vallois (1961) has reviewed studies of the pars triangularis or “cap” of the third frontal convolution as reflected on endocasts of fossil hominids. Holloway (1972b) also commented on signs of this area in the endocast of a robust australopithecine (SK 1585). But the study of this area on fossil endocasts is probably of little value for indicating the presence of language capacity, since even cytoarchitectonic studies have not yet shown differences between this area in man and other primates. An alternative method is that of Liberman (1968), who has studied the shape of the vocal tract in man and in other primates. He commented that a study of the skull of an australopithecine suggests that the pharynx may have been like that of a chimpanzee, and the Australopithew might therefore have lacked a pharyngeal region which could change its cross sectional area. As a result the vowel space would have been limited.

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TABLE I11 CRANIAL CAPACITIES IN THE HOMINIDS

Mean cranial capacity

N

(4

Australopithecus africanus

6

Australopithecus habilis Homo erectus erectus Homo erectus pekinensis Homo sapiens sapiens Homo sapiens (dwarf)

3

494 (435-540) 442 (428-485) 656 (633-684) 883 (750-1029) 1043 (915-1225) 1391 (1129-1685) 517

Species

7 5

20 1

’

Body weight (kg) 20-35 20-35 45-60 45-60 67 18

Index 1.4-1.6 1.3-1.5 1.9-2.2 2.2-2.4 2.6-2.8 3.4 1.7

The dwarf is case 9 of Seckel (1960). The estimates of cranial capacities in the upper line for Australopithecus africanus are taken from Tobias (19711, and those on the lower line from Holloway (1972a). The estimates for A . habilis, Homo erectus erectus, and H.erectus pekinensis are taken from Tobias (1971), and those for H.sapiens sapiens from Schultz (1941) for mean brain weight and Schultz (1965) for range. The estimated ranges for body weights for the fossil hominids are based on estimates in Tobias (1971) and Lovejoy and Heiple (1970). The index is a measure of how much greater the cranial capacity is than would be predicted for a pongid of the same body weight. All capacities differ significantly from those predicted.

However, although such studies may show the extent to which any vocal communication used the same range of phonemes as does the language of modern man, it is not certain that all forms of language could be excluded on the basis of this method. There is, therefore, no certain way of determining whether a particular fossil hominid possessed language. However, in view of the values for cranial capacity in the hominids discussed above, it would seem premature to argue, as does Holloway (1972a), that those models of language are discredited which suggest that a particular size of brain or neocortex is necessary for language. There is no evidence to exclude the possibility that in the hominids a certain brain size in relation to body size might be necessary, but not sufficient, for language.

I. CONCLUSION I n a paper on “Problems in the Evolution of the Mind,” Lashley (1949) claimed: “The only neurological character for which a correlation with behavioral capacity in different animals is supported by significant evidence is the total mass of tissue, or rather, the index of cephalization, measured by the ratio of brain to total body weight, which seems to represent the amount of brain tissue in excess of that required for transmitting impulses to and from the integrative centers.” The conclusion of this section of the paper must be similar, though qualified. I t must be admitted that many of the data used here are poor, and that, for example, the conclusions on association

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R. E. PASSINGHAM AND G . ETTLINGER

cortex based on the data of Shariff (1953) may have to be altered when Stephan (1972) has more data on the same topic. The available data are not always consistent. For example, calculations on the basis of the data of Stephan ( 1 969) suggest that man’s striate cortex is significantly smaller than would be expected for a primate of his neocortical volume; and it has been argued here that this finding implies that the rest of the neocortex has increased more than would be expected. Yet, on the basis of data from Solnitzky and Harman (1946), it was found that the prestriate cortex of man does not differ from that predicted; and on the basis of data of Shariff (1953), that the volume of association cortex for man fits the trends for other primates. These inconsistencies almost certainly depend on the poverty and inaccuracy of the available data, and firm conclusions will therefore have to await further data from Stephan (1972). The most striking change within the primates is an increase in size of the brain and neocortex, and attempts to correlate brain development with abilities within the primates might well start with size. However, it is important not to assume that this conclusion implies mass action, for it has been shown that, as the brain increases in size within the primates, so also the proportions of areas within, for example, the neocortex change. Since lesion studies have implicated association areas in learning (see Section 111), a correlation between size of brain and capacity to learn might reflect a relation between size of association areas and learning, since brain size and proportion of association areas are related. Man has been shown to fit the trends within the other primates for neocortex and association cortex but he has, nonetheless, proportionately more neocortex and association cortex. However, except for the size of his brain, it seems that the selection pressures affecting man and his hominid ancestors were similar to those affecting other primates. All the differences between the brain of man and those of other primates can be accounted for most parsimoniously by suggesting that the main selection pressure was for a larger brain. Holloway (1968) and others have thrown doubt on the relation between brain size and ability. They have two main arguments. They point out that there is overlap between the brain weight of the gorilla and of Australopithecus africanus, and between Homo erectus pekinensis and Homo sapiens sapiens (Tobias, 1963; Ashton, 1950). But, as can be seen from Table 111, this difference disappears when brain size is related to body weight. T h e second argument is that there is a vast range of brain sizes in modern man, and that within modern man brain size and ability seem to be poorly related (Cobb, 1965; Tobias, 1971; Weidenreich, 1948). But the range of brain weights put forward depends mainly on a few freak values from a n anecdotal literature and Tobias (1970) has pointed out how many factors have to be taken into account when removing the brain, estimating the size, and interpreting the

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results. For example, the low value of 1017 gm for Anatole France quoted by Cobb (1965) is for a n old man whose brain may well have lost weight with age. The range of 1129-1685 cm3 for cranial capacity obtained by Schultz (1965) suggests that the few extreme values quoted by Cobb (1965) and Tobias (1971) may misrepresent the true standard deviation. The latter authors also appear to misunderstand the nature of a correlation, since it is never enough to quote a few cases which do not fit in order to disprove a correlation. A correlation of even 0.7, probably much higher than any actual correlation between brain size and ability, fails to account for 51y0 of the variance. But, of course, the relation claimed is not between absolute but relative brain size and ability, and no body weights are given for the extreme cases. The Pygmies, for example, have a cranial capacity of 1108 cm3 (Dart, 1956), but they are also small. Women have a mean cranial capacity, 1330 cm3, which is smaller than that for men, 1446 cm3 (Schultz, 1965), but women are lighter and shorter than men. Until someone measures relative brain size in a large sample, and correlates it with performance on a standard test, it will not be possbile to know whether relative brain size is correlated with performance. The correlation will be bound to be low since many factors determine performance; but brain size may be one of them. Furthermore, even if brain size and ability are poorly correlated within a species, they may be more highly correlated in studies comparing species. The size of a correlation depends on the range of values sampled. Differences in ability between higher and lower primates may be due partly to differences in brain size, and until someone has done the relevant experiment it seems premature to reject this possibility. Cebus monkeys, for example, are similar to chimpanzees in encephalization (Stephan, 1972), and they are also the only primates other than great apes which have been found to be able to use stones as tools (Vevers and Weiner, 1963), make twig tools (in van Lawick Goodall, 1970), use sticks as weapons (Cooper and Harlow, 1961), and paint (Jolly, 1972). The connection between their brain size and their behavior might be spurious, but it is possible that it is genuine. Even if relative brain size was related to ability, and even if a certain value was necessary for language, it is almost certainly not a sufficient basis for it. This may be illustrated by pointing out that neither in Wernicke’s area nor in Broca’s area have differences yet been demonstrated between the left and right hemisphere, and yet anatomical specializations might be expected to account for the fact that language is localized most commonly in the left hemisphere. Nor have there yet been found anatomical changes in either the minor or major hemisphere during childhood which might parallel the development of dominance for language. Ettlinger (1971) has argued that such changes might particularly be expected in the minor hemisphere, because there is a limited period during development when this

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hemisphere can subserve language. If such differences between the hemispheres do exist, then these same differences have been missed in comparison of man with other primates. It seems a sorry conclusion that, on the present state of our knowledge, we do not know in what way man’s brain is specialized for language, since it is his possession of language which is the major behavioral difference between man and the nonhuman primates. 111. Behavior

A. SENSORY FUNCTIONS I t has been noted in the previous section that it is the neocortex which has shown the greatest increase within the primate order (Stephan and Andy, 1969). Some have been led by this fact to suggest that a process of “encephalization,” or more strictly “neocorticalization,” has taken place within the primates. However, it is essential to distinguish two different claims which might be made. First, it might be claimed that the neocortex subserves functions in primates and other mammals which are subserved partially or completely by paleocortex, archicortex, or subcortical structures in nonmammalian vertebrates. Second, it might be stated that these functions come to depend more fully on the neocortex as it increases in size within the primate order. The latter may be said to be a claim for “progressive neocorticaliza tion. ” Of the sensory systems, the claim for neocorticalization has been made most frequently for vision. If the claim is that striate cortex performs functions in primates which are subserved by non-neocortical areas in reptiles and birds, it is necessarily correct. However, Nauta and Karten (1970) have argued that the external striatum in reptiles and birds is homologous to the mammalian neocortex. If, however, the claim is that the striate cortex becomes more important for vision with increase in brain size within mammals, it is probably incorrect. The question can be decided by comparing the effects of striate lesions on vision in different animals. Marquis (1935) and Weiskrantz (1966) were forced to compare rat, cat, and monkey for lack of data on other primates, and, as Hodos and Campbell (1969) pointed out, this is not a phylogenetic series. However, for studies of neocorticalization it is not important that it should be. Nonetheless, it is not a useful series since there are projections from the lateral geniculate to the extrastriate area 18 in the cat (Glickstein et al., 1967), and therefore removal of striate cortex would not be expected to have the same effects in the cat as in primates, in which the lateral geniculate projects only to striate cortex. Comparisons may, however, validly be made between insectivores, such as the tree shrew, and members of the primate order. Hall and Diamond (1968) have reported that striate lesions in the hedgehog leave them relatively I

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unimpaired in relearning brightness discriminations but retarded in relearning discriminations between horizontal and vertical stripes. If the lesions encroach on extrastriate cortex the animals are not able to relearn. The results for the tree shrew are controversial and difficult to interpret. Snyder and Diamond (1968) originally reported that tree shrews (Tupaia glis) with total striate lesions were not impaired in relearning brightness or orientation discriminations, whereas animals with striate and extrastriate lesions were slow to relearn. But Ward and Masterton (1970) found that their tree shrews were impaired after striate lesions on brightness and pattern discriminations, and that they had poor acuity. Ware et al. (1972) were unable to find evidence of an acuity impairment, using stimuli which differ in the width of stripes and the size of the stimulus card from those used by Ward and Masterton (1970). Further research will be needed to clarify the differences in results obtained by the two groups. However, under certain conditions Diamond and co-workers have found a deficit on pattern discriminations if an annulus surrounded each stimulus in a pattern discrimination (Killackey et al., 1971) and if an irrelevant color is shown as a background to the stripes in a n 0rientatio.n discrimination (Killackey and Diamond, 1971). Nonetheless, the most striking claims are that removal of striate cortex may leave pattern vision unimpaired in situations in which there are no distracting stimuli, and this finding is related by Snyder and Diamond (1968) to the large size of the superior colliculi. The effects of striate lesions on the vision of primates has been studied only for the rhesus monkey and man. The results for both are controversial. Here only the effect of total striate lesions will be considered: those of partial lesions are discussed by Weiskrantz and Cowey (1970) for rhesus monkeys and by Teuber (1968) for man. Weiskrantz (1963) reported that a rhesus monkey with a nearly complete striate ablation could still discriminate between different amount of contours, but could not discriminate horizontal from vertical. Humphrey and Weiskrantz (1 967; Humphrey, 1970) found that another monkey with a striate lesion could detect and reach for stationary lights, and Humphrey (1972) has since found that it could detect and avoid obstacles. However, Humphrey has never been able to teach this animal a form discrimination. O n the other hand, Pasik and Pasik, (1971) have demonstrated with a large series of rhesus monkeys that striate lesions alone do not prevent the animals relearning flux, brightness, form and even color discriminations. I t is not possible to account for the differences in outcome at the moment. Complete lesions have been published only by Pasik and Pasik (1971), although the completeness of the lesion of the animal studied by Humphrey (1970, 1971) remains to be determined. Pasik and Pasik (1971) have shown that if a prestriate lesion is added to a striate, the animals are unable to relearn any but flux discriminations. I t is not known

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how much prestriate cortex was damaged incidentally in making the striate lesions in the experiments of Weiskrantz (1963, 1972) and Humphrey and Weiskrantz (1967). The data on man have been briefly reviewed by Humphrey and Weiskrantz (19671. Brindley et al. (1969) have reported on two patients who could tell when the light was turned on or off, but could not detect the steady state. However, in neither was there any evidence that the lesions were confined to the striate cortex, and indeed it is stated only that they involved the occipital lobes. Similarly, the patient of Ter Braak et al. (1971) was found at postmortem to have cysts in the occipital lobes and extensive softening in the occipital lobe, pulvinar, and other areas. Similarly, in other cases it is difficult to see how striate cortex alone could be involved if the lesion was space occupying or vascular. T h e human cases almost certainly have both striate and prestriate damage and can be validly compared only with those animals studied by Pasik and Pasik (1971) which had similar damage. There is therefore no evidence for the effect of total striate lesions in man, and no reason to think that the effects of striate and prestriate lesions differ in monkey and man. The question of progressive neocorticalization for visual functions within the primates cannot be answered on the basis of such limited data. T h e effects of total striate lesions sparing prestriate cortex have been studied in only one primate, the rhesus monkey. O n the other hand, the effects of striate and prestriate lesions appear to be similar in the two primates studied and may turn out to be similar in the two insectivores studied. However, for the moment it can only be said that the thesis of progressive neocorticalization for vision has not been proved. B. MOTORFUNCTIONS The hypothesis of progressive neocorticalization has also been put forward for motor functions. Noback and Moskowitz (1963), for example, gave as evidence for this claim the statement of Fulton (1947) that lesions of the motor area cause only a mild paresis with quick recovery in lemurs, pottos (Perodicticusbotto), bushbabies, and marmosets; that the paresis is more enduring in spider monkeys (dteles), mangabeys (Cercocebus), and macaques ; and that in chimpanzees and man the disturbance is profound. Neither Noback and Lfoskowitz (1963) nor Fulton (1947) cited the study on which this conclusion is based. Noback and Moskowitz (1963) quoted only the study of IValker and Fulton (1938) on the effects of hemidecortication in chimpanzee, baboon (Papio), macaque, potto, cat, and coati (Nasua nasua), in which the recovery was found to be greater in the baboon and macaque than in the chimpanzee. Fulton and Keller (1932) reported that after lesions of the leg area of the motor cortex, chimpanzees recovered more slowly

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than baboons or macaques. Only two other studies appear to provide relevant evidence. Campbell et al. (1966) reported quick recovery from a lesion of motor cortex in the slow loris (Nycticebus cougang), but their lesions were not complete; Zuckerman and Fulton (1941) ablated some of the left motor cortex of a bushbaby (Galago demidovii) and found that the animal was still dragging its right foot after 9 days, but again there was no evidence that the lesion was complete. However, the claims for progressive encephalization of motor functions in primates could only be supported by a large-scale comparative study of primates in which lesions are made in homologous areas. Such a study could only be carried out if information was obtained on the boundaries of motor cortex (4, FA) and of the agranular cortex in front of it (6, FB, FC). Such information is now available for a small series of 6 primates (von Bonin and Bailey, 1961), but it was not available when Fulton (1947) discussed neocorticalization for the motor system. Unless the boundaries of FA, FB, and FC are known, lesions that are intended to include only FA may include varying amounts of FB and FC. Just as it is necessary for vision to know the effects of ablating striate cortex alone, and of striate and prestriate cortex together, so it is necessary in the study of motor functions to determine separately the effects of ablating FA (4) and the effects of ablating all frontal agranular cortex. I t is quite possible that the lesions in the animals referred to by Fulton (1947) were less complete in the lower than the higher primates. Furthermore it is not possible to find any human cases with lesions large enough to involve all motor cortex, and yet not so large as to encroach on other areas (Bucy, 1944). An additional method to ablation of motor cortex is the sectioning of the pyramidal tracts either unilaterally or bilaterally, although the pyramidal tract contains many fibers from areas other than motor cortex. Tower (1944) has reviewed the effects of section of the pyramidal tract in primates, and concludes that unilateral section of the pyramidal tracts in the rhesus monkey produces more severe hypotonia and paresis than that seen in the cat, and that the paresis is yet more severe in the chimpanzee. However, as Philips (1971) pointed out, a more critical comparison is of the effects of bilateral pyramidal lesions, since after unilateral lesions the limbs opposite the side affected may compensate for the motor disability. However, no comparative evidence is available. There is only the study of Lawrence and Kuypers (1968) on rhesus monkeys, in which recovery was found for all but precision movements of the hand. There are no confirmed cases of bilateral pyramidal section in man, and no comparisons can therefore be made. On the other hand, Bucy et al. (1964) reported the case of a man with partial surgical destruction of one pyramidal tract. Histological examination showed that 83% of the fibers had been cut. He recovered to the extent that even

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fine hand and finger movements were possible. Other reports of unilateral pyramidal destruction in man have failed to demonstrate that the damage is confined to the pyramidal tract, particularly in cases of hemorrhage causing destruction of the internal capsule. Since no adequate studies have been done, the question of progressive neocorticalization for motor functions within the primates must remain unanswered until further studies have been carried out. It seems possible from the published studies that fine movements of the digits may be more disturbed in higher than in lower primates, but even if that were true it would not prove neocorticalization. For higher primates have much finer control of their hands than do lower (Bishop, 1964), and the inability to make fine movements is more crippling in higher primates. But in lower primates motor control of the hands is still carried out by the neocortex. The fact that the motor cortex is capable of carrying out finer and more skilled movements in higher than lower primates in no way proves neocorticalization.

C. LEARNING Human sensory and motor capacities do not depend on language. Verbal instructions or responses may certainly be utilized in assessing for instance flicker fusion thresholds or motor skills. However, recourse to language here represents merely a convenience: precisely the same capacities would be revealed without any call upon language (or by a hypothetical human being devoid of language in the absence of brain injury). In contrast, language does normally contribute in a fundamental sense to more complex human performance. Attempts to devise so-called nonverbal tests of performance have not been entirely successful. (Only in infants aged less than 12 months can the role of language be confidently discounted.) As will be discussed in Section 111, G, on language, man’s linguistic competence far exceeds that of any other primates. Therefore, in comparing complex behavior in man and other species, the contribution of language to human performance must be carefully evaluated, otherwise differences will emerge that merely reflect the all-pervasive benefits to man of speech. But is our task to compare a hypothetical human devoid of language with the real nonhuman primates? Probably not, when dealing with complex behavior: our hypothetical speechless man would learn (i.e., classify, store, think, solve abstract or concrete problems, give evidence of remembering, etc.) in a way so different from that of any real man that our comparisons would become more hypothetical than real. Therefore we can do no more than compare real (talking) human beings with other primates, and assess as best we may whether any differences are independent of man’s possession of language.

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1. Discrimination

Sensory inflow is differentiated as part of the process of perception even before it is formally categorized or has acquired significance. This differentiation allows different kinds of inputs to be distinguished (even if both are totally unfamiliar) :particular stimuli come to stand out (or be discriminated) from the vast array of other environmental stimulation. I n man, such discrimination between different kinds of stimulation seems to take place mainly in the minor (nondominant) cerebral hemisphere, provided the inflow is “nonverbal.” This basic conclusion seems secure as a result of the pioneering observation by Milner (1958) in vision, by Weinstein (1962) in touch, and by Kimura (1961) in audition. [There remains, however, some conflict of evidence, partly reviewed by Blakemore et al., (1972).] Moreover, for vision and audition the temporal lobe within the minor hemisphere has been particularly implicated. (Recent work with patients whose cerebral commissures have been divided affords independent evidence for the importance of the minor hemisphere in nonverbal discriminations; but study of these patients cannot further delimit the regions within a hemisphere that are concerned with particular tasks.) Therefore we know which hemisphere (and to a lesser extent which region in that hemisphere) is especially concerned with nonverbal discriminations in man. The nature of the discriminative (or perceptual) process, nonetheless, cannot yet be described in neural terms. The clinical evidence points approximately to where discriminations take place and supports the suggestion that in man discrimination is an independent stage in the learning process (since it can perhaps be independently disordered), but it does not point to how, in the intact brain, one kind of sensory inflow is distinguished from another. I t is not certain whether discrimination performance as conventionally assessed in nonhuman primates represents the same process as in man. With nonhumans reward is made available (or errors are punished) to achieve reliable “discrimination.” So the monkey or ape has to learn to attach differential significance to stimuli during formal training at the same time as it learns to perceive nonidentities. There are exceptions to this difficulty: C 6 equivalence testing,” “stimulus generalization,” “dimensional learning,” and so forth are more closely analogous to human tests of discrimination. I t is also possible that discrimination tasks are solved by humans by recourse to higher cognitive abilities (e.g., abstraction) but by nonhuman primates in a more direct way. Normative studies have shown that prosimians and insectivores are capable of discrimination performance comparable to those achieved by simians on those few tasks which have been given in common. Thus the tree shrew ( Tupaia glis), a n insectivore, can distinguish vertically inverted

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triangles even when each triangle is surrounded by a circle (Diamond and Hall, 1969). There have been innumerable attempts to define the cortical regions concerned with discrimination performance in the rhesus monkey; and not a fcw in other primate species (except that little relevant information is available for the apes). Until about 1968 the evidence from ablation studies indicated that the temporal neocortex was the only region specifically concerned with visual pattern discrimination. More recently, it has been established by the work of Butter (1969, 1972), of Gross et al. (1971), and of others that prestriate and striate cortical removals can also give rise to defective visual discrimination, especially when the visual test objects are “masked” in some way. The impairment is considered to be independent of the field defects produced by some of the removals. Very similar observations have been made for the tree shrew by Killackey (in Diamond and Hall 1969) : a failure after striate cortical removals to discriminate triangles surrounded by circles, but normal performance when the surround is removed or when other tasks require a reversal of a previously learned response. Killackey et al. (1972) have extended the observations on the tree shrew and claimed a disorder of “sejective visual attention” after striate lesions, a disorder in “shifting attention” after temporal lobe lesions. [This latter claim requires confirmation by use of extradimensional instead of nonreversal shifts (Slamecka, 1968).] Thus there is a common pattern throughout the primates of impaired visual discrimination performance with lesions of the temporal lobes (though in man the minor temporal lobe is specifically implicated whereas in other primates the removals must generally be bilateral) ; and, in addition, a more selective defect with complex visual patterns when the removals involve prestriate or striate cortex in nonhuman primates. As discussed in Section III, A, it is not yet certain whether man will be found to have a similar perceptual disorder after striate cortical lesions. I n any casc one major obstacle to any valid comparison between man and nonhuman primates arises in this (and almost every other) context: with rare exccptions (e.g., cerebral gunshot wounds) brain lesions in man are extensive so as to involve at least a major proportion of a lobe; but in experimental animals removals are generally made of restricted brain regions. I t is therefore not possible as yet to decide whether the association cortex of man is organized in the same way as in other primates. For example, man might not have separate cortical regions concerned with visual, auditory, and tactile discrimination, whereas such a segregation by sense-modality is clearly established for the monkey and many other nonhuman animals.

2. Classijication All mammals classify their discriminated inputs. Thus they distinguish between objects known to be edible and others known to be inedible;

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between dangerous and safe; and so forth. Man also has abstract classifications (e.g., beautiful and ugly, generous and mean, just and unjust). Certain human classes, e.g., just and unjust, can be confidently regarded as totally dependent on language at least for their formulation. Others (e.g., beautiful and ugly) have been shown to have some counterpart in the monkey -even if simplified perhaps to a more primitive nice and not-nice. Thus, Humphrey (1971) reported preferences for colors and intensities in the rhesus monkey. These preferences remained stable in time and were consistent across animals. Yet other human classifications may prove untestable in nonhuman animals : for instance, generous and mean have significance beyond nice and not-nice for man, but could hardly be distinguished from nice and not-nice without language, should they occur in the monkey. And other human classifications (e.g., numbers) may be linguistically based after a certain age, but when present in nonhuman animals (even birds can count) are the outcome of perceptual categorization. We shall consider only those classifications that are presumed not to depend on language in man, that occur in nonhuman primates, and that have been subjected to study in neuropsychological investigations. De Renzi et al. ( 1966) reported performance on a modified version of the Weigl block-sorting test, which is regarded as largely “nonverbal.” In an unselected series of patients, those with dominant hemisphere lesions who also suffered from dysphasia performed more poorly than any other group, for example, nondysphasic patients with dominant hemisphere lesions. This finding could mean that impaired sorting even on a “nonverbal” task is secondary to dysphasia; or that in man the systems concerned with sorting overlap those concerned with speech, and are situated in the middle third of the dominant hemisphere. [To the extent that Holmgren’s skein-sorting test can also be regarded as primarily a test of sorting, de Renzi et al. (1972) have confirmed an association between dysphasia and impairment of %onverbal” categorization. However, the authors are cautious in their interpretation of their findings.] Suggestive evidence that classification by sorting need not be linked to human language comes from the work of Hayes and Nissen (1 971) with their chimpanzee, Viki. Although never able to communicate with language, Viki spontaneously initiated sorting of various objects. On more formal tests of sorting, her performance was highly proficient, even for objects varying within different classes. Moreover, either unprompted or with prompting, she was able to shift between categories of sorting of the same objects (e.g., sorting the same set by shape, then by color or by size). It remains possible of course, that sorting is dependent upon language in man, but not in the apes. Meanwhile it is safer to assume that sorting is a nonverbal skill not unique to man.

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The other primates have been tested for their ability to sort only indirectly; by “matching from sample,” i.e., choosing from a selection of objects the one identical with a separately presented object (the sample) ; by “oddity” training, i.e., choosing the odd subject from among three or more simultaneously presented objects; or by “conditional” training, i.e., responding to a feature of the test situation, for example the color of the test tray, which indicates to the animal whether it is to select the large, small, dark, or light objects. It is known that apes may master such “sorting” problems by learning to make a different response to every possible stimulus array (e.g., by learning 20 separate responses) instead of by learning the underlying principle, as discovered by Farrer ( 1967). However, Milner (1973) has shown that rhesus monkeys will transfer a learnt matching-tosample response from one to another visual dimension, for instance from color to shape. Earlier, Levine and Harlow (1959) had reported significant oddity learning over many one-trial problems by rhesus monkeys. If then apes and monkeys can be trained to sort on tasks that are similar or analogous to those in clinical use with man, are the neuropsychological findings comparable? In brief, we do not know. The great majority of work on such complex tasks with nonhuman primates has not involved direct study of brain function. No relevant information exists for apes. Only seldom have the effects of restricted cortical removals on sorting been investigated in the rhesus monkey. Harlow et al. (1951) reported impaired solution of oddity problems after frontal or other large cortical removals; Iversen and Humphrey (1971) observed impairment on the oddity task after standard (but not after small posterior) inferotemporal removals; and Wegener (1968) recorded defects on different conditional tasks with lesions to either frontal or posterior association cortex. Unfortunately, these (and a few other relevant) investigations do not answer the question at issue: is one (or more) sector of the monkey brain especially concerned with classification ? All the lesions found to yield impaired performance on tasks involving classification are known also to give rise to disorder on less complex tasks. Therefore it remains uncertain whether classification itself or merely some more elementary contributory process has been disordered. [In man, Milner (1963) observed defective card sorting by patients with frontal lobe lesions: but their failure is recognized to arise not primarily from a disorder in the process of sorting itself but in shifting from one to another category of sorting.] Although we know so little at this time about the neural basis of classification, it remains possible that future work might indicate that man and the other primates differ in one or more ways. First, in man all classification (even of “nonverbal” material) may perhaps be found to be ultimately dependent on language ; second, in man classification may be accomplished by a unitary system irrespective of modality of inflow, whereas separate

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systems for different sense-modalities may accomplish classification in other primates (see Sections 111, C, 1 and 111, F). Answers to such speculation must await further work. Meanwhile there is no evidence at this time for differences within the primates. 3. Spatial Perception I n man it is clearly established that spatial discriminations can be severely disordered (by posterior parietal or parieto-occipital lesions) in the presence of intact nonspatial performance. The evidence has recently been reviewed by HCcaen (1972), who concluded that the “minor” hemisphere is specialized for spatial performance, although not to the exclusion of some contribution from the “major” hemisphere, and that all modalities of spatial performance are involved. Such a severe selective disorder of spatial performance remains to be identified in other primates. So far there is only limited evidence (e.g., Orbach, 1959; Bates and Ettlinger, 1960; Pohl, 1973) that a similar but mild disorder might also follow bilateral striate, prestriate, or posterior parietal ablations in the rhesus monkey. In certain respects (e.g., construction, topographic memory, recognition of faces) it is difficult to assess the monkey adequately, but more subtle tests of spatial perception in simians will undoubtedly be devised. Ettlinger and Kalsbeck (1962) cautioned against too ready an acceptance of apparently similar inaccuracies of reaching in man and monkey as reflecting genuinely similar spatial defects; and Hartje and Ettlinger (1974) have extended the earlier observations of Ettlinger and Kalsbeck without bringing about any obvious rapprochement between the findings in man and monkey. Even if in some respects the spatial disorders in man and monkey were to be found comparable, what appears to be an important constituent element in the human spatial disorder, visual neglect as opposed to inattention to one of two simultaneous stimuli (Heilman et al., 1971), has so far never been observed in the monkey. The responsible lesion for the neglect (as also for the wider spatial disorder) is generally in the right parieto-temporal-occipital region as again recently confirmed in 56 patients who showed the disorder from 179 with right-sided lesions (HCcaen, 1972). The lesions were established surgically or postmortem. In this series there was no indication for any spatial disorder to be associated with frontal lesions, as reported in the monkey by various authors. Therefore, a t present it seems unlikely that spatial perception is organized in the same way in man and monkey. 4. Problem Solving As with classification, there is a great deal of relevant normative information for different primate species, but little neuropsychological evidence (except for man and the monkey) dealing with brain organizations. However,

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in the case of problem solving, unlike the other aspects of learning previously discussed, the normative results suggest marked differences between various primate species. In man, language may often contribute to the solution of even “nonverbal” problems, and this contribution must be taken into account when human performance seems preeminent. Thus Bryant (1971) has demonstrated “transitivity’’ and “conservation” (in Piaget’s terminology) to be present in children aged 4-6.5 years, a level of achievement so far not attained by any ape. (Admittedly, the children might have solved the “nonverbal” tasks with the aid of language.) Then the chimpanzee can modify tools in ways not reliably established for monkeys (van Lawick Goodall, 1970); also it can substitute tokens for edible rewards (Wolfe, 1936) and can achieve self-recognition in mirrors (Gallup, 1969), performances not yet observed in monkeys. The rhesus monkey can achieve greater proficiency of learning set formation than the squirrel monkey or marmoset in that order, according to Warren (1965). Similarly, the bushbaby, tree shrew and hedgehog can be graded in that order for their ability to solve delayed spatial alternation (Masterton and Skeen, 1972). (It may be objected to this argument that all the primates should be compared on a single behavioral measure. Such an objection could be answered in various ways. Perhaps most compelling is the following; if there genuinely exist differences in problem solving ability, then adjacent species should be tested on tasks more likely to prove sensitive to such differences, i.e., tasks that tax their abilities maximally; whereas a single test might fail to reveal actual differences between those several species which could each attain maximal proficiency, or between those that all failed on this task.) Despite our confident assertion of graded differences within the primates in the ability to solve problems, we cannot at the present demonstrate any differences in brain organization relevant to this ability. I n man, for example, a variety of brain lesions can impair different kinds of problem solving, according to McFie (1960) and to Piercy (1964), but in no case is the nature of the disturbance clearly understood. For instance, it remains uncertain whether any lesion impairs problem solving selectively. Apes have not yet been studied in this respect. For the rhesus monkey there is substantial evidence that bilateral temporal removals are followed by impairment of visual learning set formation (e.g., Chow, 1954; Brush et al., 1961). However, there is also evidence that (in rhesus and squirrel monkeys) removals of frontal cortex can have a quantitatively similar, although qualitatively different, effect on learning set tasks (e.g., Mishkin et al., 1962; Miles, 1964). Masterton and Skeen (1972) have proposed on the basis of phylogenetic differences in the development of the prefrontal cortex that this area is selectively concerned with “intelligence,” as assessed on delayed spatial alternation in bushbabies, treeshrews, and hedgehogs. This contention cannot

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however, be accepted as proposed: other indices of brain development may yet correlate more closely with phylogenetic differences on alternation (see Section 11), and conversely a wider range of behavioral tasks should be sampled. A more detailed discussion of these findings is not needed as temporal lesions are considered likely to impair learning set formation, and frontal lesions alternation, indirectly: the temporal removals by impairing visual discrimination and/or learning of the association between stimulus and reward; the frontal removals by accentuating a normal tendency to respond in some preferred way or if more laterally placed by producing a rather selective disorder of spatial memory. No other lesion is known to impair problem solving in the monkey, whether this ability be regarded as unitary, or as seems more likely comprising a number of “independent” abilities, each specialized to some extent for the solution of different kinds of problem; and to the extent that the temporal and frontal removals act indirectly (as already suggested), it seems likely that no cortical area is selectively implicated in such abilities. Empirically, therefore, we cannot draw conclusions regarding the neural mechanisms responsible for problem solving in different primates. (Theoretically it can be argued that the normative evidence for differences in capacity between species imply at least quantitative differences in brain organization.)

5. Associative Learning Stimuli discriminated as different often acquire significance: the onset of a red light might signify danger, the onset of a green light that it is safe to proceed (or look for a food reward). More is known about this than any other aspect of learning within the primates; and in particular there is a substantial body of neuropsychological evidence. The normative studies indicate not only quantitative differences (e.g., in the rate of learning and in the amount that can be learned) by different primates, but also qualitative differences. Thus to take only one example of quantitative differences, Milner (1968) described 5 compound-stimulus memory tasks (as devised for animals by Konorski, 1959) with 5 nonverbal-stimulus values on each task. She stated: “These tasks, even with a 60 sec interval and an interpolated distraction, proved extremely easy for normal (human) subjects . . .” (p. 342). This task would prove difficult for other primates. To take one example of qualitative differences, Rumbaugh (1971) found that gorillas learned discrimination reversals in a way qualitatively different from that adopted by gibbons and talapoins ; in contrast, the orang-utan (Pongo) and chimpanzee, both anthropoid apes, were shown in a different study (Rumbaugh and Gill, 1971) to achieve comparable levels on discrimination reversals without evidence of qualitative differences in the mode of solution. Considering the lesion effects, Milner (1968) has reviewed the evidence

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that nondominant temporal lobe lesions in man are more likely to impair nonverbal learning than are lesions situated elsewhere. Nonetheless, this finding is insufficient: first, the evidence for a n association between temporal lobe pathology and nonverbal learning of tactile tasks is less compelling; and second, the temporal lesions also impair other performances (see Section 111, C , 1). [The evidence for man i s k t h e r complicated: Samuels et al. (1972), found impairment on short-term visual retention tasks after parietal but not temporal lobe lesions.] Once again there is no relevant evidence for the apes. However, it is amply clear from many experiments on the rhesus monkey and other simians that the ventral temporal areas are involved in visual associative learning. (Indeed the evidence for the monkey anteceded the evidence for man.) More recently it has been shown by Cowey and Gross (1970) and by others that within the ventral temporal area there exists a gradient of differential function-the anterior subarea being more involved in learning than the posterior. Even the tree shrew has an area on the temporal surface that may function analogously to this anterior temporal subarea in the rhesus monkey (Killackey et ad., 1972). In the rhesus monkey it is established that auditory associative learning is impaired by superior temporal removals, and tactile learning by posterior parietal ablations. Recently, Jones and Mishkin ( 1972) have proposed that associative learning of all modalities ultimately takes place in the region of the temporal pole and amygdala. So there is as yet no evidence for differences within the primates in the neural system involved in associative learning. Nonetheless, we must conceive of the possibility that in the course of further work such differences might emerge to correlate with the known differences, both quantitative and qualitative, apparent in the normative studies.

6. Summary This section on learning has proved disappointing in various ways. First, when considering certain aspects of learning there has been little or no relevant neuropsychological work on any species other than man. Our inferences regarding brain organization then becomes indirect ; it is almost tautologous to suppose there will be found to be differences in brain function when species differ qualitatively in their performance. What we really would like to know is whether brain organization is similar or different even when normative studies indicate no differences in behavior. But such knowledge must wait on further neuropsychological work. Second, it has become evident that many of the neuropsychological investigations that exist do not contribute specifically to the central issue of brain organization. The reason for this is clear: a t present brain lesions yield information on which sector of the brain is involved in a given performance

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(i.e., on “localization”), but mostly not on how this sector is involved (i.e., on “mechanisms”). Until we are able to infer the nature of the brain processes underlying specific performances as well as their location within the brain we cannot contribute to the central theme of this review. Last, it is clear that the apes have been neglected in neuropsychological research. The reasons (e.g., conservation, cost, management, etc.) are obvious. However, this deficiency will ultimately have to be made good before a truly comparative survey can be achieved. O n the positive side we have tentatively proposed that differences between species exist (or might in the future be shown to exist) in brain function relating to certain aspects of learning. I n no instance, however, is it yet possible to make a confident assertion regarding the nature of such differences.

D. MEMORY There are many ingenious experiments on various aspects of memory in human subjects, for example by Broadbent (1970). This work invoked concepts such as “registration,” “short- or long-term storage,” and “retrieval” for stages of the memory process. Nonetheless, we know little of how the brain achieves each stage. For example, there is so far no agreement on the nature of structural, chemical, or other change underlying storage of learned information (see Zippel, 1973). Given our present ignorance of brain mechanisms in memory, comparative evaluations are difficult. 1. Lsion Studies

I n all species lesion studies have contributed mainly to knowledge of localization, not to process. Moreover, in considering restricted cortical lesions there is always uncertainty concerning the nature of the defect: the human patient with damage to the right temporal lobe and the monkey with inferotemporal removals is impaired at aspects of visual performance. Although in each species performance on tasks involving memory may be disturbed, the fundamental process impaired by such lesions remains unknown (particularly since performance is also impaired on visual perceptual tasks). Nevertheless, one clinical condition has (almost by accident) permitted some insight into the memory process. This is the “amnesia” which is associated with nutritional deficiencies (e.g., in alcoholics), with bilateral surgical removal of the hippocampus and of other parts of the medial temporal lobe, or with disease (e.g., encephalitis) of the temporal lobe. (Amnesia may have other etiology-for instance, trauma. Moreover, in combining alcoholics and patients with medial temporal lobe damage we do

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not wish to imply that their memory disturbances are identical. This is probably not the case. Nonetheless, their disorders of memory are sufficiently similar to be grouped together.) Talland ( 1965), Milner ( 1968), and Weiskrantz ( I 97 1) have described in detail the findings in man. Briefly, the amnesic patient cannot recall (and often cannot recognize) material perceived even a few minutes earlier through any sense (unless he is permitted to rehearse verbally in the interval). Such a patient does not normally know where he is, whom he saw a few minutes previously or that he has a defective memory (although in surgical cases there may be some insight into the disorder). Recognition may be less impaired than recall, and recognition of verbal material may be poorer than of pictorial material, as shown by Piercy and Huppert (1972). Nonetheless, the amnesia is clearly not confined to verbal material. Such amnesic patients are sufficiently cotnmon for a “typical” clinical picture to emerge. (Other kinds of specific clinical memory defect, e.g., defects on tests of immediate auditory mernory, are rare so that their interpretation must remain in doubt; nonspecific memory disorder accompanies many kinds of cerebral disease but has not contributed to our understanding of the memory process.) Clinical amnesia, when precipitated by alcoholism, is associated with bilateral pathology in various areas, most frequently in the medial Dorsal and medial Pulvinar nuclei of the thalamus and in the medial Mammillary nuclei of the hypothalamus (Victor et al., 1971). Surgical intervention in epileptic or psychotic patients on the medial surfaces of the temporal lobes has also been followed by various degrees of amnesia, in relation to the extent of the bilateral removals for 5-8 cm backward from the pole (Scoville and Milner, 1957). Disease processes other than alcoholic conditions implicate various regions, but especially the hippocampal formation and gyrus (van Buren and Borke, 1972). Many experiments have attempted to reproduce in the rhesus monkey the memory defects reported by Scoville and Milner (1957) to follow bilateral medial temporal lobe removals in man; and a few have studied the behavioral effects of destruction of the Mammillary nuclei or of the medial Dorsal nuclei. Overall, these investigations have been negative in the sense that no disorder of memory as severe as that in man has been produced in any lesioned animal. This is not to claim that the surgical procedures in monkeys have been without behavioral effect. Various disturbances have indeed been observed after medial temporal lesions in rhesus monkeys or in baboons, for instance, by Orbach et al. (1960), Correll and Scoville (1965), and Iversen and Weiskrantz as reviewed in 1970. However, such disturbances are observed under either of two conditions; when the possibility of interference between items that have to be remembered is maximized but delay may be quite short (Correll and Scoville, 1970); or when alternations or

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reversals are required between spatial alternatives, but not between objects (Mahut, 1971; Jones and Mishkin, 1972). [The chief exception is the defect in recognition memory observed by D. Gaffan (personal communication) in 3 rhesus monkeys after bilateral section of the fornix: in one task the impairment seems to be related to time interval per se.] The importance of interference in enhancing the defect in monkeys is widely accepted. Weiskrantz (1971) has proposed that the amnesic patient suffers from a qualitatively similar condition. i.e., memory is impaired due to “interference” from potentially incorrect responses. (‘The resemblance may well be more apparent than real, on theoretical grounds; in the monkey interference arises from interpolated tasks or response requirements ; in man it is supposed to arise from items earlier committed-perhaps weeks or months previously-to memory.) The evidence for the role of interference in human amnesia is not compelling, and has been contraindicated by the observation of Piercy and Huppert (1972). Moreover, there is no known parallel in human patients to the selectively spatial reversal disorder described by Mahut (1971) in rhesus monkeys. Last, the amnesic patient fails to remember material presented through any sense-modality whereas the defect appears to be exclusively visual in baboons (Iversen, 1967). Isaacson (1972) has recently attempted to reconcile the discrepant findings from man and other primates by reference to the preexisting clinical disease in man. Isaacson (1972) argued first that epilepsy can impair behavior more severely than a clean removal of the epileptic system or structure. Next, he noted that many (but not all) of the cases of amnesia consequent upon medial temporal lobe removals were epileptic before surgery. He then concluded that it is the epileptiform discharge arising from the remaining areas of the temporal lobe (not the absence of the temporal lobe structures removed at the time of surgery) which is responsible for the memory changes in man. This view suffers from various difficulties; epileptic discharges are frequently less disturbing to behavior than ablations (see next section) ;not all amnesics were epileptics; and why do the surgical removals greatly increase the amnesia without increasing the amount of the electrical discharges ? I t might then be answered that hippocampal removals produce amnesia only if there has been damage in man (due to whatever condition gave rise to epilepsy) to structures which can subserve memory in animals subjected to surgical removals of the medial temporal lobe. For example, entorhinal cortex may mediate normal memory in the lesioned monkey but not after similar surgery in man if the entorhinal cortex was already epileptic. Isaacson’s (1972) views remain controversial, but they could be assessed empirically by suitable surgery in epileptic monkeys. It could also be argued that amnesia in man is compounded from two constituents : verbal memory defect (from left-sided damage) and nonverbal

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memory defect (from right-sided damage). Then the mild defect in the monkey might be similar to the disorder of nonverbal memory in patients with right hemispheric lesions (see Section 111, C). However, it remains unlikely that in man separable mild verbal and nonverbal disorders of memory would, in combination, produce the devastating symptoms of amnesia. Last, there is D. Gaffan’s view (personal communication) that both man and monkey can have the same basic defect: the inability to recognize as familiar a previously experienced stimulus or response. He believes that man, monkey, and the rat show similar disturbances of memory after lesions of the hippocampal system, but that their testing has not been comparable. It is too early to evaluate his findings, which nonetheless deserve serious consideration. Overall, the lesion studies point to a substantial difference between the effects of thalamic or medial temporal damage in man and other primates. Amnesia as known clinically has so far not been observed in monkeys. (Apes have not been subjects in this kind of experiment.) Weiskrantz (1971) has rightly emphasized the recent work indicating that at least some information is stored in patients that fail to recognize or recall. Moreover, he has made more plausible the existence of a common effect of interference. Nonetheless, no lesioned monkey has yet been demonstrated to fail on tasks (e.g., learning a new discrimination across days) that most amnesic patients could not perform (but see Gaffan, 1972). At the moment the differences between man and other primates can still be regarded as qualitative.

2. Electrical Stimulation In all species, electrical excitation of cortex (by imposed currents or by the application of a chemical agent which produces epileptiform discharges) can sometimes prove more, but at other times less, disruptive than a removal of the relevant tissue. Isaacson (1972), as already discussed, has quoted the evidence that such electrical excitation can be more disruptive. However, many other observations, for example Stamm and Pribram (1960), Chow (1961), and Nie et al. (1 973) indicate a lesser degree of behavioral change, or at least no more severe changes than would be expected to follow removals. I n all species an electrical stimulation leading to widely propagated discharge over the brain surface (i.e., an electroconvulsive stimulus) can impair retention of material exposed immediately prior to the onset of the electric stimulus. Again, no differences between species emerge. (In man, this procedure is used only on patients suffering from mental illness.) Therefore at the present it appears that factors relating to the nature of the electrical excitation (e.g., the extent of its propagation, the voltage of the paroxysmal discharges, etc.) are more important than species differences in their effects on memory. It may be even supposed that electrical excitation (being

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disruptive or normal functioning) may never give rise to species differences that would not also be observed with destructive lesions.

E. LATERALIZATION OF HEMISPHERIC FUNCTION AND EXCHANGE OF INFORMATION BETWEEN THE HEMISPHERES The problem of asymmetrical function is separable from man’s possession of language. First, language could be laid down symmetrically in the brain (as may well be the case in the young child and in some ambidextrous subjects). Second, cerebral asymmetry is now known to extend in man to many aspects of behavior additional to language, so that in one and the same individual the left hemisphere may lead in the control of some, but the right in the control of other, kinds of behavior. Third, the origins of cerebral asymmetry remain unknown. Although the other asymmetries could derive from the lateralization of language, it is now recognized that man’s asymmetrical brain organization could equally follow from lateralization of nonverbal functions to the right hemisphere during infancy; or from the development of hand preferences during early childhood. Anatomical asymmetries have already been discussed in Section 11, F. 1. Lateral Preferences

A congenital and genetic basis for human hand preferences has been proposed, for example, by Annett (1972)’ whereas acquired factors have been emphasized, for example, by Provins (1967). On either view there could be a n association between hand preference and cerebral lateralization (see also Semmes, 1968). The nature of any such association remains undecided at present, except for agreement that in a minority of adults there may be an anomalous correspondence : either a normally asymmetrical cerebral organization with a reversal of the expected hand preference; or a weakening of the normal cerebral asymmetries in some left-handers or ambidexters. On the available evidence, man is generally supposed to differ from other primates in his lateral preferences (and, it is suggested, for noncultural reasons). In no other species is the preference for the use of the same hand (e.g., always the right) by different individuals thought to be as strong as in man. As a rough approximation, about 66-687, of human adolescents consistently prefer to use the right hand (i.e., on 1 0 0 ~ of o tests) whereas less than 507, of chimpanzees and monkeys prefer that (or the left) hand on 90% of trials on a single test (Annett, 1972). [In chimpanzees (Finch, 1941) and monkeys (Beck and Barton, 1972), the left and right hands are preferred about equally often over a variety of tasks, even by individuals with strong

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preferences for the one or the other.] Nonetheless, in such comparisons between man aad nonhumans, preferences have usually been assessed differently. In man performance is generally assessed once on a number of tasks and is then cumulated to give a composite index of laterality (but consistency on the same task is usually disregarded) ; in animals it is usual for performance to be repeatedly measured on the same task [but only infrequently has the generality of preferences across tasks been taken into account, as by Warren et af. (1967) and Beck and Barton (1972)l. The available evidence suggests that test-retest reliability measures in man are significant, at least for the right hand (Provins and Cunliffe, 1972); and intertest measures of preferences in the rhesus monkey are also frequently significant (Beck and Barton, 1972), supporting the original supposition of differences between man and other primates. [However, Schaller (1963) has claimed that all of 8 gorillas used the right hand first more often than the left hand in chest-beating displays. Overall, in 69 instances the right hand was used first and in 13 the left hand. Therefore, in this small sample all, i.e. lo%, gorillas preferred the right hand for one task on 82% of occasions, giving a higher incidence of individuals preferring the same hand than for man but using a weaker index of preference and a task which may not be found to correlate with more meaningful tests of hand preferences. Further study is needed to confirm these lateral preferences.] Additional support for the supposition that man differs in his lateral preferences comes from the finding that only about 29y0 of human subjects are ambidextrous, i.e., do not prefer either hand consistently (Annett, 1972)) whereas the proportion of ambidextrous subjects similarly defined would be greater in other species. Last, in most human adults there exists an association between lateral preference and cerebral asymmetry. Such a correspondence is unknown (apart from the three exceptional reports discussed below) in nonhuman primates, even in individuals with strong lateral preferences. 2. Cerebral Asymmetry of Function Although human and nonhuman primates differ in their lateral preferences, as already discussed, there are many individuals in every species with strong lateral preferences. Thus Milner (1969), using a different definition than Annett (1972), observed only 22% of 58 monkeys to show mixed hand preferences on his second task; and Beck and Barton (1972) reported 5 of 10 monkeys as having significant preferences (left or right) on at least 25 of 31 measures on their 17 tasks. Since the great majority of humans with strong preferences are known to have marked cerebral asymmetries, and since such asymmetries might also be independent of language,

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it is not unreasonable to investigate whether apes or monkeys with strong lateral preferences have evidence of cerebral asymmetries. Overall the findings are negative. For example, Ettlinger and Gautrin (1 971) studied visual discrimination performance in monkeys and failed to obtain differential effects (taking hand preferences into account) between left- and right-sided ventral temporal ablations. This negative outcome cannot be attributed to insensitive tests since the combined group of 7 animals with either left- or right-sided unilateral ventral temporal removals was significantly impaired on 2 tasks. Other workers (e.g., Dewson et al., 1970) have used different tasks and different unilateral ablations, but with the same outcome for the monkey. However, Gazzaniga (1963) studied 3 right-handed monkeys before and after brain bisection. Animals were trained with each hand and eye combination both pre- and postoperatively. After surgery, only the left hand and right eye combination gave evidence of good retention. Gazzaniga’s (1963) findings suggest that visual learning can take place exclusively in the right hemisphere of monkeys that naturally prefer the right hand. Ettlinger et al. (1968) described 4 rhesus monkeys which preferred the use of the right hand during tactile training in the dark and the left hand during visual training in the light. Both of these reports might be taken to indicate a preferential involvement of the right hemisphere in visual performance. However, Hamilton and Lund (1970) found a different effect namely, superiority of the left over the right hemisphere, when assessing the rate of learning of 4 discriminations involving the direction of movement by 4 split-brain monkeys. This asymmetry, favoring the left hemisphere, was not observed when the same animals were trained on two pattern tasks or one discrimination task between moving and stationary dots. [The hemisphere ipsilateral to the preferred paw is reported by Webster (1972) to be more strongly involved than the contralateral hemisphere in 3 of 8 visual tasks in the cat. However, procedural factors make it difficult to interpret these findings.] I n the face of such conflict of evidence, caution dictates the position that cerebral asymmetries in the monkey so far remain in doubt. Further work is obviously needed. If Gazzaniga’s (1963) or Hamilton and Lund’s (1970) observations are confirmed, we could at least conclude that language is not fundamental to asymmetrical cerebral organization. If the incidence of asymmetry were higher in the ape than in the monkey (as might well prove to be the case), and if also the range of asymmetrically organized behaviors were wider in the ape, cerebral lateralization might seem to be related to the level of cognitive development. Meanwhile it is clear that the frequency and range of cerebral asymmetry in man surpasses anything likely to be found in other primates. This then constitutes a substantial difference among the primates,

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but one difficult to interpret until the origins of cerebral asymmetries are understood for man. 3. Exchange of Information between the Hemispheres The present authors know of no differences between species in the overall anatomical pattern of commissural projection that might be independent of differences in the relative distributions of cortical areas and subcortical systems. However, the massa intermedia is present more frequently in the monkey than in man. [Only in one monkey of more than 50 prepared for division of the massa intermedia has it been found to be absent (J.J. Maccabe and G. Ettlinger, personal communication).] Moreover, Glees and Wall (1948) observed an interthalamic commissure in the monkey which seems to be absent in man. Since in man the commissures mediate the transmission of information to unilateral language systems, their functional organization could be different from that of the commissures in nonhuman animals. As more comes to be known of the regional specialization within the commissures, many more functional differences between species may be found. As already discussed, the left and right hemispheres of nonhuman animals are in general functionally equivalent (but see Gazzaniga, 1963; Hamilton and Lund, 1970; Webster, 1972). It might therefore be supposed that learning in such animals will proceed as rapidly when the input is restricted to either hemisphere as when it is available to both hemispheres. For the cat (and rat), this is clearly not the case. Following the earlier work on tactile learning by Meikle et al. (1962), both Sechzer (1970) and Robinson and Voneida (1970) have reported retarded visual learning when input was restricted to one eye of the cat after section of both the corpus callosum and optic chiasm (but not after section of only the callosum or chiasm) . Brain bisection retards even binocular discrimination learning in cats, according to Larsen et al. (1969). It seems then that for a normal rate of learning two interconnected hemispheres are needed in nonprimates, and that a single hemisphere, or two with their connections cut, are insufficient. This implies that a critical “mass” of cortex may be necessary for rapid learning. After brain bisection in man, comparable observations are more difficult to make. Many functions are lateralized, and then it seems apriori less likely that access to the nonrelevant hemisphere could improve performance. Also, preexisting pathology in one hemisphere may reduce its functional capacity in patients who have undergone brain bisection. However, if one considered a function, e.g., visual-spatial ability, which is known to be mediated by both hemispheres (although asymmetrically), then there is no evidence that performance with bilateral inputs is impaired in the brain-bisected patient. Similarly, calculation, memory, etc., are generally unimpaired. [In fact, the

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only instance of what might be called “mass action” effects known to us in man is the utilization of both hemispheres for the development of language in the child. This instance is not, however, strictly comparable to the evidence from the cat. In the child, both hemispheres seem to be needed for the normal development of a basic capacity, speech, but not in its application after 14 years of age. I n the cat both hemispheres seem to participate in the application to a specific problem of the basic capacity of discrimination learning. Another situation has been described by Dimond and Beaumont (1971) : better performance under conditions of overload if material is briefly presented independently to both hemispheres in man.] We can now ask whether the ape or monkey more resembles man or the cat. The optimal method for vision is that of Larsen et al. (1969): a comparison of binocular learning scores in animals with brain bisection, or commissure section alone, or chiasm section alone, or unoperated. No such data are available. The optimal method ‘for touch is to compare unimanual learning (with the preferred hand) in split-brain and unoperated animals. Ebner and Myers (1962) have not presented their findings in this way. However, a comparison of their Figs. 3 and 4 suggests no difference between the performance with the first hand (not necessarily the preferred) of 4 split-brain and 3 unoperated monkeys. Semmes and Mishkin (1965) describe a trend for higher learning scores by 4 monkeys trained after callosal section (mean 134 trials) than by 4 other animals trained before callosal section (mean 92 trials) or by 4 trained as unoperated controls (mean 111 trials). (The first hand to be trained is not stated to be preferred.) M. Hunter and G. Ettlinger (personal communication) have, however, found that 4 split-brain rhesus monkeys learn more slowly with their preferred hand than 5 unoperated controls on 2 tactile tasks, but more rapidly on 2 other tactile tasks. (Each animal was trained alternately with the preferred or nonpreferred hand on successive tasks, in a balanced design: findings have been included only for the preferred hand, giving 2 or 3 scores per group on each task.) Therefore, the weight of evidence assigns a place for the monkey closer to man than to the cat, that is, as learning more rapidly only in a very few situations with two than with one cerebral hemisphere. Clearly more work is needed along these lines, especially in vision. Meanwhile it appears that the mass of cortex available to a functional system in one hemisphere is generally sufficient in man, even if the system normally calls upon both cerebral hemispheres for optimal performance ; whereas in nonprimates the two hemispheres may need to be interconnected for optimal performance. The rhesus monkey, although lacking the cerebral asymmetries of man, nonetheless appears to learn as rapidly with one as with two hemispheres in most situations. (These conclusions receive support from consideration of a different line of evidence: large unilateral lesions, including hemispherectomies, in the different species.)

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F. THEEXCHANGE OF INFORMATION BETWEEN SENSE-MODALITIES As with other kinds of behavioral performance, we must again seek to exclude the contribution to human cross-modal capability that is derived from man’s possession of language. Although there is little direct evidence on this point, it is generally assumed that information is readily exchanged between sense-modalities in man through the mediation of language. A seen object, if identified as a “fork” or even merely as a “metal strip with 3 prongs at one end and a flat handle a t the other,” seems more likely to be recognized from among a series of palpated objects as soon as the tactile information fits the verbal label.

*

1. Cross-Modal Matching and Recognition It is evident from a number of investigations that children and even infants can match seen and felt objects, in the absence of verbal labeling. For example, Bryant et al. (1972) have found significant cross-modal recognition in infants below 12 months of age. They made use of the finding that their subjects prefer to reach for the one of a pair of visible objects that had previously emitted sound. An object emitting sound was placed in the infant’s hands. When this and a second object (neither now emitting sound) were then exposed visually to the infants, they reached significantly more often for the one that previously emitted sound while held in the hand. Their visual choice must have been based on a comparison of the visible object qualities with the remembered tactile qualities of the sound-emitting object. With young children or infants as subjects performance is never perfect. This is also true for the apes (chimpanzees and orang-utan) of Davenport and Rogers (1970, 1971), which were reported to match significantly between vision and touch but which averaged only about 75y0 correct performance. Attempts to demonstrate cross-modal matching in rhesus monkeys have so far failed (Ettlinger and Blakemore, 1967; Milner, 1973). However, these attempts with monkeys used different training procedures than those reported to be successful with apes. Moreover, apes might also fail when trained with the methods that proved unsuccessful with monkeys. Therefore, at the present it appears that no valid differences can be demonstrated between primates at cross-modal matching. [Butters and Brody (1968) and Butters et al. (1970) suggested that in man the left parietal region may be selectively concerned with cross-modal matching. However, verification of the site and extent of the lesions was not achieved surgically or a t postmortem; the left- and right-sided cases were studied successively; and the relationship of cross-modal defect to dysphasia was not evaluated except by clinical rating within the left parietal group.]

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2 . Cross-modal Transfer a. General Learning It is probable that even without verbal mediation children can exchange (or transfer) general learning between sense-modalities. [Various technical terms exist for different kinds of such general learning, e.g., “dimensional learning,” “learning of a principle,” “learning set formation.”] For example, Blank and Klig (1970) reported transfer of such general learning from touch to vision in children aged under 5. If the children were first trained in touch on a task in which differences between the stimuli in texture were relevant (but differences in shape were irrelevant), they performed better than did other children trained first in touch with shape relevant (but texture irrelevant) when new visual stimuli were given with texture (but not shape) relevant. We know of no comparable experiments with apes. Milner and Ettlinger (1970) failed to observe the transfer of another kind of general learning between sense-modalities in rhesus monkeys (provided that account was taken, as had not been done in the earlier studies with monkeys, of the slight nonspecific improvement which occurs during the lengthy training required for this kind of investigation). Even when reported as positive, the earlier work with monkeys showed only extremely slight transfer of general learning. If then Milner and Ettlinger’s (1970) findings and conclusions were to be confirmed on replication, it would be possible to point to a different outcome for man and monkey.

b. Spec@ Learning Curiously, the evidence for man is ambiguous regarding the sequential exchange (or transfer) of specific learning between sense-modalities when verbalization is precluded. Ettlinger (1973) has argued in a review that such specific cross-modal transfer does not occur in man without recourse to language (except possibly in one instance where special training conditions prevailed and the outcome was positive only in one direction). Certainly further observations are needed on this point. Apes have not so far been tested for cross-modal transfer. Of the many experiments conducted with monkeys, the great majority have been negative. One recent exception is the report of Frampton et al. (1973), but even then only one measure (errors on first 10 trials) indicated significant transfer. Another exception is the earlier report by Ward et al. (1970), who studied the prosimian Galago senegalensis. Using a method of training which teaches the animal to avoid an electric shock given for a n incorrect response, the authors trained the animals to discriminate between two rates of intermittence of a light or of a sound. A significant effect of the previous training in the first sense-modality could be detected during 100

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trials in the new (second) sense-modality. [However, the tree shrew failed to show transfer on the same task (J. Ward, personal communication), apparently because it strongly prefers one rate of intermittence.] The positive outcome in the bushbaby confirms a number of investigations in rats, mice, and rabbits, all showing specific cross-modal transfer between the modalities of audition and vision (see Ettlinger, 1973). These findings cannot yet be interpreted with confidence. If one accepts Ettlinger’s (1973) view that specific cross-modal transfer has so far not been demonstrated in man without the aid of language, then there is no substantial discrepancy between man and monkey. The apparent paradox would, however, remain that nonprimates and one prosimian can achieve a n exchange between senses not available to man or monkey. This paradox cannot be resolved by reference to the sense-modalities tested, since monkeys have been found to fail also on auditory-visual tasks. Ettlinger (1973) viewed the transfer in nonsimians as a form of wide stimulus generalization, most probably mediated by subcortical systems, in which information regarding modality was lost. Such systems were thought to survive in the monkey (and perhaps in man), but be suppressed by higher cognitive processes which take modality into account.

G . LANGUAGE Human language permits communication about the remote past, present, and distant future, about the concrete and abstract, about the inner and the outer worlds of experience, and so forth. Without language, man can still communicate, but at a more primitive level, as when we express our needs in a foreign country where no one speaks our language (though even then linguistic competence might contribute). It is claimed that primates other than man can communicate in a simple, natural way. Thus Menzel (1971) has described a n experiment in which one chimpanzee is thought to transmit to others information concerning the occurrence (and possibly the location) of pleasant and unpleasant events. From this report it remains unclear as to when this information is thought to have been exchanged: at the time that the chimpanzee possessing the information rejoined its group, or at the time the leader-animal was released with the others. T h e latter alternative could have been excluded by maintaining the leader in confinement after it had rejoined its group. If further work indicates that the leader must be released with the group for successful performance, it remains possible that the group is reacting merely to the early movements of the leader after their release. Miller (1971) has shown that rhesus monkeys also can communicate the occurrence of unpleasant events. I t is not clear whether this represents a level of communication in any way

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superior to the communication of affect. Little is known about the underlying neural systems, and no brain lesion is known to produce a loss of this communication. [However, Suomi et al. (1970) have elegantly shown that monkeys with bilateral frontal lobectomies have different social preferencesalbeit consistent ones-from unoperated control animals. Differential responding to the existence in other monkeys of brain lesions is best regarded as a subtle social perception, without intention by the stimulus animals to communicate. The frontal lobectomies only changed, but did not abolish or reduce, the social preferences.] The Gardners (1971) and Premack (1971a) have summarized their observations, first reported in 1969, that the chimpanzee can achieve a level of communication superior to that so far discussed. The Gardners (1971) trained one chimpanzee, Washoe, to communicate by use of gestures which form part of the American Sign Language for the Deaf (ASL), i.e., which belong to a standard human form of communication. Fouts (1972) has since shown that such signs can be readily learned by Washoe, providing a method of “molding” (i.e., physically placing the hands and arms into the appropriate positions) is adopted; and that a t least some such signs could also be acquired by each of four additional chimpanzees (Fouts, 1973). Premack (1971a) trained one chimpanzee, Sarah, to communicate with plastic tokens on a board. Premack‘s claims, if substantiated, would prove the more remarkable. Gestures are often used spontaneously by chimpanzees in the wild and in captivity. So the ASL could represent a gradual addition to a normal aspect of behavior; whereas the use of tokens in the way taught to Sarah is totally unnatural to the chimpanzee. Also, performances are reported to have reached a surprising level of proficiency (e.g., her “feature analysis,” or description of the token for apple, a blue plastic triangle, was the same as for a real apple). In neither investigation were brain processes studied directly. However, both groups are continuing with their efforts to train chimpanzees to communicate. Although no direct evidence is available on this point, it is likely that primates other than apes would not be able to attain the performance achieved by Washoe and Sarah. The argument is indirect: since even apes can fail to acquire the proficiency shown by Washoe and Sarah; and since other evidence (some already reviewed) indicates that chimpanzees have cognitive ability superior to that of monkeys, it has not been thought worthwhile to attempt to teach ASL to the monkey. (This indirect argument could prove fallacious: given better methods of training, perhaps most apes and some monkeys, e.g., the Cebus, could acquire ASL. Nonetheless, there are no grounds for supposing that any monkey could ever match the average level of achievement of the ape. However, despite such a hierarchical trend within the primates, it remains possible that a nonprimate like the dolphin may by

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convergent evolution achieve a level of communication superior to that of the monkey.) O n the present evidence, then, we must conclude that there may be differences between man and the chimpanzee, and probably between the chimpanzee and other primates, in their ability to use gestures or tokens to communicate. An attempt must be made to assess the performance of Washoe and Sarah, in order to identify the greater discontinuity in communication as between man and apes or apes and monkeys. Lenneberg (1971) has been inclined to doubt the relevance of such communication to human language. Nonetheless, in discussing what might be the minimal criteria for communication in apes to be regarded as related to human language, he has moved from his earlier position (Lenneberg, 1967) that, in principle, language is unique to man. Even more recently, he has pointed to the difficulties of delimiting what we call language and of determining the moment a t which a baby “has language” (Lenneberg, 1973). So that there is here, perhaps, a n implicit recognition that, with further advances in the training of chimpanzees or other apes, a level of communication might be attained that could qualify as equivalent to a primitive stage in the development of language in the human child. Bronowski and Bellugi (1970) based their analysis on a fully documented account of the earlier 2 years of training with Washoe (but do not consider her later achievements or those claimed for Sarah). While accepting that humin language can no longer be distinguished by the use of names for things (e.g., Washburn, 1968), they proposed two new distinguishing features : human communication can be detached or disengaged from the context in a way the chimpanzee’s gestures rarely are; and the child spontaneously “reconstitutes” his utterances (i.e., has the ability “. . . to analyze out regularities in the language, to segment novel utterances into component parts as they relate to the world, and to understand these parts again in new combinations.”) This analysis by Bronowski and Bellugi (1970) assumes that the limits of chimpanzee communication had been achieved at the time of their writing. This assumption is no longer tenable in the light of subsequent publications. Brown ( 1970) was given access to observations on Washoe up to the age of 36 months. By this age she controlled about 34 signs, many of which were used in sequences of 2 or 3 signs. Brown (1970) emphasized that the human child generally combines its words in certain orders to express a structural meaning (e.g., agent-action or agent-object) : word order is correct in the great majority of examples, whereas Washoe’s sign combinations were not known to be ordered preferentially. T o Brown this implied that as yet there was no evidence of (or against) Washoe’s possession of syntactic capacity. The most up-to-date opinion has been expressed by McNeill (1973), who

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a t the time he was writing (October 1972) had access to observations on Washoe and Sarah, then still unpublished. Emphasizing the variable order of Washoe’s sign combinations and the absence of questions and of negatives in her sign combinations (although these occurred in single word messages), he concluded that her word strings do not encode conceptual relations (which, however, are themselves more or less shared by man and chimpanzee). Nonetheless, by more formal instruction Sarah was able to master several syntactic forms (e.g., “same” and “not same”) that Washoe did not use, McNeill (1973) even suggested that the chimpanzee may have accomplished communication along a different evolutionary line to that followed by man: Washoe (and perhaps other chimpanzees in the wild) may use a linguistic system based on personal and social interaction whereas ours is based on objects and their relationships. It may, therefore, be impossible to assess at this time the relative discontinuities in communication between man, ape, and monkey. (So many attempts to demonstrate language in chimpanzees failed completely prior to 1969 that prudence requires a cautious assessment of the future.) Nonetheless, it is now clear that apes can achieve performances that may be significant for our understanding of human language and that had remained unknown until some 4 years ago. Washoe’s and Sarah’s communication could not be expected to be identical with human language. However, gestures or finger language are used by human deaf-mutes as their chief mode of communication. Moreover, Sarno et al. (1969) and Critchley (1970, p. 321 et seq.) have provided clear evidence that in man brain lesions can impair communication by gesture and finger spelling in a manner very similar to the dysphasia of ordinary speaking adults. Whatever status will eventually come to be assigned to such performance in the chimpanzee, the differences between species should help to specify what is unique to human language; whether a primitive language can contribute to other cognitive processes as fundamentally as does human language; what brain properties permit the development of language in the child; and why human language is generally confined to the one hemisphere in the adult. Such questions cannot be answered at present, but during the next decade work along these lines in apes could have a greater impact than any other kind of neuropsychological investigation for our understanding of human brain organization.

H. CONCLUSION Space has not permitted discussion in S.ection I11 of various important aspects of cerebral organization, e.g., similarities within primates, including man, of nonspecific effects on behavior of cerebral lesions, or of the

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similarities of cerebral development after birth. I n discussing the behavioral evidence (Section IrI), comparisons between species have been based on two lines of behavioral evidence: the performance levels achieved by unoperated (i.e., normal) animals, and the consequences of brain lesions. Each approach has its own difficulties. However, no others exist for the comparative neuropsychologist, for whom behavior is the chief focus of interest. I n comparing animals belonging to different species, the comparative psychologist rightly ensures that the conditions, instead of being standard, are optimal for successful (but not perfect) performance by each species. Thus with simians it may be most meaningful to test for cross-modal transfer of training between vision and touch with solid objects, whereas with nonsimians it may be more appropriate to train in vision and audition with different levels of intensity or with different rates of intermission. Since monkeys happen to fail under both conditions, but nonsimians succeed at least under the latter, no problem of interpretation arises: there appear to be differences. However, it has also been described how sorting can be directly assessed in man and the chimpanzee, whereas classification by monkeys and nonsimians must be assessed indirectly. If then on indirect tasks the monkey requires many hundreds of trials whereas on direct tests the chimpanzee sorts spontaneously, how different are their abilities at classification ? How can differences between test procedures be isolated from differences between species? There is no simple answer. And yet we cannot neglect the standard approach of the comparative psychologist. Given these, and further, limitations, it must not be considered surprising that we have been unable to draw clear-cut conclusions for many kinds of performance. Nonetheless a n attempt has been made in this section to draw upon the evidence from normal animals for a n overall comparison between species. When dealing with brain lesions it was pointed out that there do not occur naturally in man areas of cortical damage or disease as restricted as these produced by the experimenter in nonhuman animals. The more extensive brain lesions in man do not permit fine separations of behavioral consequences (since the larger lesions, by involving more than one neural system, are less likely to impair only a restricted aspect of behavior). In acdition it was emphasized that neuropsychological research on apes is virtually nonexistent. Again, such problems have imposed severe limitations on our conclusions. But a substantial deficiency of the lesion method would remain even if comparable lesions were to exist for man and nonhuman primates; and even if there were already in existence a body of neuropsychological research on apes. At present, neuropsychology points to brain regions that are involved in different performances (i.e., tells us “where” in the brain), but generally

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does not succeed in revealing the functions of these regions (i.e., tells us little of “how” that brain region functions). This deficiency is more transient than permanent because the aim of neuroljsychology is ultimately to delineate function, not merely locus; and its increasingly closer relationships to physiology, biochemistry, and pharmacology promise an understanding of the mechanisms of behavior. Nonetheless this stage has not yet been attained. And in comparative studies we must take care not to exclude the possibility that similar brain mechanisms exist in different species, albeit located in nonhomologous regions of the brain (i.e., present-day neuropsychology might point to a spurious difference between species by taking only “locus” of lesions into account). Despite the difficulties, the neuropsychological approach is the only one that could, even in principle, succeed in the comparative study of brain organization in behavior. Already now it can (to some extent) dissociate different kinds of behavior: if differently placed lesions reliably result in different behavioral defects (i.e., blindness and somatic sensory loss; or amnesia and spatial neglect), it becomes possible that the unimpaired performances (e.g., vision and somatic sensitivity; or memory and spatial attention) are separately mediated in the brain. Moreover, it can be argued that only the clinical evidence (of cerebral dominance,’ dysphasia, separate speech areas, etc.) can sustain the view that language represents a major qualitative difference between man and nonhuman primates. (The reader need only consider the current difficulty in connection with “intelligence” : is there some aspect that is unique to man? The answer would become clear if “intelligence” in man, but not in other primates, were lateralized, or if man, but no other animal, had a large sector of the brain devoted only to “intelligence.”) Therefore ultimately we can hope to find answers to the questions we have raised, even if in the meanwhile we must be content to advance but slowly. Progress can be expected from further collaboration across disciplines (e.g., electrophysiology combined with behavior study; neuroanatomy guided by behavioral findings) ; from study of many additional species beyond the standard laboratory animals (rat, cat, and rhesus monkey) ; from refinement of present methods (e.g., tests that are “purer”; lesions that are smaller and more closely related to anatomical and physiological boundaries) but perhaps most of all from the “far-shot” class of experiment (e.g., that which against all the then availabie evidence was successful in teaching prelinguistic skills to the chimpanzee ; or that which examined the then supposedly remote possibility of differential functioning within a supposedly homogeneous region like the inferotemporal cortex of the monkey). Man’s brain will surely prove capable of elucidating its own organization and that of its nonhuman relatives.

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IV. Discussion

This paper has reviewed comparisons of the brain and behavior of living primates, using both the correlational and experimental methods. I n both methods a study is made of a pseudo-evolutionary series of living primates, to help in the interpretation of the fossil record, and to provide indirect evidence where the fossil record could never provide direct evidence. The problems arising from the use of such a series have been briefly discussed in Section I. Since no single species can be taken as “representative” of the larger group to which it belongs, comparisons of only a few species as representatives of either prosimians, New or Old World monkeys, or apes may provide results which are misleading. Given the variations in both brain and behavior within, for example, the New World monkeys, choice of the marmoset and the cebus monkey as the representative species would present a very distorted view of the brain or behavior of the New World monkeys as a whole. If attempts are made to correlate the brain with abilities, this can be done only if large numbers of different species are used, so that the means and standard deviations for each major group can be assessed. A large series is also needed if differences in brain or behavior between man and the great apes are to be validly assessed, since these differences must be interpreted in the light of the trends within the nonhuman primates. When experimental methods are used, such as producing brain lesions, it may be impractical to collect a large series. It is therefore all the more important that information be available on the great apes, and yet there is still little experimental study of the brain and behavior of these animals. The correlational method can only suggest possible relations between behavior and particular parts of the brain, but these suggestions can be confirmed by the experimental method. Comparisons of the effects of lesions on behavior can only be interpreted where the lesions are similar in the primates studied. It will be apparent from the previous sections that there are often no comparable data on the effects ofparticular lesions in man and the nonhuman primates, for example, for restricted lesions of the temporal cortex. KO one disputes that there are marked differences in the abilities of man and other primates. There have, however, been claims that certain of these differences are qualitative. I t has been argued in Section I1 that man is not unique either in tool-making or in transmitting cultural traditions of behavior; but he is unique in naturally possessing language, even though great apes may be shown in the laboratory to have abilities that are preadaptive for language. Language provides an efficient means for teaching toolmaking, and for passing on information and traditions of behavior. Unfortunately, we do not have the means to determine when language evolved in the hominids, and we can rely only on indirect evidence from the

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tool cultures found and the evidence of cooperative hunting (Pfeiffer, 1972). Evidence such as this suggests that Homo erectus may have possessed some form of language. I t is further possible that the selection pressure for the development of language increased when man’s ancestors started to hunt large game, and required both specialized tools and means of planning and coordinating group hunts. I n view of the differences in abilities between man and the other primates differences might be expected in the anatomy of their brains, particularly in the neocortex. I t has been shown that man’s brain has changed in a way that fits many of the trends within the other primates; for example, as brain size increases so does the percentage of it formed by the neocortex, and as neocortex increases so does the percentage of it formed by association cortex. But man differs from other primates in having a brain and a neocortex which are roughly three times as great as would be expected for a primate of his body size. Sacher (1959) has shown that there is a complex relationship in mammals between body size, life-span, and brain size. Larger mammals tend to live longer, and both brain and body size are related to length of life. Compared with mammals of the same body weight, man lives longer and has a much larger brain. As yet, however, it has not proved possible to relate man’s possession of language to any clear difference between his brain and that of other primates. Such a difference might lie either in the number of cells needed over and above those required for sensory analysis and motor commands, or in some structural specialization of particular areas. Geschwind’s (1965) theory of the importance of the angular gyrus for cross-modal association and language in man has already been discussed. Neither the anatomical evidence (Section 11) nor the behavioral evidence (Section 111) on other primates is consistent with this theory. It seems more likely, not that man is specialized for cross-modal associations per se, but that he is specialized in his capacity to use and comprehend sound in communication. However, apart from the possibility that man is unique in having slight asymmetry between the hemispheres in the supratemporal plane (Geschwind and Levitsky, 1968), hope of finding the structural specializations subserving language rests on studies of the fine structure of each hemisphere. Comparative studies of the effects of lesions on the behavior of different primates suggest three major apparent differences between man and other primates. First, in man lesion studies have demonstrated asymmetry of function between the two hemispheres, such that the effects of lesions differ according to the hemisphere damaged. As yet there are no convincing demonstrations of such asymmetry of function in other primates (see Section 111.) Second, in man language tends to be subserved by the left hemisphere in right-handers. Although lateralization of language and specialization of

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nonverbal functions between the hemispheres are related in man, it is theoretically possible that there might be asymmetry of function in other primates which lack language. The third apparent difference is that between the effects of bilateral hippocampal lesions in man and in other primates. It is possible that this difference may be resolved by the demonstration of common features in the effects of such lesions in man and other primates (Weiskrantz, 1971). But it would also be rash to ignore those respects in which the results for man and rhesus monkeys still appear to differ. When these differences have been fully documented, a n attempt might then be made to account for them, perhaps as the result of specialization of the left- and right-sided hippocampal regions in man for verbal and nonverbal functions (reviewed by Milner, 1971). The issues discussed here have been largely ignored for many years, and it is only recently that there has been a revival of interest among neuropsychologists in the evolution of the brain and behavior of man and his ancestors. This revival of interest is due, more than to anything else, to the work of Diamond and his colleagues (Diamond and Hall, 1969; Masterton and Skeen, 1972). Much of the work has to be carried out on living primates, and requires that a distinction be made between those characters which have been inherited from a common ancestor, and those which have been independently acquired. A proper understanding of the evolution of the brain and behavior of man and his ancestors can come only from a description of those characteristics which he has inherited in common with other primates, and those which are unique to the hominids. ACKNQWLEDCMENTS

We are grateful to Professor H. Stephan for providing the data on which Fig. 2 is based, and to Professor A. H. Schultz for providing the data for the great apes used in Fig. 7; also to Professor C. G. Philips for advice on Section 111, B, and to both Dr. A. Cowey and Dr. A. D. Milner for their comments on an early draft; and to Mrs J. Bewry for her expert secretarial assistance. REFERENCES

Annett, M. (1972). Brit.J . Psychol. 63, 343. Ashton, E. H. (1950). Proc. Zool. Soc. London 120, 715. Bailey, P., and von Bonin, G. (1951). “The Isocortex of Man.” Univ. of Illinois Press, Urbana. Bailey, P., von Bonin, G., Garol, H. W. C., and McCulloch, W. S. (1943). J . Neurophysiol. 6, 129. Bailey, P., von Bonin, G., and McCulloch, W. S. (1950). “The Isocortex of the Chimpanzee.” Univ. of Illinois Press, Urbana. Bates, J. A. V., and Ettlinger, G. (1960). Arch. Neurol. (Chicfgo) 3, 177. Beck, C. H. M., and Barton, R. L. (1972). Cortex 8, 339.

CORTICAL FUNCTIONS IN PRIMATES

293

Bishop, A. (1964). In “Evolutionary and Genetic Biology of Primates” (J. BuettnerJanusch, ed.), Vol. 2, p. 133. Academic Press, New York. Blakemore, C., Iversen, S. D., and Zangwill, 0. L. (1972). Annu. Rev. Psychol. 23, 413. Blank, M., and Klig, S. (1970). J. Ex). Child Psychol. 9, 166. Brindley, G. S., Gautier-Smith, P. C., and Lewin, W. (1969). J. Neurol., Neurosurg. Psychiat. 32, 259. Broadbent, D. E. (1970). Proc. Roy. Soc., Ser. B 175, 333. Broadbent, D. E. (1971). “Decision and Stress.” Academic Press, New York. Brodmann, K. (1912). Anat. Anz. 41, Suppl. 157. Bronowski, J., and Bellugi, U. (1970). Science 168, 669. Brown, R. (1970). In “Psycholinguistics: Selected Papers” (R. Brown, ed.), p. 208. Free Press, New York. Brush, E. S., Mishkin, M., and Rosvold, H. E. (1961). J. Comp. Physiol. Psychol. 54, 319. Bryant, P. E. (1971). Brit. Med. Bull. 27, 200. Bryant, P. E., Jones, P., Claxton, V., and Perkins, G. M. (1972). Nature 240, (London) 303. Bucy, P. C. (1944). In “The Precentral Motor Cortex” (P. C. Bucy, ed.), p. 353. Univ. of Illinois Press, Urbana. Bucy, P. C., Keplinger, J. E., and Siqueira, E. B. (1964). J. Neurosurg. 21, 385. Butter, C. M. (1969). Brain Res. 12, 374. Butter, C. M. (1972). Neuropsychologia 10, 241. Butters, N., and Brody, B. A. (1968). Cortex 4, 328. Butters, N., Barton, M., and Brody, B. A. (1970). Cortex 6, 174. Campbell, C. B. G., Yashon, D., and Jane, J. A. (1966). J. Comp. Neurol. 127, 101. Cartmill, M. (1970) Ph.D. Dissertation, University of Chicago, Chicago, Illinois. Cartmill, M. (1972). In “The Functional and Evolutionary Biology of Primates” (R. Tuttle, ed.), p. 97. Aldine, Chicago, Illinois. Charles-Dominique, P., and Martin, R. D. (1970). Nature (London) 227, 257. Chow, K. L. (1950). J. Comp. Neurol. 93, 313. Chow, K. L. (1954). J. Comp. Physiol. Psychol. 47, 194. Chow, K. L. (1961). J. Neurofihysiol. 24, 391. Chow, K. L., and Hutt, P. J. (1953). Brain 76, 625. Cobb, S. (1965). Arch. Neurol. (Chicago) 12, 555. Cooper, L. R., and Harlow, H. F. (1961). Psychol. Rep. 8, 418. Correll, R. E., and Scoville, W. B. (1965). J. Comp. Physiol. Psychol. 60, 175. Correll, R. E., and Scoville, W. B. (1970). J. Comp. Physiol. Psychol. 70, 464. Count, E. W. (1947). Ann. N . Y. Acad. Sci. 46, 993. Cowey, A. (1965). J. Neurophysiol. 27, 366. Cowey, A., and Gross, C. G. (1970). Exp. Brain Res. 11, 128. Critchley, M. (1970). “Aphasiology, and other Aspects of Language.” Arnold, London. Daitz, H. M., and Powell, T. P. S. (1954). J. Neurol., Neurosurg. Psychiat. 17, 75. Dart, R. A. (1956). S. Afr. J. Med. Sci. 21, 23. Davenport, R. K., and Rogers, C. M. (1970). Science 168, 279. Davenport, R. K., and Rogers, C. M. (1971). Behaviour 39, 318. Davis, R. T. (1971). Proc. Znt. Congr. Primatol., 3rd, 1970 Vol. 3, p. 150. de Renzi, E., Savoiardo, M., and Vignolo, L. A. (1966). Cortex 2, 399. de Renzi, E., Faglioni, P., Scotti, G., and Spinnler, H. (1972). Cortex 8, 147. Dewson, J. H., Cowey, A., and Weiskrantz, L. (1970). Exp. Neurol. 28, 529. Diamond, I. T., and Hall, W. C. (1969). Science 164, 251. Diamond, I. T., Snyder, M., Killackey, H., Jane, J., and Hall, W. C. (1970). J. Comp. Neurol. 139, 273. Dimond, S., and Beaumont, T. G. (1971). Nature (London) 232, 270.

294

R. E. PASSINGHAM AND G. E’ITLMGER

Drewe, E. A., Ettlinger, G., Milner, A. D., and Passingham, R. E. (1970). Cortex 6, 129. Ebner, F. F., and Myers, R. E. (1962). J . Neurophysiol. 25, 380. Ettlinger, G. (1971). Klin. Wochenschr. 49, 786. Ettlinger, G. (1973). ZR “Memory and Transfer of Information” (H. P. Zippel, ed.). p. 42. Plenum, New York. Ettlinger, G., and Blakemore, C. B. (1967). Neuropsychologia 5, 147. Ettlinger, G., and Gautrin, D. (1971). Cortex 7, 317. Ettlinger, G., and Kalsbeck, J. E. (1962). J . Neurol., Neurosurg. Psychiat. 25, 256. Ettlinger, G., Blakemore, C. B., and Milner, A. D. (1968). Nature (London) 218, 1276. Farrer, D. N. (1967). Percept. 6’M o f . Skills 25, 305. Finch, G. (1941). Science 94, 117. Flechsig, P. (1901). Lancef 2, 1027. Fouts, R. S. (1972). J . Comp. Physiol. Psychol. 80, 515. Fouts, R. S. (1973). Science 180, 978. Frampton, G. G., Milner, A. D., and Ettlinger, G. (1973). NeuropsychoZogiu 11, 231. Fulton, J. F. (1947). In “Howell’s Textbook of Physiology” (J. F. Fulton, ed.), p. 273. Saunden, Philadelphia, Pennsylvania. Fulton, J. F. and Keller, A. D., (1932). “The Sign of Babinski.” Thomas, Baltimore. Gaffan, D. (1972). Neuropsychologia 10, 327. Gallup, G. C. (1969) Science 167, 86. Gardner, B. T., and Gardner, R. A. (1971). Zn “Behavior of Nonhuman Primates” (A. M. Schrier and F. Stollnitz, eds.), Vol. 4, p. 117. Academic Press, New York. Gazzaniga, M. S. (1963). Exp. Neurol. 8, 14. Geschwind, N. (1965). Bruin 88, 237 and 585. Geschwind, N. (1971). In “Cognitive Processes of Nonhuman Primates” ,(L. E. Jarrard, ed.), p. 149. Academic Press, New York. Geschwind, N. (1972). Sci. Amer. 226, No. 4, 76. Geschwind, N., and Levitsky, W. (1968). Science 161, 186. Giolle, R. A., and Tigges, J. (1970). IR “The Primate Brain” (C. R. Noback and W. Montagna, eds.), Vol. 1, p. 29. Appleton, New York. Glees, P.. and Wall, P. D. (1948). J . Cornp. Neurol. 88, 129. Glickstein, M.,King, R., Miller, J., and Berkely, M. (1967). J . Conip. Neurol. 130, 55. Gross, C. G., and Weiskrantz, L. (1964). In “The Frontal Granular Cortex and Behavior” (J. M. Warren and K. Akert, eds.), p. 74. McGraw-Hill, New York. Gross, C . G., Cowey, A., and Manning, F. J. (1971). J. Comp. Physiol. Psychol. 76, 1. Hall, W. C., and Diamond, I. T. (1968). Brain, Behavior, €8 Evolution 1, 215. Hamilton, C. R., and Lund, J. S. (1970). Science 170, 1482. Harlow, H. F., Uehling, H., and Maslow, A. H. (1932). J. Cornp. Psychol. 13, 313. Harlow, H. F., h‘feyer, D., and Settlage, P. H. (1951). J . Comp. Physiol. Psychol. 44, 320. Harmsn, P. J. (1957). “Paleoneurologic, Neoneurologic and Ontogenetic Aspects of Brain Phylogeny.” Janies Arthur Lecture, Amer. Mus. Natur. Hist. Hartje, W., and Ettlinger, G. (1974). Cortex 9, 344. Haug, H. (1956). J. Comp. Neurol. 104, 473. Hayes, K. J., and Nissen, C. H. (1971). IR “Behavior of Nonhuman Primates” (A. M. Schrier and F. Stollnitz, eds.), Vol. 4, p. 59. Academic Press, New York. Htcaen, H. (1972). “Neuropsychologie de la perception visuelle.” Masson, Paris. Heilman, K. M., Pandya, D. N., Karol, E. A., and Geschwind, N. (1971). Arch. Neurol. (Chicago) 24, 323. Hodos, W., and Campbell, C. B. G. (1969). Psychol. Rev. 76, 337. Holloway, R. L. (1968). Brain Res. 7, 121. Holloway, R. L. (1970). Science 168, 966.

CORTICAL FUNCTIONS IN PRIMATES

295

Holloway, R. L. (1972a). In “The Functional and Evolutionary Biology of Primates” (R. Tuttle, ed.), p. 185. Aldine, Chicago, Illinois. Holloway, R. L. (197213). Amer. J. Phys. Anthropol. 37, 173. Hopf, A. (1965). J. Hirnforsch. 8, 25. Humphrey, N. K. (1970). Brain Behavior €8 Evolution 3, 324. Humphrey, N. K. (1971). Nature (London) 229, 615. Humphrey, N. K. (1972). New Sci. 54, 682. Humphrey, N. K., and Weiskrantz, L. (1967). Nature (London) 215, 595. Isaacson, R. L. (1972). Neuropsychologia 10, 47. Iversen, S. D. (1967). Exp. Neurol. 18, 228. Iversen, S. D., and Humphrey, N. K. (1971). Brain Res. 30, 253. Iversen, S. D., and Weiskrantz, L. (1970). Neuropsychologia 8, 21. Jarrard, L. E., and Moise, S. L. (1971). In “Cognitive Processes of Nonhuman Primates” (L. E. Jarrard, ed.), p. 3. Academic Press, New York. Jarvik, M. E., Goldfarb, T. L., and Carley, J. L. (1969). J. Ex). Psychol. 81, 1. Jerison, H. J. (1955). Science 121, 447. Jerison, H. J. (1963). Hum. Biol. 35, 263. Jolly, A. (1972). “The Evolution of Primate Behavior.” Macmillan, New York. Jones, B., and Mishkin, M. (1972). Exp. Neurol. 36, 362. Jones, E. G., and Powell, T. P. S. (1970). Brain 93, 793. Jiirgens, U., and Ploog, D. (1970). Exp. Brain Res. 10, 532. Kaas, J., Hall, W. C., and Diamond, I. T. (1970). J . Neurophysiol. 33, 595. Kaas, J., Hall, W. C., and Diamond, I. T. (1972). J. Comp. Neurol. 145, 273. Killackey, H., and Diamond, I. T. (1971). Science 131, 696. Killackey, H., Snyder, M., and Diamond, I. T. (1971). J. Comp. Physiol. Psychol. 74, 1. Killackey, H., Wilson, M., and Diamond, I. T. (1972). J. Comp. Physiol. Psychol. 81, 45. Kimura, D. (1961). Can. J . Psychol. 15, 156. Konorski, J. (1959). Bull. Acad. Pol. Sci. 7, 115. Konorski, J. (1967). “Integrative Activity of the Brain.” Chicago Univ. Press, Chicago, Illinois. Krantz, G. S. (1968). Curr. Anthropol. 9, 450. Krompecher, S., and Lipack, J. (1966). J. Comp. Neurol. 127, 113. Kroustov, G. F. (1970). Sou. Psychol. 9, 6. Kummer, H. (1971). “Primate Societies: Group Techniques of Ecological Adaptation.” Aldine, Chicago, Illinois. Kuypers, H. G. J. M. (1958a). J. Comp. Neurol. 110, 221. Kuypers, H. G. J. M. (1958b). Brain 81, 364. Larsen, J. W., Winans, S. S., and Meikle, T. H. (1969). Brain Res. 14, 717. Lashley, K. S. (1949). Quart. Rev. Biol. 24, 28. Lawrence, D. G., and Kuypers, H. G. J. M. (1968). Brain 91, 1. le Gros Clark, W. E. (1971). “The Antecedents of Man,” 3rd ed. Edinburgh Univ. Press, Edinburgh. Lenneberg, E. H. (1964). In “New Directions in the Study Language” (E. H. Lenneberg. ed.), p. 65. M I T Press, Cambridge, Massachusetts. Lenneberg, E. H. (1967). “Biological Foundations of Language.” Wiley, New York. Lenneberg, E. H. (1971). In “Early Childhood: The Development of Self Regulatory Mechanisms” (D. N. Walcher and D. L. Peters, eds.), p. 157. Academic Press, New York. Lenneberg, E. H. (1973). Abstr. Pap., 17th Int. Neurop@chol. Symp., 1972 (to appear in Neuropsychologia). Leonard, C. M. (1969). Brain Res. 12, 321.

296

R. E. PASSINGHAM AND 0. ETT’LMCER

Levine, M., and Harlow, H. F. (1959). A m . J. Psychol. 72, 253. Liberman, P. (1968). J. Acoust. SOC.Amer. 44, 1574. Lovejoy, C. O., and Heiple, K. G. (1970). Amer. J. Phys. Anthropol. 32, 33. McFie, J. (1960). J. N u v . Ment. Dir. 131, 383. McNeill, D. (1973). In “Competence in Infancy” (K. Conolly and J. Bruner, eds.). Academic Press, New York (in press). Mahut, H. (1971). Neuropsychologia 9, 409. Maier, N. R. F., and Schneirla, T. C. (1964).,“Principles of Animal Psychology,’’ 2nd ed. Dover, New York. Marquis, D. G. (1935). Arch. Newol. Psychiat. 33, 807. Martin, R. D. (1968a). 2. Tietpsychol. 25, 409. Martin, R. D. (1968b). Man 3, 377. Maslow, A. H., and Harlow, H. F. (1932). J. Comp. Psychol. 14, 97. Masterton, B., and Skeen, L. C. (1972). J. Comp. Physwl. Psychol. 81, 423. Masterton, B., Hefner, H., and Ravizza, R. (1969). J. Acourf. Soc. A m . 45, 966. Mathers, L. H. (1971). Brain Res. 35,295. Meikle, T. H., Sechzer, J. A., and Stellar, E. (1962). J. Neurophyrwl. 25, 530. Menzel, E. W. (1971). Folia Primtol. 15, 220. Miles, R.C. (1964). In “The Frontal Granular Cortex and Behavior” (J. M. Warren and K. Akert, eds.), p. 149. McGraw-Hill, New York. Miller, R. E. (1971). Primate Behau. 2, 139. Milner, A. D. (1969). Neuropsychologia 7, 375. Milner, A. D. (1973). J. Comp. Physwl. Psychol. 83, 278. Milner, A. D., and Ettlinger, G. (1970). Neuropsychologia 8, 251. Milner, B. (1958). Res. Publ., Ass. Res. N m . Ment. Dis. 36,244. Milner, B. (1963). Arch. Nncrol. (Chicago) 9, 90. Milner, B. (1968). In “Analysis o f Behavioral Change” (L. Weiskrantz, ed.), p. 328. Harper, New York. Milner, B. (1971). Brit. Med. Bull. 27, 272. Milner, B., Branch, C., and Rasmussen, T. (1964). In “Disorders of Language” (A. V. S. de Rueck and M. O’Connor, eds.), p. 200. Churchill, London. Mishkin, M. (1972). In “The Brain and Human Behavior” (A. G. Karczmar and J. E. Eccles, eds.), p. 187. Springer-Verlag, Berlin and New York. Mishkin, M., Prockop, E. S., and Rosvold, H. E. (1962). J. Comp. Physiol. Psychol. 55, 178. Morris, D. (1967). In “Primate Ethology” (D. Morris, ed.), p. 1. Weidenfeld & Nicolson, London. Myers, R. E. (1972). Acta Neurobiol. Exp. 32, 568. Nauta, W. J. G., and Karten, H. J. (1970). In “Neurosciences” (F. C. Schmidt, ed.), Vol. 2, p. 7. Rockefeller Univ. Press, New York. Nie, V., Upton, A., and Ettlinger, G. (1973). Exp. Neurol. 40, 632. Noback, C. L., and Moskowitz, N. (1963). In “Evolutionary and Genetic Biology of Primates” (J. Buettner-Janusch, ed.), Vol. 1, p. 131. Academic Press, New York. Oakley, K. P. (1967). “Man, the Tool-maker.’’ Brit. Mus. (Natur. Hist.), London. Orbach, J. (1959). A M A Arch. Neurol. Psychiaf. 81, 49. Orbach, J., Milner, B., and Rasmussen, T. (1960). Arch. Neurol. (Chicago) 3, 230. Pakkenberg, H., and Voigt, J. (1964). Acfa Anat. 56, 297. Pandya, D. N., and Kuypers, H. G. J. M. (1969). Brain Res. 13, 13. Pasik, T.,and Pasik, P. (1971). Vision Res. 3, Suppl., 419. Passingham, R. E. (1973). Brain, Behavior, &? Evolution (in press). Penfield, W., and Roberts, L. (1959). “Speech and Brain Mechanisms.” Princeton Univ. Press, Princeton, New Jersey.

CORTICAL FUNCTIONS IN PRIMATES

297

Pfeiffer, J. E. (19721. “The Emergence of Man,” 2nd ed. Harper, New York. Philips, C. G. (1971). Proc. Int. Congr. Primatol. 3rd, 1970 Vol. 2, p. 2. Piercy, M., and Huppert, F. A. (1972). Nature (London) 240, 564. Piercy, M. F. (1964). Brit. J. Psychiat. 110, 310. Pohl, W. (1973). J. Camp. Physiol. Psychol. 82, 227. Powell, T. P. S., Guillery, R. W., and Cowan, W. M. (1957). J. Anat. 91, 419. Premack, D. (I971a). In “Cognitive Processes ofNonhuman Primates” (L. E. Jarrard, ed.), p. 47. Academic Press, New York. Premack, D. (1971b). In “Behavior of Nonhuman Primates” (A. M. Schrier and F. Stollnitz, eds.), Vol. 4, p. 185. Academic Press, New York. Provins, K. A. (1967). Aust. J. Psychol. 19, 137. Provins, K. A., and Cunliffe, P. (1972). Neuropsychologia 10, 199. Radinsky, L. B. (1967). Science 155, 836. Radinsky, L. B. (1970). Zn “The Primate Brain” (C. R. Noback and W. Montagna, eds.), Vol. 1, p. 209. Appleton, New York. Radinsky, L. B. (1972). In “The Functional and Evolutionary Biology of Primates” (R. Tuttle, ed.), p. 175. Aldine, Chicago, Illinois. Rensch, B. (1956). Amer. Nafur. 90, 81. Robinson, B. W. (1967). Physiol. d Behav. 2, 345. Robinson, B. W. (1972). In “Perspectives on Human Evolution” (S. L. Washburn and P. Dolhinow, eds.), Vol. 2, p. 438. Holt, New York. Robinson, J. S., and Voneida, T. J. (1970). Exp. Neural. 26, 72. Romer, A. S. (1966). “Vertebrate Paleontology,” 3rd ed. Chicago Univ. Press, Chicago, Illinois. Rose, J. E., and Woolsey, C. N. (1949). Elctroencephalogr. Clin. Neurophysiol. 1, 391. Rumbaugh, D. M. (1971). J. Camp. Physiol. Psychol. 76, 250. Rumbaugh, D. M., and Gill, T. V. (1971). Proc. Int. Congr. Primatol., 3rd, 1970 Vol. 3, p. 158. Sacher, G. (1959). In “The Lifespan of Animals” (G. E. W. Wolstenholme and M. O’Connor, eds.), p. 115. Churchill, London. Samuels, I., Butters, N., and Fedio, P. (1972). Cortex 8, 283. Sarno, J. E., Swisher, L. P., and Sarno, M. T. (1969). Cortex 5, 398. Schaller, G. B. (1963). “The Mountain Gorilla; Ecology and Behavior.” Univ. of Chicago Press, Chicago, Illinois. Schultz, A. H. (1941). Amer. J . Phys. Anthropol. 28, 273. Schultz, A. H. (1965). In “Homenaje a Juan Comas,” Vol. 2, p. 327. Mexico. Scoville, W. B., and Milner, B. (1957). J . Neural., Neurosurg. Psychiat. 20, 11. Sechzer, J. A. (1970). Science 169, 889. Seckel, H. P. G. (1960). “Bird Headed Dwarfs.” Karger, Basel. Semmes, J. (1968). Neuropsychologia 6, 1 1. Semmes, J., and Mishkin, M. (1965). In “Functions of the Corpus Callosum” (E. G. Ettlinger, ed.), p. 60. Churchill, London. Shariff, G. A. (1953). J. Camp. Neural. 98, 381. Sholl, D. A. (1956). “The Organization of the Cerebral Cortex.” Methuen, London. Simons, E. L. (1962). Ann. N . Y. Acad. Sci. 102, 282. Simons, E. L. (1969). Ann. N . Y. Acad. Sci. 167, 319. Simons, E. L. (1972). “Primate Evolution; an Introduction to Man’s Place in Nature.” Macmillan, New York. Simpson, D. A. (1952). J . Anat. 86, 20. Slamecka, N. J. (1968). Psychol. Bull. 69, 423. Snyder, M., and Diamond, I. T. (1968). Brain, Behavior, B Evolution 1, 244.

298

R. E. PASSINGHAM AND G . ETTLINGER

Solnitzky, O., and Harman, P. J. (1946). J. Comp. Neurol. 84, 339. Stamm, J. S., and Pribram, K. H. (1960). J. Neurophysiol. 23, 552. Stephan, H. (1969). Proc. Int. Congr. Primafol., 2nd, Vol. 3, p. 34. Stephan, H. (1972). In “The Functional and Evolutionary Biology of Primates” (R. Tuttle, ed.), p. 155. Aldine, Chicago, Illinois. Stephan, H., and Andy, 0.J. (1969). Ann. N . Y. Acad. Sci. 167, 370. Stephan, H., and Andy, 0. J. (1970). In “The Primate Brain” (C. R. Noback and W. Montagna, eds.), Vot. 1, p. 109. Appleton, New York. Stephan, H., Bauchot, R. V., and Andy, 0. J. (1970). In “The Primate Brain” (C. R. Noback and W. Montagna, eds.), Vol. 1, p. 289. Appleton, New York. Strong, P. N. (1959). J . Comp. Physiol. Psychol. 52, 333. Suomi, S. J., Harlow, H. F., and Lewis, J. K. (1970). J. Comp. Physiol. Psychol. 70, 448. Talland, G. A. (1965). “Deranged Memory.” Academic Press, New York. Ter Braak, J. W. G., Schenk, W. D., and van Vliet, A. G. M. (1971). J. Neurol., Neurosurg. Psychiat. 34, 140. Teuber, H. L. (1968). I n “Analysis of Behavioral Change” (L. Weiskrantz, ed.), p. 274. Harper, New York. Tilney, F. (1927). J. Comp. Neurol. 43, 371. Tobias, P. V. (1963). Nature (London) 197, 743. Tobias, P. V. (1970). A m . J. Phys. Anthropol. 32, 3. Tobias, P. V. (1971). “The Brain in Hominid Evolution.” Columbia Univ. Press, New York. Tower, D. B. (1965). J. Comp. Neurol. 101, 19. Tower, S. S. (1944). In “The Precentral Motor Cortex” (P. C. Bucy, ed.), p. 150. Univ. of Illinois Press, Urbana. Vallois, H. V. (1961). In “The Social Life of Early Man” (S. L. Washburn, ed.), p. 124. Aldine, Chicago, Illinois. van Buren, J. M., and Borke, R. C. (1972). Bruin 95, 599. van Lawick Goodall, J. (1970). Aduan. Study Behau. 3, 195. Vevcrs, G. M., and Weiner, J. S. (1963). Symp. Zool. Soc. London 10, 115. Victor, M., Adams, R. D., and Collins, G. H. (1971). “The Wernicke-Korsakoff Syndrome.” Blackwell, Oxford. von Bonin, G . (1937). J . G e m . Pqchol. 16, 379. von Bonin, G. (1941). J. Comp. Neurol. 75, 287. von Bonin, G. (1952). A M A Arch. Neurol. Psychiat. 67, 135. von Bonin, G. (1962). In “Interhemispheric Relations and Cerebral Dominance” (V. B. Mountcastle, ed.), p. 1. Johns Hopkins Press,Baltimore, Maryland. von Bonin, G. (1963). “The Evolution of the Human Brain.” Chicago Univ. Press, Chicago, Illinois. von Bonin, G., and Bailey, P. (1947). “The Neocortex of Macaca mulatta.” Univ. of Illinois Press, Urbana. von Bonin, G., and Bailey, P. (1961). Primatologiu ZI 10, 1. von Economo, C. (1929). “The Cytoarchitecture of the Human Cerebral Cortex.” Oxford Univ. Press, London and New York. von Rose, M. (1935). In “Handbuch der Neurologie” (0.Bumke and 0. Foerster, eds.), p. 541. Springer-Verlag, Berlin and New York. Walker, A. E. (1938a). “The Primate Thalamus.” Univ. of Chicago Press, Chicago, Illinois. Walker, A. E. (1938b). J. Anat, 73, 37. Walker, A. E. (1966). In “The Thalamus” (D. P. Purpura and M. D. Yahr, eds.), p. 1. Columbia Univ. Press, New York.

CORTICAL FUNCTIONS IN PRIMATES

299

Walker, A. E., and Fulton, J. F. (1938). J. Nerv. Ment. Dis. 87, 677. Ward, J. P., and Masterton, B. (1970). Brain, Behavior, B Evolution 3, 421. Ward, J. P., Yehle, A. L., and Doerflein, R. S. (1970). J. Comp. Physiol. Psychol. 73, 74. Ware, C. B., Casagrande, V. A., and Diamond, I. T. (1972). Brain, Behavior, B Evolution 5, 18. Warren, J. M. (1965). In “Behavior of Nonhuman Primates” (A. M. Schrier, H. F. Harlow, and F. Stollnitz, eds.), Vol. 1, p. 249. Academic Press, New York. Warren, J. M. (1972). Acta Neurobiol. Exp. 32, 581. Warren, J. M. (1973). In “Comparative Psychology: A Modern Synthesis” (D. A. Dewsbury, ed.), p. 471. McGraw-Hill, New York. Warren, J. M., Abplanalp, J. M., and Warren, H. B. (1967). In “Early Behavior. Comparative and Developmental Approaches” (H. W. Stevenson, ed.), p. 73. Wiley, New York. Warrington, E. K., and Weiskrantz, L. (1970). Nature (London) 228, 628. Washburn, S . L. (1959). In “The Evolution of Man’s Capacity for Culture” (J. N. Spuhler, ed.), p. 21. Wayne State Univ. Press, Detroit, Michigan. Washburn, S. L. (1968). “The Study of Human Evolution.” Univ. of Oregon Press, Eugene. Webster, W. G. (1972). Neuropsychologia 10, 75. Wegener, J. G. (1968). Cortex 4 , 203. Weidenreich, F. (1941). Trans. Amer. Phil. Soc. 31, 321. Weidenreich, F. (1948). Sci. Mon. 67, 103. Weinstein, S . (1962). In “Interhemispheric Relations and Cerebral Dominance” (V. B. Mountcastle, ed.), p. 159. Johns Hopkins Press, Baltimore, Maryland. Weiskrantz, L. (1963). Neuropsychologia 1, 145. Weiskrantz, L. (1966). In “Current Problems in Animal Behaviour” (W. H. Thorpe and 0. L. Zangwill, eds.), p. 30. Cambridge Univ. Press, London and New York. Weiskrantz, L. (1971). In “Cognitive Processes of Nonhuman Primates” (L. E. Jarrard, ed.), p. 25. Academic Press, New York. Weiskrantz, L. (1972). Proc. Roy. SOC.,Ser. B. 182, 427. Weiskrantz, L., and Cowey, A. (1970). In “Progress in Physiological Psychology” (E. Stellar and J. M. Sprague, eds.), Vol. 3, p. 237. Academic Press, New York. White, L. A. (1959). In “The Evolution of Man’s Capacity for Culture” (J. N. Spuhler, ed.), p. 74. Wayne State Univ. Press, Detroit, Michigan. Wolfe, J. B. (1936). Comp. Psychol. Monogr. 12, No. 5, 1. Woolsey, C. N. (1958). In “The Biological and Biochemical Basis of Behavior” (H. F. Harlow and C. N. Woolsey, eds.), p. 63. Univ. of Wisconsin Press, Madison. Zippel, H. P., ed. (1973). “Memory and Transfer of Information,” Plenum, New York. Zuckerman, S., and Fulton, J. F. (1941). J . Anat. 75, 447.

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PORPHYRIA: THEORIES OF ETIOLOGY AND TREATMENT By H. A. Peters, D. J. Cripps, and H. H. Reese Departments of Neurology and Dermatology, University of Wisconsin Medical School, Madison, Wisconsin

I. Introduction and Scope . A. History: Early Studies and Theories B. Incidence . 11. Classification of Disturbed Porphyrin Metabolism . 111. Clinical Symptoms and Signs in Hepatic Porphyrics IV. Gastrointestinal Signs . X-Ray Findings . V. Genitourinary Signs VI. Cardiorespiratory Signs VII. Dermatological Signs . VIII. Precipitating Factors. A. Pregnancy B. Hormones C. Sedatives and Drugs D. Other Factors . IX. Pathology . A. Renal Studies B. Neuropathology . C. Vasculitis D. Hepatic Adenoma X. Biochemistry . A. Porphyrin Pathways . B. Enzymes XI. Pathogenesis of Attacks . A. Trace Metals, Zinc and Copper B. Tryptophan Metabolism C. Electrolytes . D. Amino Acid Excretion E. Hydrocortisone . F. Lipid Metabolism . G. Macroamylasemia . H. GrowthHormone XII. Therapy A. Chelation . B. Phlebotomy . C. Chloroquine D. Cholestyramine E. Pyridoxine 301

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1. Introduction and Scope

A. HISTORY: EARLYS T ~ I EAND S THEORIES The first demonstration of porphyrin substance was by Scherer (1841), who added concentrated sulfuric acid to dry powdered blood and then removed the iron by washing. This iron-free residue was treated with alcohol and took on a blood-red color. A similar observation was made by Mulder (1844), who named the substance hematin. It was renamed “cruetine” by Thudichum (1867), who further defined its spectrum and noted that the substance fluoresced. Hoppe-Seyler (1871) noted that the substance was really two ; he named the main constituent hematoporphyrin after the Greek word meaning purple. Important clinical observations were subsequently made by Schulz (1874) and Baumstark (1874), who described cases of probable congenital porphyria and associated this pigment in the urine with the disease in man. MacMunn (1880) described a dark pigment excreted in the urine of a patient with subacute rheumatism who was taking sodium salicylate, and he renamed the pigment urohematoporphyrin because of its striking resemblance to hematoporphyrin. LeNobel (1887) confirmed this finding and listed a number of conditions, including lead poisoning, in which he found this pigment. Stokvis (1889) reported the first case of Sulfonal-induced porphyria in an elderly woman who had taken this drug and later died. He felt that the pigment noted was similar to, but not identical with hematoporphyrin. Harley (1890) reported the case of another woman who developed many of the neurological features of acute intermittent porphyria after taking Sulfonal. Later Ranking and Pardington (1890) described two women who excreted red urine and showed neurological, psychiatric, and gastrointestinal symptoms, but neither woman had taken Sulfonal. It remained for Giinther (1911, 1922) to record a number of cases in which often Sulfonal or related drugs had been taken for long periods before the onset of symptoms. After barbiturates were introduced into clinical medicine in 1903, Dobrschansky (1906) described a case of acute porphyria without paralysis that followed prolonged use of diethyl barbituric acid. I n two publications Gunther (191 1, 1922) first classified the diseases of porphyrin metabolism, many of which were associated with the ingestion of Sulfonal, Trional, or Veronal. He also named for the first time “congenital porphyria,” in which the predominating symptoms included skin photosensitivity. Garrod (1923) in the second edition of his classic monograph

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credited Gunther with the first recognition that this disease was a n “inborn error of metabolism.” One of Gunther’s cases was a man called Petry; extensive studies of Petry by Hans Fischer between 1915 and 1945 resulted in tremendous contributions to porphyrin chemistry. The historical background is especially well documented by Goldberg and Rimington ( I 962). Since the porphyrins were first classified by Gunther, the search has been €or the delineation of a toxic porphyrin or a derivative that might explain the myriad of neurological and psychiatric and dermatological manifestations of this disorder. Yet all attempts to demonstrate toxicity in the porphyrin precursors or the porphyrin molecules themselves have been without avail. Porphyrin precursors and porphyrin molecules have been injected and ingested; even the progeny of porphyric mothers in whom the fetal circulation is constantly exposed to these substances in high concentration have failed to demonstrate a clinical sign in the newborn. Kluver (1944) reported that free porphyrins of the central nervous system normally show red fluoresence on exposure to near-ultraviolet light. The fluorescence maxima and solubility properties indicate that the porphyrin present in greatest amount is coproporphyrin, with a smaller amount of protoporphyrin. These findings were confirmed by Blanshard (1953), who succeeded in isolating coproporphyrin, especially the type I11 isomer. Kluver (1955) noted that the fluorescence was present in warm-blooded animals, but not in reptiles, amphibians, and fish, and he postulated a relationship between fluorescence and temperature regulation. He further described an ascending porphyrinization reminiscent of the ascending myelinization in the central nervous system. This porphyrinization first appears over the ventral aspects of the cord and then in the fiber masses of the brain stem, cerebellum, and cerebrum, and finally in the commissures of the cerebral hemispheres. I n the rat, fluorescence is absent at birth but appears in the spinal cord at the age of 25 days. The same period is also particularly significant in the development of myelin, vascularization, and motor activity in the rat. Porphyrins occur prominently in oligodendroglia. Kluver also noted an inverse relationship between the amount of free porphyrin and the amount of cytochrome that exists in various areas of the central nervous system and alluded to the theory advanced by Keilin (1 945) that coproporphyrin itself was a derivative of cytochrome. Kluver’s spectroscopic examination of the peripheral nerves revealed that porphyrins were present in the optic, trigeminal, facial, and auditory nerves, but he could not detect the presence of these substances in the oculomotor group of nerves or in the spinal nerves. Solomon and Figge (1958) noted that the red fluorescence of the porphyrins in the nervous system was not uniformly distributed. Quantitative determination revealed a higher concentration of coproporphyrin

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I11 in the medulla oblongata and brain stem than in the cerebellum, cerebrum, or spinal cord. Solomon and Figge thought that, since porphyrins were abundant in the conductive areas of the central nervous system and found to be most concentrated in the important medullary region, porphyrins might be involved in the process of nerve impulse conduction. Feeling that this hypothesis would not be tenable if porphyrins were absent in peripheral nerves, they decided to determine whether porphyrins were present in peripheral nerves and in what amounts. Their studies showed that both protoporphyrin and coproporphyrin I1I were relatively abundant in peripheral nerves and that they contained approximately two molecules of coproporphyrin to each molecule of protoporphyrin. Because coproporphyrin I I1 and protoporphyrin are major constituents of the conductive centers of the brain and coproporphyrin I11 appears in the urine in patients suffering from lead intoxication and acute poliomyelitis, as well as in porphyria, the possibility seems to exist that the condition of symptomatic acute porphyria itself may be due to a withdrawal of porphyrins from the central and peripheral nervous systems or a failure of the body to replenish a needed supply of these important substances. Peters (1954) and Peters et al. (1957, 1958, 1966) have postulated that an enzymatic block along the pathway of porphyrin metabolism could possibly be due to, or aggravated by, zinc, copper, or some other heavy metal and could explain the development of symptoms of acute, chronic, or mixed porphyria. Since the porphyrins are so intimately involved in myelin metabolism, this could explain the fleeting or vague symptoms noted. The focal electroencephalographic findings seen in exacerbation of some of their patients as well as transverse myelitis suggested that the disturbed metabolism could affect local tissue levels while hypothalamic areas similarly involved could explain schizophrenic and other psychiatric symptoms. The reported success of chelating agents as well as other mechanisms for withdrawal of cations from the body in the therapy of acute chronic and mixed porphyria suggests that heavy metals could be playing a vital role in the production of symptomatic porphyria. These and other theories will be explored further in this review. B. INCIDENCE The porphyrias are not the rare and exotic maladies they were once thought to be. Largely reflecting considerable clinical interest in this biochemical aberration, the incidence of porphyria has been as high as 1 :100 in South Africa according to Dean and Barnes (1958) ; 1 : 1000 to 1.5:IO0,OOO in Sweden, according to Waldenstrom (1957) and Waldenstrom and Vahlguist (1939); 1 :80,000 in Ireland according to Fenton (1955); 1 :60,000 in Leeds, according to Astley and Williams (1956); 1.5 :100,000 in Australia, according to Saint et a!, (1 954). Since the disease in inherited as a Mendelian

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dominant, with variable penetrance, the finding of one patient may often lead to other active or latent cases in the same family. In South Africa, Dean and Barnes noted that a single colonist, arriving in 1688, had given rise to most of their current variegate porphyria cases. The quantitative estimations of porphobilinogen and 8-aminolevulinic acid by the method of Granick and Vanden Schrieck (1955; Mauzerall and Granick, 1956) aided greatly in the detection of both active and latent cases. In the mixed, chronic, and variegate forms of the disorder, as well as hereditary coproporphyrinuria, quantitative estimation of fecal porphyrin has been a useful screening device. At University Hospitals, in Madison, Wisconsin, over 100 cases of porphyria have been diagnosed since 1953. In addition, experience with hexachlorobenzene-induced porphyria in Turkey has demonstrated that cutanea tarda form of porphyria can be induced in nonporphyric individuals exposed to this fungicide, the numbers in epidemic regions between 1955 and I959 having been estimated at around 3000 cases, according to Cam (1957, 1959),Dogramaci et al. (1962), Cetingil and Ozen (1960), Schmid (1960), and Cam and Nigogosyan (1963). II. Classification of Disturbed Porphyrin Metabolism

Since the original classifications of Gunther (1911, 1922),numerous other classification systems have been proposed by Watson (1960, 1963), Waldenstrom (I957), Schmid et af. (1954), and Roman (1967a). The various porphyrias have been summarized by Goldberg and Rimington (1962). They may be distinguished as hepatic or erythropoietic, depending on the primary site of porphyrin synthesis. 1. Congenital (erythropoietic) porphyria Erythropoietic protoporphyria (Magnus et al., 1961) 2. Acute intermittent porphyria (hepatic) Hereditary coproporphyria (Berger and Goldberg, I 955) 3. Cutaneous hepatic porphyria a. Hereditary forms i. Porphyria cutanea tarda hereditaria, or protocoproporphyria (Waldenstrom, 1957) ii. Mixed porphyria (Watson et al., 1951a; Watson, 1954) iii. Porphyria variegata (Dean and Barnes, 1959) b. Acquired forms i. Porphyria cutanea tarda symptomatica (Waldenstrom, 1957) ii. Bantu porphyria (Barnes, 1959) iii. Turkish porphyria (Cetingil and &en, 1960) iv. Porphyrin-producing hepatic adenoma (Tio et al., 1957) v. Experimentally produced porphyrias

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In this tabulation we have included under the acute intermittent porphyria the hereditary coproporphyria of Berger and Goldberg (1 955), also called idiopathic coproporphyria by Watson et al. (1949). The congenital erythropoietic porphyria includes also the erythropoietic protoporphyria of Magnus et al. ( 1961 ) . A final category of symptomatic porphyrinuria would include lead poisoning, coproporphyrinuria secondary to liver disease, malignancies, and malnutrition. In congenital (erythropoietic) porphyria the metabolic error resides in the bone marrow, with onset in infancy of mutilating photosensitive dermatitis, and it is transmitted as a recessive trait or in some instances as a dominant, Acute intermittent porphyria hepatica is inherited as a Mendelian dominant with variable penetrance and is characterized by recurrent gastrointestinal, psychiatric, and neurological symptomatology occurring singly or in combination. Hereditary coproporphyria has marked symptomatic similarity to acute intermittent porphyria. Cutaneous hepatic porphyria includes porphyria cutanea tarda hereditaria (protocoproporphyria of Waldenstrom) manifested by the onset of photosensitive cutaneous lesions. Mixed porphyria incorporates a combination of cutaneous and acute symptoms in which dermatologic as well as neurological, psychiatric, and abdominal symptomatology appear in varying combinations. Latent forms also exist. Porphyria variegata may present itself with cutaneous symptoms alone, with acute intermittent porphyric symptoms alone, or all these symptoms may be combined. Presence of a n X-porphyrin in the feces provides further identification in this condition, in which latent forms also exist. In the United States the differences between the Swedish and the South African type of porphyria established by Waldenstrom and also by Dean and Barnes do not seem to be clearly demarcated. We summarize in tabular form the main categories of porphyria. Eythropoietic porphyria Photosensitivity : Severe. Melanosis hypertrichosis bullae; scarring sclerodactyly Other Features : Hemolytic anemia, splenomegaly, erythrodontia Intidencc: Rare; recrssive inheritance; onset infancy, 62 cases (32 males and 30 females) Porphyrins RBC: Copro and Uro raised; Proto moderate Stool: Copro and Cro raised; Proto moderate Urine: Copro and Uro raised l Abbreviations used: PCT, porphyria cutanea tarda; AIP, acute intermittent porphyria; 8-ALA, 6-aminolevulinic acid; PBG, porphobilinogen; Uro S , uroporphyrinogen synthetase; Proto, protoporphyrin; Copro, coproporphyrin; Uro, uroporphyrin; AIA, allylisopropyl barbituric acid.

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Eythropoietk protoporphyria Photosensitivity : Severe. Burning pain usually within 30 minutes after exposure, edema, dermatitis, purpura Other Features: Cholecystitis, cholelithiasis, hepatic periportal fibrosis Incidence :Relatively common ; dominant inheritance (variable penetrance) ;onset infancy, 100 cases (1961-1967), 64 males and 36 females. Porphyrins RBC : Proto raised Stool: Proto and Copro usually raised Urine: Normal Porphyria cutanea tarda Photosensitivity : Moderate. Melanosis hypertrichosis morphea; fragility (bullosa actinica et mechanica) Other features :Polycythemic, hepatomegaly Incidence: Relatively common onset at age 40-60 years; usually males; acquired, rarely hereditary Porphyrins RBC: Normal Stool: Uro raised, Copro moderate Urine: Uro raised, Copro moderate, precursors normal Acute intermittent porphyria Photosensitivity :None Other features : Intermittent episodes neuritis, abdominal pain, psychosis Incidence: Onset puberty, females and males; 1:lo00 to 1:100,000 population Porphyrins RBC: Normal Stool: Normal or moderately raised Urine: 8-ALA and PBG raised (in attack), Uro raised (formed from PBG) Hereditay coproporphyria Photosensitivity :None Other features :Similar to A1P Incidence: Onset after puberty, 56 cases, 15 active (10 females and 5 males), 41 latent Porphyrins RBC: Normal Stool: Copro raised (high), Proto increased Urine: I-ALA and PBG raised (in attack), Copro raised Porphyria variegata Photosensitivity :similar to PCT Other features: Acute episodes as in AIP Incidence: Common in South Africa, 1:100, dominant inheritance, 8000 cases Porphyrins RBC: Normal Stool: Proto high, Copro raised, presence of X-porphyrin Urine: S-ALA and PBG during attack

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In the acquired forms of porphyria, the Bantu porphyrics (South African) usually are accompanied by a reversal of the A:G ratio, according to Mentz and Bersohn (1959), who found 65y0 positive tests for increased urinary excretion of porphyrin in the Bantu compared to 15y0 of the European population including normal and hospitalized nonporphyric individuals. Turkish porphyria was induced by the ingestion of hexachlorobenzenetreated seed wheat that was consumed by a population of Turkish peasants who, running out of winter food supplies, had turned to the seed wheat for food. Manifestations of this disturbance were chiefly cutaneous, as in PCT, but included evidence also of adrenal insufficiency and some mixed features, such as neuropathy and psychosis. Bleiberg (1964) has shown that workers exposed in the manufacture of the fungicides 2,4-dichlorophenol (2,4-D) and 2,4,5-trichlorophenol (2,4,5-T) develop a cutanea tarda type of porphyria similar to that seen in Turkey (see Section V I I I on precipitating factors). The urinary excretion of excessive amounts of porphyrin is common in porphyria; excretion is also increased, but to a lesser extent, in liver disease, including cirrhosis and hepatitis, according to Lageder (1934), Nesbitt (1943), Nesbitt and Snell (1942), Watson et al. (1951a), Dobriner (1937), and Zeile and Brugsch (1934); in cardiac failure, according to Vannotti (1954); in toxemia of pregnancy, according to Fikentscher (1935); in vitamin deficiencies, such as pellagra, according to Vannotti (1 954), Ellinger and Dojmi (1 935) ; in the recovery of the bone marrow after hemorrhage, according to Delangen and Grotepass (1941) ;in pernicious anemia, according to Watson (1935) ; in pyrexial conditions, according to Dobriner (1937) and Watson (1946) ; and after Sulfonal, Veronal, or lead poisoning, according to Zeile’s work in 1934 and Giinther (1922). Tio et al. (1 957) and Thompson et al. (1970) reported cases of a porphyrinproducing hepatic adenorna with porphyrinurea symptomatica that were relieved by resection of the adenoma. Age at onset of symptoms is usually after puberty, according to Waldenstrom (1957). Goldberg (1959) noted that 5 out of 50 patients were over 40 and that in twenty-four symptoms occurred between the ages of 20 and 29. I n most series, incidence is higher in females than in males, varying from 3: 2 to 2 :1. The severity of the disease in both sexes appeared similar. A case of acute intermittent porphyria in a 5-year-old boy has been described by Kreimer-Birnbaum et af. (1971). 111. Clinical Symptoms and Signs in Hepatic Porphyrics

Summarizing the clinical symptoms seen in cases of acute intermittent porphyria (AIP), Peters et al. (1958) described bulbar symptoms in the form of dysphagia, diplopia, anisocoria, dysarthria, facial paresis, and laryngeal

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palsy, to which may be added two recent cases with internuclear opthalmoplegia. Tracheostomy was often a life-savingprocedure. Grand ma1seizures are not infrequent, and in addition Jacksonian seizure activity, accompanied by focal electroencephalographic localization and homonomous hemianopsia. Status epilepticus and syncopal attacks were noted. Cerebellar ataxia and extrapyramidal symptoms have also been recorded. Peripheral neuropathy to the extent of tetraparesis is not unusual, particularly in those subjected to Pentothal anesthesia. Dysesthesias and pain in the distribution of the trigeminal nerve have been seen. Brachial plexitis and mononeuritis is recorded. The peripheral neuropathy can be as often distal in onset as proximal and, when severe and progressive, is accompanied by recurring myoclonic-typejerks of the muscles plus involuntary asynchronous muscular contractions. There may be delayed response to command for motor activity, and a positive “Hoover sign” has been seen in those developing peripheral neuropathy, leading to the erroneous impression that the patient is suffering from hysteria. Muscle and joint pains may be severe both during the onset of paralysis and again in the recovery phase. Sensory changes may be absent or involve all modalities. In one patient, the clinical picture was that of a Guillain-Barre syndrome with increased spinal fluid protein. Suspicions were raised when the Guillain-Bard syndrome recurred after one year. The decreased level of consciousness in another postpartum patient reported by Peters (1956) had been sufficient to obscure the presence of a fracture dislocation of the right shoulder, until the lowered level of consciousness was improved 8 hours after initiation of dimercaptopropanol treatment. Psychiatric symptoms occurred in 58% of the patients in the form of a toxic psychosis, characterized by bizarre catatonic excitement, misidentification, dejh vu, and auditory and visual hallucinosis. Consciousness varied from torpor to coma. In remission, it was not unusual to note psychoneurotic symptoms in the form of phobic neurosis often associated with hyperventilation. These symptoms were best handled with psychotherapy along with breathing and relaxing exercises and needed to be differentiated from porphyric attacks in which hyperventilation could also be a prominent finding. Some of the patients showed a predominantly schizophrenic picture with flattened affect, paranoid ideation, sufficient to diagnose them as schizophrenic on both Rorschach and clinical evaluation. Since some patients manifested only schizophrenic symptoms, it was the belief that a porphyricschizophrenic syndrome existed, the symptoms of which could be reversed by the use of chelating agents alone. Addiction to meperidine HCl and opiates is a frequent finding, particularly in those having had pol ysurgery, and abuse of sedatives, including barbiturates, chloral hydrate, paraldehyde, chlordiazepoxide,meprobamate, and glutethimide has also been seen.

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IV. Gastrointestinal Signs

Abdominal symptoms may be associated with severe obstipation, and fecal impaction should always be looked for as a n early complication. One patient had no defecation for 4 weeks and the rock-hard impacted fecal material was finally lysed with rectal infusions of “Adolph’s Tenderizer” (papaya). The abdominal pain is often not accompanied by muscle spasm and may be migratory, and severe, the patient will often adopt a knee-chest position. When polysurgery has resulted in adhesions, however, distention and obstruction occurs. At other times, the pain may be referred to the back, which when accompanied by drop-foot, can give rise to the false impression of a herniated intervertebral disc syndrome. Although obstipation was noted at various times in over half of the patients, severe diarrhea was seen in three cases of AIP, one of whom, a male, had frequent bloody stools. I n a second attack of bloody diarrhea 5 years later, this patient revealed with proctosigmoidoscopy a picture compatible with ulcerative colitis. After intravenous ethylenediaminetetraacetic acid (EDTA) therapy, the bowel’s appearance and symptoms reverted to normal. X-RAYFINDINGS Calvy and Dundon (1 952) reported X-ray findings in 17 cases of AEP. Their study included a review of the literature and indicated that at least 50y0 of the patients had segmental gaseous dilatation of the intestine; the findings varied from day to day with no characteristic pattern. Calvy and Dundon also noted fluid levels and found that the cecum might be dilated. Spasm and gaseous dilatation of the gastrointestinal tract could explain the abdominal pain. V. Genitourinary Signs

Oliguria, dysuria, incontinence, and urinary retention sufficient to require catheterization has been frequently reported. The Burgundy wine or brownish urine described as typical in books were seldom mentioned. Even in severe cases, the urine may be normal in color and may not change color after acidification and exposure to sunlight despite the presence of large amounts of abnormal porphyrins in the urine. The urinary odor has been described as offensive during attacks, as noted by the nurses and the patients. VI. Cardiorespiratory Signs

The cardio vascular system during exacerbations revealed a tachycardia well over 100 during attacks. Hyperpnea often reflected deteriorating respiratory function or florid hallucinosis. Messert and Baker (1967) have emphasized the marked central neurogenic hyperventilation seen in an attack of severe porphyric polyneuropathy.

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31 1

Increased blood pressure was noted during a number of attacks. On the other hand, orthostatic hypotension with acute exacerbations of porphyria has been recorded by Schirger et al. (1962). Blindness of cerebral origin has been reported by Goldstein et a1 (1957). Severe cardiomegaly was seen in a 23-year-old patient by Bray (1962) and by Olson (1971). The electrocardiogram has been evaluated by Krouch and Hermann ( 1955), who described in eight patients normal electrocardiograms except for the presence of sinus tachycardia. One of their patients who expired did not show any pathology, and the death was due to respiratory paralysis. VII. Dermatological Signs

Dermatological symptoms in acute porphyria may include a malar flush over the butterfly area. Other patients have shown a dusky, diffuse complexion described as metallic, while several cases have diffuse melanosis. In porphyria cutanea tarda (PCT) and variegate and mixed porphyrics, cutaneous bullae as well as milia and a positive Nikolsky sign have been observed. One patient during attacks of AIP showed marked periorbital and parotid swelling, associated with pain, psychotic behavior, abdominal colic, and urine revealed a zinc diuresis. It later developed that this patient had been self-administering oral mercurial diuretics over a 4-year period. In AIP many patients described aggravated symptoms, and even induction of symptoms, after exposure to paints and solvents. Diet drinks containing saccharine and sucaryl, both sulfa derivatives, likewise seemed to precipitate symptoms in others. Prolonged fasting for diet purposes also seemed to figure in exacerbation of symptoms. VIII. Precipitating Factors

Gunther (191 1) referred to toxic porphyria because of the frequency with which AIP attacks were produced by the ingestion of Sulfonal and Trional. Goldberg (1954) showed that various barbiturate hypnotics, notably those containing ally1 groups, induced porphyric changes in the rabbit. He stated that barbiturates could inhibit some of the oxidative processes of brain tissue, A. PREGNANCY Waldenstrom (1937) reported that two of his patients had died from AIP during pregnancy and suggested that the hypnotic and other drugs given during that time may have been responsible. He also thought that pregnancy itself could precipitate an attack. Whittaker and Whitehead (1956) demonstrated a Mendelian dominance in one family and precipitation of acute attacks under the influence of barbiturates in pregnancy. Neilson and Neilson (1957) reported 3 cases of acute porphyria complicated by pregnancy,

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2 of which were fatal. One of the fatal cases was treated with guanine and adenine without effect. None of the patients was treated by chelation. The third patient recovered, however, after barbiturates were stopped and chlorpromazine was used for restlessness. They reviewed the occurrence of pregnancy in porphyric individuals and concluded that it was rare. They found descriptions of 37 cases in the world literature, to which they added 3. Of the 40 patients, only 1 was of the erythropoietic type; 4 were cutanea tarda (PCT) or mixed type, and 35 suffered from AIP. A total of 76 pregnancies occurred; 60.5% of the pregnancies progressed to the stage of viability, 607, occurred in primigravidas, and 40% in multigravidas. Ninetyfive percent had exacerbation of the disease during pregnancy. Of the seventeen maternal deaths, fourteen were found among the primigravidas, to account for an 8"J, death rate among the primigravidas and 42.50/, for the total series. Twenty-five abortions occurred, I0 being therapeutic. A few died undelivered, before the period of viability. The experience of Peters' group would seem to contradict the above statistics since many successful pregnancies were recorded in their patients, often prior to the development of attacks of AIP. One patient, for example, experienced a fracture dislocation of the shoulder postpartum during an AIP attack, during which the lowered level of consciousness completely masked the pain of the orthopedic injury. She responded within hours to the administration of dimercaptopropanol, then first complained of pain. She later went on to deliver successfully two other infants without difficulty. We would suggest that drugs used during pregnancy of the sulfa and barbiturate series may have contributed to the statistics noted by Neilson. B. HORMONES Meyer et d. (1954) reported the occurrence of an acute attack of porphyria following the injection of 25 mg of progesterone. Levit et al. (1957) described a woman with AIP in whom studies demonstrated that progesterone, either endogenous or exogenous, would induce attacks. Perlroth et al. (1965) noted that female patients with AIP maintained on constant diet and administered estrogens revealed an increase in urinary 8-aminolevulinic acid and porphobilinogen. Vail (1967) noted that PCT could be induced by estrogens in 2 patients with improvement following estrogen withdrawal. Becker (1 965) described 3 patients who developed cutaneous lesions of PCT after nearly a year or more of diethylstilbestrol administration for carcinoma of the prostate. Two of these patients benefited by protection of their skin from heat and friction, and the third was free of lesions after discontinuance of the hormone, though the excessive excretion of uroporphyrin and coproporphyrin in the urine and feces continued for a year in one patient after discontinuance of the synthetic

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estrogen. Perlroth et al. (1965) described 3 patients with AIP whose clinical symptoms occurred during their menstrual cycle; they were given oral contraceptives with relief of signs and symptoms. They proposed that inhibition of gonadotropin secretion with stabilization of endogenous ovarian steroid production at low levels was a possible mechanism for therapeutic effect. Thus, the effects of hormones may be opposite in PCT and AIP. AND DRUGS C. SEDATIVES

1. Barbiturates Levine et al. (1972) presented evidence that an active metabolite of certain barbiturates containing ally1 groups will cause breakdown of cytochrome P-,,, heme. With and Peterson (1954) demonstrated that uroporphyrin could be found in conditions other than genetically determined porphyria when the patient was suffering from serious or potentially fatal disease and further was stressed with drugs, such as the barbiturates. Patients reported by Peters’ group and others have also underlined the importance of exposure to Pentothal and other quick-acting barbiturates in the precipitation of severe, acute, and mixed porphyric symptoms. However, they also studied a patient with AIP who tolerated excessive amounts of Seconal €or years without an acute crisis, but merely a subacute course. After exposure to Seconal was discontinued and then restarted months later, acute symptoms in the form of colic, weakness, and psychiatric symptomatology immediately became evident.

2. Other Sedatives So-called nonbarbiturate hypnotics especially glutethimide (Doriden) and ethchlorvynol (Placidyl) have been associated with exacerbations in AIP. The effects of diazepam (Valium) and chlordiazepoxide (Librium) are unpredictable. Porphyria patients with an abuse potential may misuse any and all drugs with sedative properties. Even paraldehyde and alcohol are high on the list. Bromide intoxication has been seen in a patient with AIP, though the porphyric disturbance was not exacerbated and the bromide psychosis vanished after the blood bromide levels returned to normal, according to Peters. 3. Tranquilizers and Sedatives Cripps and Peters (1970) described a case of hereditary coproporphyria in which acute neurological symptoms were precipitated by various unrelated tranquilizers including meprobamate and chlorpromazine.

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4. Anticonvulsants The anticonvulsant drugs, including primidone, diphenylhydantoin, and phenobarbital, have also been suspected of precipitating AIP.

5. Suya Drugs and Derivatives Peters has noted precipitation of acute symptoms of A.I.P. on exposure to sulfisoxazole, chlorothiazide diuretics, and other sulfa derivatives. Included in this latter category are the artificial sweetening agents saccharine and sucaryl, which seemed to precipitate symptoms in AIP. Griseofulvin may precipitate symptoms of AIP, according to Redeker et al. (1964). On the other hand, Ziprkowski et al. (1966) noted no effect in PCT, and Dogramaci likewise noted no effect of griseofulvin on patients with hexachlorobenzene-induced Turkish PCT. Chloroquine (see Section XII) may induce acute porphyria in some patients but may alleviate symptoms when given in low dosage to patients with PCT.

FACTORS D. OTHER 1. Liver Disease

Brunsting et al. (1951) pointed out that in PCT damaging influences on the liver either by diseases, such as cirrhosis, syphilis, diabetes, chronic amebiasis, or by hepatotoxic drugs, poisons such as arsenic, and particularly alcohol, seem to offer the precipitating insult to chronic or mixed porphyrias. Three of their patients had diabetes, two of whom had diabetic neuropathy which preceded the development of porphyria by 10 years.

2. Ethanol Welland (1964) noted that 7 patients with AIP maintained on normal diet and given ethanol showed a significant increase in urinary &aminolevulinic acid synthetase in the liver. Gitlin (1969) described a case of “Zieve syndrome” in which there was hyperlipemia, jaundice, and hemolytic anemia associated with ingestion of large amounts of alcohol and improved once alcohol was withdrawn. Associated symptoms included anorexia, nausea, diarrhea, vomiting, and weight loss. The presenting feature was pain in the upper abdomen of varying intensity and location. Since Gitlin observed in this case abnormal porphyrin metabolism, including coproporphyrin, uroporphyrin, and slight elevation of porphobilinogen, he suggested that other similar cases could be related to disturbed porphyrin metabolism secondary to the chronic ingestion of alcohol.

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The finding of abnormal porphyrin excretion in some alcoholics and the absence of family history in acute intermittent porphyrics suggests that either new mutations are taking place or undefined toxic factors may give rise to this genetic disturbance. Indeed, Waldenstrom (1957) stated that cases of porphyria could result from chronic alcoholism. 3. Chemicals Hexachlorobenzene, a wheat fungicide whose ingestion proved to be responsible for an epidemic of acquired PCT in Turkey, has been matched by 2,4-dichlorophenol (2,4-D) 2,4,5-trichloropheno1 (2,3,5-T) which seemed to produce a Turkish-type porphyria in workers in a chemical factory (Bleiburg, 1964). Siyali (1972) examined the concentration of pesticides in the blood of 237 people, 185 of whom had some occupational exposure. Whereas the commonly used pesticides were not found in many of the group surveyed, hexachlorobenzene was found in 95y0 of the subjects in concentrations averaging 22 ppb. 4. Diet Welland (1964) noted that porphyric patients who used dieting in the form of fasting could precipitate acute attacks. A caloric reduction between 60 and 80y0 produced symptoms associated with an increase of both &aminolevulinic acid and porphobilinogen. An increase of porphyrin precursors followed an isocaloric substitute of fat for protein alone or protein together with carbohydrate. Welland’s group held the effect of diet to be related to protein and carbohydrate content rather than to the total caloric content. Knudsen et al. (1967) also reported a case of AIP precipitated by fasting.

5. Paints and Solvents Peters’ group emphasized the important role of exposure to paints and solvents, which seemed to play a key role in tbe precipitation of severe AIP in 2 patients and was associated with exacerbation in others. 6. Miscellaneous Factors Goldberg et al. (1969) studied the urinary excretion of 17-oxosteroids in 6 patients with AIP. They noted significant elevation of etiocholanolone glucuronide in 2 patients, of dehydroepiandrosterone glucuronide and sulfate in 2 patients, and of epiandrosterone sulfate in 3 patients. Of the 4 patients who showed abnormalities, 2 were in relapse, 2 had had severe attacks 8 months and 2.5 years earlier, and those with normal patterns had been in remission for 15 years and 4.5 years. Goldberg et a1:injected dehydroepiandrosterone intraperitoneally into rats every 24 hours and caused

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significant elevation of 8-aminolevulinic acid synthetase. They felt that this represented an endogenous factor that partially explained the age of onset after puberty and the occurrence of attacks in which an exogenous factor could not be identified. IX. Pathology

There are no constant histological lesions in AIP, and findings outside of the central nervous system are minimal. In most patients the neuropathy is attended by demyelinization of peripheral nerves sometimes accompanied by axonal degeneration, according to Gibson and Goldberg (1956), H'ierons (1957), and Schwarz and Moulton (1954). However, Prunty (1946) noted changes in anterior horn cells which he thought were due to retrograde degeneration from the peripheral nerves although the neuronal damage seemed disproportionately great and primary damage to the anterior horn cells had not been excluded. There was a minimum of lesions in the cerebrum even in patients with mental symptoms; in patients dying of paralysis, the nervous system could appear normal. Although vascular lesions are not usual, a vascular basis has been suggested by Denny-Brown and Sciarra (1945). In this regard, blindness of cerebral origin was reported by Goldstein et al. (1957). One could postulate vascular spasm as being etiologic. Gibson et 01. (1957) noted in their postmortem material that the liver in AIP showed alterations that were slight in comparison to those in PCT, but abnormal albumin :globulin ratio and flocculation tests were not uncommon Gibson et al. also noted on two occasions a fine nodularity of the liver. A. RENALSTUDIES Schley et al. (1970) studied renal functions in 7 patients with AIP, and used the phenolsulfonphthalein test and Cln,C,,,, Ccr.Four of their patients with oliguria and azotemia were examined repeatedly over a period of 1 4 years during or shortly after the acute phases of AIP and during intervals when symptoms were absent. In 2 of these cases renal function was disturbed but improved after the acute phase; in 1 patient renal function returned to normal when the patient was in remission but became impaired again in an additional attack. Three cases without oliguria and azotemia during the acute phases showed normal values for Cln, C,,,, C,,, and phenol-red test during asymptomatic intervals. Those with impaired function had a longer history of the illness and a higher frequency of exacerbation. Serum concentrations of sodium, potassium, and chlorine were normal in all cases or only slightly altered. Schley et al. postulated that during the acute phases of AIP renal ofiguria and azotemia w p e caused by an unknown toxic agent. Peters et al. (1957) reported a patient with severe AIP who experienced

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severe oliguria before and during treatment with EDTA and whose marked edema was interrupted on the addition of hydrocortisone. In a study of 9 fatal cases of AIP by Ten Eyck et al. (1961), the autopsy in most cases revealed congestion, centrolobular necrosis, and fatty degeneration granules of iron-free lipochrome pigment in the hepatic cells of the patients. Pulmonary infarction was seen in 3 of these cases. Also noted was atrophy of the testes in all patients who had symptomatic acute porphyria at the time of death.

B. NEUROPATHOLOCY Neuropathologic changes in the 9 fatal cases of AIP reported by Ten Eyck et al. (1961) showed patchy demyelinizarion and degeneration of the axis cylinders in the peripheral nerves, dorsal roots, cauda equina, and autonomic nervous system. Degenerated nerve cells were found in the anterior horn cells of the spinal cord, dorsal root ganglia, cerebellum, dorsal nucleus of the vagus nerve; and the celiac plexus. Neurogenic atrophy and hyaline degeneration were seen in the nucleus of half of the patients. Only minor nonspecific alterations were present in the cerebral cortex of half of the patients who were free from associated diseases. Ten Eyck et al. noted a family history of porphyria in only 1 of the 9 patients; 7 had associated potentially fatal disorders. Gibson and Goldberg (1956) studied the neuropathology of AIP and noted that myelin disintegration could be observed prominently 2 weeks after onset of paralysis with all cases showing small perivascular areas of demyelinization. Even patients who had fatal attacks after illness of short duration revealed peripheral nerves affected only to a minor degree. Gibson and Goldberg also noted retrograde degeneration of nerve cells, especially in anterior horn cells and the medulla oblongata. Neither the study of porphyrin fluorescence of sections nor the literature supported the supposition that the nerve lesions in nerve biopsy specimens in a patient 12 months after the development of quadriparesis, were due to a toxic action of porphobilinogenor uroporphyrin. Drury (1956) found a reduction in the number of myelin sheaths and chronic interstitial changes, but little residual cellular reaction; the nerve fibers seemed to be undamaged, and the clinical course of his patient showed that 30 months after the biopsy the patient had completely recovered mentally as well as physically.

C. VASCULITIS Becker (1961) called attention to the similarity or sister relationship between the symptoms and signs of periarteritis nodosa and acute porphyria. Muller et al. (1956) described a case of AIP associated with panarteritis. Peters also described a fatal case of AIP associated with polyarteritis nodosa.

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A vasculitis in the form of Wegener’s granulomatosis was reported by Hansotia et al. (1 969) in which increased urine copper levels as well as borderline levels of uroporphyrin excretion were noted. The patient responded for several years to intermittent intravenous infusions of EDTA. ADENOMA D. HEPATIC Tio et al. (1957) have described the unique occurrence of a porphyrinproducing benign hepatic adenoma in a n 80-year-old Dutch woman, without a family history. She first complained of blisters on the dorsum of her hands and itching of her face as a result of indirect sunlight. The skin of her hands and face were thickened, pigmented, and excoriated. O n abdominal examination, she was found to have a round, smooth tumor about the size of a fetal head at the right subcostal region. T h e urine was rich in porphyrin, predominantly uroporphyrin, and her serum fluoresced strongly in ultraviolet light. The stool coproporphyrin and protoporphyrin were markedly increased. Six months after onset of symptoms, removal of a tumor from the right lobe of the liver resulted in rapid cessation of symptoms and a reduction in porphyrin excretion. It was thought that the porphyrins were synthesized within the tumor cells of this benign-appearing adenoma. Thompson et al. (1970) reported a comparable patient. X. Biochemistry

The metabolism of porphyrins has been well discussed by Goldberg and Rimington (1962) and will only be summarized here. Since the ability to synthesize the tetrapyrrole nucleus appears to be almost a fundamental property of living matter, it is not surprising that free porphyrins are found in minute quantities in nearly all tissues of the higher organisms. Contrary to earlier speculation, we now know that porphyrins represent stages in the synthetic pathways toward heme and are not, as previously supposed, products of heme or hemoglobin destruction. Hans Fischer had previously speculated that coproporphyrin was derived &om protoporphyrin b y the process of carboxylation, and uroporphyrin from coproporphyrin in the same way. This was thought to be a detoxification process for urinary elimination. Although Fischer first proposed that 4 different uroporphyrins and 4 different coproporphyrins could exist, and no fewer than 15 different protoporphyrins and mesoporphyrins, his subsequent degradation and synthetic studies revealed that only 2 of the 4 possible coproporphyrins occurred in nature, namely isomers I and 111, and the natural porphyrin was always protoporphyrin I X arising from coproporphyrin 111. It was uroporphyrin I which he first isolated from pathological urine, and later uroporphyrin 111. He also discovered that protoporphyrin or heme of hemoglobin, myoglobin, cytochrome b, catalase, peroxidase, and all naturally occurring hemoproteins is

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always isomer IX derived from the I11 series. Although coproporphyrin I occurs in normal urine and feces together with smaller quantities of coproporphyrin 111, no physiological importance could be found for the I series of porphyrins, which were thought to be unnatural and useless. According to Booij and Rimington (1957), in congenital (erythropoietic) porphyria there is an enzymatic defect manifesting itself as a failure to direct the change of porphobilinogen into uroporphyrin 111. Instead, in congenital porphyria, uroporphyrinogen I is produced and excretion of uroporphyrin 1 and coproporphyrin I is greatly increased as a consequence. Protoporphyrin is not formed since the enzyme directing the step from coproporphyrinogen to protoporphyrin is specific for the I11 series isomer. It is uroporphyrin I and coproporphyrin I that are deposited in bones and teeth in this condition, suggesting, according to Sveinsson et al. (1949), ready absorption of these parts of heme synthesis by calcium phosphate. In congenital porphyria, the bone marrow likewise shows extensive fluorescence. According to Turner (1937), the American fox squirrel in particular demonstrates a physiological porphyria and usually shows a high content of porphyrin in the urine and feces. There are other examples of congenital porphyria in domestic animals, including cattle, swine, and cats. Glenn et al. (1968) called attention to a congenital porphyria appearing in the domestic cat, inherited along dominant lines as compared to congenital porphyria in man, which is recessive in inheritance. A. PORPHYRIN PATHWAYS The pathway of porphyrin and heme synthesis may be summarized as follows, according to Rimington (1959). Glycine and succinyl-CoA, the latter derived from the Krebs cycle involving substrates like acetate and cofactors such as magnesium and lipothiamide, combine through a system requiring pyridoxal or pyridoxal phosphate and possibly ferrous iron, according to Brown (1958). Unstable ketonic acid and a-amino-p-ketoadipic acid is the first product. With the loss of carbon dioxide, S-aminolevulinic acid is formed as a result of the action of an enzyme, a-aminolevulinic acid dehydrase. There results the condensation of two molecules of S-aminolevulinic acid to form porphobilinogen. '

B. ENZYMES The enzyme responsible for the formation of S-aminolevulinic acid is S-aminolevulinic acid synthetase, which is a mitochondria1 enzyme that initiates and also limits the synthesis of protoporphyrin and heme, according to Shemin and Rittenburg (1946) and Granick and Urata (1963). This enzyme is present in nucleated red cells and in reticulocytes as well as in liver cells. Overactivity of the enzyme in acute porphyria was

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described by Tschudy et al. (1965) as representing “the first overproduction disease localized to a specific enzyme.” The enzyme was further shown to be induced in erythropoietic porphyria by Miyhei and Watson (19721, who also showed that the administration of multiple transfusions in erythropoeitic porphyria, in which there was marked elythropoiesis believed to be related to the presence of many fluorescing normoblasts in the peripheral blood, caused a disappearance of the fluorescing normoblasts along with striking reduction of the porphyria. The plasma 6-aminolevulinic acid synthetase activity declined to 1.4yoof the pretransfusion value. Heightened activity of 6-aminolevulinicacid synthetase has also been brought about, for example, by the administration of various drugs, estrogens, increased lead stores, barbiturates. Tschudy demonstrated a great elevation of 6-aminolevulinic acid synthetase activity in a homogenate of liver removed at autopsy shortly after the death of a patient with AIP. Nakao et al. (1966) demonstrated increased activity of the enzyme in mitochondria in 2 gm of liver taken surgically from a patient with AIP. Dowle et at. (1967) extended the study of 6-aminolevulinic acid synthetase activity to porphyria variegata and considered that in variegate porphyria and AIP the activity of the enzyme could be correlated with the clinical severity of the case. Kappus et al. (1969) demonstrated in the plasma of several patients with AIP a substance that strongly induced the synthesis of porphyrin in chick embyro liver cells growing in primary culture. They did not find this substance in normal individuals or in porphyric patients in remission whereas the ingestion of drugs or contraceptive steroid mixtures in normal subjects did significantly induce activity of 6-aminolevulinicacid synthetase. Four molecules of the pyrrole porphobilinogen are normally combined to give an unsymmetrical I11 seriespigment. Bogorad (1958) was able to separate two factors concerned in enzymatic synthesis of uroporphyrinogen 111. The first is a relatively stable enzyme which he called porphobilinogen deaminase, and the second is uroporphyrinogen isomerase. The porphobilinogen deaminase works alone and converts porphobilinogen into uroporphyrinogen I and ammonia, but if the two are present together, they modify the action so that uroporphyrinogen I11 results. Uroporphyrinogen synthetase is now thought to be the enzyme that catalyzes the conversion of porphobilinogen to porphyrin. It was at this stage of porphyrin synthesis that Peters (1954, 1956, 1961) and Peters et al. (1957, 1958) postulated an enzymatic block, possibly due to zinc or some other heavy metal, that could give rise to the symptoms in acute intermittent porphyria by depriving the nervous system of essential porphyrin building blocks. The overactivity of 6-aminolevulinic acid synthetase did not seem to be consistent with his hypothesis. The recent observation by Meyer and Marver (1971) that uroporphyrinogen synthetase is diminished by 50y0 in

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cases of AIP, however, fits in very well with the concept of an enzymatic block that could deprive the nervous system of essential coproporphyrin 111 and protoporphyrin only if uroporphyrinogen synthetase is rate limiting. Meyer et al. further analyzed the disposition of orally administered &aminolevulinic acid given to a large kindred with AIP. Like their affected relatives, unaffected siblings excreted increased amounts of porphobilinogen, but, unlike the former, their conversion of porphobilinogen to porphyrin was not decreased. A lack, then, of uroporphyrinogen synthetase could be the fundamental defect in acute porphyria. Whereas Kaufman and Marver (1970) noted in a case of hereditary coproporphyria that there was increased enzymatic synthesis of &aminolevulinic acid in the liver leading to increased excretion of porphyrins and their precursors with a similar enzymatic abnormality in AIP, this was not found in a patient with PCT in which, despite massive porphyrinuria, hepatic aminolevulinic acid synthetase was not detectably elevated. Meyer et al. (1972) were able to demonstrate that uroporphyrinogen synthetase activity was decreased also in AIP by approximately 50% of normal in red cell hemolysates. Nonporphyric subjects did not show this abnormality. Meyer et al. studied a family with AIP in which 5 members were found to have increased uroporphyrinogen synthetase activity as compared to unaffected relatives or normal controls. However, two unaffected siblings who showed normal urinary excretion of porphyrin precursors also had low uroporphyrinogen synthetase activity. When given a single dose of d-aminolevulinic acid to mimic the endogenous overproduction of aminolevulinic acid that occurs in affected family members, the conversion of exogenous aminolevulinic acid to porphyrins excreted in urine and stool was decreased in these two siblings in a manner similar to the pattern seen in patients with AIP. One of these two siblings developed a typical case of AIP 6 months after the studies. Two siblings with normal uroporphyrinogen synthetase activity in red cell hemolysates revealed normal conversion of aminolevulinic acid to porphyrins after a loading dose of 6-aminolevulinic acid. They raised the question whether this might not be a helpful test for the detection of the carrier state. It might also be inferred that this might prove to be a good method for estimation of clinical activity of the porphyric process in patients undergoing various therapies. The main biosynthetic pathway of the porphyrins is completed by the decarboxylation of uroporphyrinogen I11 to coproporphyrinogen 111. Between coproporphyrinogen I11 and protoporphyrin IX, there occurs not only a decarboxylative but also a desaturation step, two vinyl groups ultimately taking the place of two propionic acid residues. The introduction of iron to form heme would appear to take place enzymatically with protoporphyrin as substrate, not protoporphyrinogen. The participation of ascorbic acid,

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glutathione, and cysteine as cofactors has been demonstrated by Goldberg et al. (1956), Golbderg (1959), Schwartz et al. (1959), Lockhead and Goldberg (1961), and Grinstein et al. (1959). Further discussion of heme synthesis seems unwarranted in this review. Suffice it to say that anemia is rarely seen in the hepatic porphyrias. Blood contains small quantities of porphyrin mostly in normal serum, and this has been confirmed by others. Since coproporphyrin is eliminated in the urine it is not surprising that it is found in transit in the blood plasma. In congenital erythropoetic porphyria, on the other hand, large increases are observed in the coproporphyrin content of both erythrocytes and plasma as well as red cell protoporphyrin, and the plasma also contains uroporphyrin I in considerable quantity. Iber et al. (1956) studied porphyrin excretion and hemoglobin synthesis in a case of AIP and noted that the rate of hemoglobin synthesis was changed first by venesection and later by transfusion, but this did not modify the urinary excretion of porphobilinogen, uroporphyrin, or coproporphyrin. They took this to mean that the protoporphyrin of hemoglobin and the abnormal porphyrins in acute AIP arise independently. They proposed that all the porphyrins arise from porphobilinogen. Another alternative explanation is that protoporphyrin and the abnormal porphyrins are formed at different sites in the body from nonlimiting precursors. Porphobilinogen is formed at two sites, but once it escapes extracellularly it cannot reenter cells but must be excreted. Each site thus functions independently. It was noted by Goldberg ( 1955) that porphobilinogen does not cross membranes readily, and Watson (1952) concurred. On the other hand, in a case of porphyria erythropoetica where splenectomy was used to correct the anemia, the porphyrin excretion decreased. In this situation it was felt that the production of red cells and the appearance of abnormal porphyrins were related (Grinstein et al., 1951; Schmid el al., 1955). XI. Pathogenesis of Attacks

For years investigators attempted to demonstrate that a toxic porphyrin substance must exist to give rise to the multitude of clinical symptoms seen in acute hepatic porphyria. All the porphyrins and their precursors have been injected and ingested without demonstration of toxic effects. The only exception is hematoporphyrin, which is not a naturally occurring substance, but which produces photosensitivity on a tissue level. Meyer-Betz (1913) in a unique experiment injected 200 mg of hematoporphyrin into his veins and demonstrated marked skin photosensitivity to sunlight 4 days later. Diamond et al. (1 972) demonstrated that glioma cells in vitro and in vivo would take up hematoporphyrin and that exposure to light then produced massive destruction of the glioma. Schwartz et al. (1955) were able to sensitize non-

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radiosensitive tumors to the effects of X-ray therapy by administering hematoporphyrin to their patients. A. TRACE METALS, ZINC,AND COPPER Peters (1954, 1956, 1961) and Peters et al. (1957, 1958) suggested that zinc or some other trace metal present in excess because of a deficiency or depletion of the body’s natural chelating defenses may be responsible for a n enzymatic block in porphyrin synthesis causing subsequent withdrawal or failure to form enough coproporphyrin 111 and protoporphyrin in the peripheral and central nervous system as an underlying mechanism in AIP. These authors called attention to an increase in zinc, and at times of copper, excretion in the urine during acute attacks of porphyria when the patient was developing severe neurological and psychiatric symptoms. Zinc excretion varied from normal to levels as high as 36 times normal in one patient. These levels gradually fell to normal during and following the diuresis of zinc induced by chelating agents, and Peters et al. thought that the urinary excretion of zinc more closely paralleled the clinical symptomatology than did the excretion of abnormal porphyrin metabolites. The above theory in the light of the block of uroporphyrinogen synthetase would explain the marked accumulation of aminolevuljnic acid and porphobilinogen in the liver of patients dying from AIP. According to Nesbitt (1944), however, hepatic zinc measured in a fatal case was found to be normal. Gibson et al. (1958) pointed out that the 8-aminolevulinic acid dehydrase enzyme is enhanced in its activity by dimercaptopropanol (BAL), presumably by protecting sulfhydryl groups, but the enzyme is inhibited by ethylenediaminetetraacetic acid. Penicillamine is also a powerful inhibitor of 8-aminolevulinic acid synthetase. Roman (1967b, 1969) also recorded an increased urine zinc level during exacerbations, and the blood of porphyrics contained elevated zinc. On the other hand, Roman suggested that, since the porphyrins chelate with zinc, a deficiency of this metal is brought about. This might withdraw zinc as an available cofactor in enzymatic reactions on some tissues. He hypothesized that if, for example, lactic dehydrogenase of the smooth muscles requiring zinc is deprived of zinc, there will be a decrease of the enzymatic activity with accumulation of lactic acid in the muscle, which could account for pain experienced in the porphyric. The role of trace metals is discussed in Section

XIII. B. TRYPTOPHAN METABOLISM Price et al. (1959) noted an abnormality of tryptophan metabolism in 13 of 18 patients with AIP, characterized by increased urinary excretion of

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kynurenine, acetylkynurenine, kynurenic acid, hydroxykynurenine, and occasionally xanthurenic acid or other metabolites. A similar metabolic response was found in 6 nonporphyric patients with a variety of psychotic disturbances. I n contrast, a number of patients with other neurological conditions metabolized tryptophan normally. Although the abnormality suggested a pyridoxine deficiency, neither chemical nor clinical improvement resulted from pyridoxine supplementation. Both clinical and biochemical improvement were often observed after treatment with chelating agents. They suggest that increased production of these metabolites provide the patient with additional quantities of natural chelating agents, which could then aid in the restoration of a normal balance of polyvalent cations. Several of the urinary metabolites of tryptophan are indeed excellent chelating agents. This especially is true of 2-carboxy : and 8-hydroxyquinoline derivatives, which include quinaldic acid, xanthurenic acid, xanthurenic acid 8-methyl ether, and 8-hydroxyquinaldic acid, all of which have been found in mammalian urine. The administration of chelating agents, such as ethylenediamine tetraacetate or dimercaptopropanol, may merely supplement the action of natural chelating agents when the natural system has been overwhelmed in illnesses such as porphyria and scleroderma. Huszak and Durko (1962, 1968) noted that in the urine of schizophrenics, besides indoles, 6-carboxylic and other porphyrins could be demonstrated in increasing amounts with and without tryptophan loading, as noted by Price et al. (1959). Simons (1970) was able to demonstrate that the pathway of tryptophan metabolism in the direction of serotonin production was also affected by drugs that induce aminolevulinic acid synthetase activity. They injected three chemically unrelated compounds, 1,3-dicarbethoxy- 1,4-dehydrocollidine, pregnanedioldione, and pentobarbital separately into the brains of 13-day-old chick embryos and demonstrated a n increase in serotonin activity lasting 12 hr as well as a n elevation in liver ALA synthetase activity.

C. ELECTROLYTES Prunty (1946) first described the diminished plasma and chloride levels in AIP, which he attributed to renal tubular pathology. Whittaker and Whitehead (1956) found 8 cases of acute porphyria in a patient population of 150,000 and noted on two occasions during acute attacks that the serum sodium level fell to 103 meq/liter (normal = 148). Patients were considerably improved by infusions of normal saline. Hellman et al. (1962) demonstrated abnormal electrolyte and water metabolism in AIP, which they interpreted as indicating transient inappropriate secretion

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of antidiuretic hormone (ADH). This was confirmed by Ludwig and Goldberg (1963) and Nielson and Thorn (1965). Nielson, likewise noted hyponatremia, water retention, and hypocalcemia with tetany. A diabetic type of glucose tolerance curve was present in all patients with AIP studied during acute attacks, according to Waxman et al. (1967). Perlroth et al. (1967) suggested that ADH release in AIP could be due to hypothalamic damage. They noted the report of Wayne et al. (1964), who found, on postmortem examination of a patient who died of AIP, destructive lesions involving the supraoptic and paraventricular nuclei and their fibers as well as the median eminence. Serious water retention which failed to improve during EDTA therapy was also noted in a severe case of acute porphyric polyneuropathy by Peters et al. (1957). The oliguria and edema were altered dramatically by the addition of hydrocortisone. However, hyponatremia is not a specific symptom in AIP, but may be seen also in a variety of central nervous system diseases as reported by Posner et al. (1967). These authors described 4 patients with severe hyponatremia accompanying infectious polyneuropathy. They concluded that this was due to the syndrome of inappropriate secretion of ADH, that their case with polyneuropathy might have reflected abnormalities of peripheral autonomic afferent fibers arising from vascular stretch receptors.

D. AMINOACIDEXCRETION Mellinkoff et al. (1959) demonstrated striking differences in amino acid excretion, between normal urines and those from patients with AIP. They were uncertain about the changes in blood level of amino acids. Although some of the results may have reflected an inadequate diet in their very sick patients, they also found similar abnormalities in asymptomatic individuals with AIP who were eating normally. Goldberg and Rimington (1955) also found abnormal amino acid excretion in rabbits suffering from drug-induced porphyria.

E. HYDROCORTISONE Waxman et al. (1969) reported a case of AIP in a 38-year-old man who demonstrated a decreased ability to secrete hydrocortisone. They treated this man and 3 others who were suffering from severe pain with a high carbohydrate intake and insulin and noted dramatic disappearance of pain in 3 of the 4 patients.

F. LIPIDMETABOLISM Schwartz (1955) first observed the presence of disturbed lipid metabolism in experimental porphyria, e.g., an increase of total lipid and phospholipid

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fraction of rat liver after administration of sedormid. Labbe (1961) reported that the synthesis of fatty acids by the liver of allylisopropylbarbituric acid porphyric rats doubled and postulated the existence of some type of relationship between heme and lipid transport metabolism in the liver. Lees ct al. (1970) also have demonstrated a hyper-p-lipoproteinemia in AIP. Total plasma cholesterol in 10 patients raiiged from 236 to 416nig/100 mi. in 5 out of 10 subjects. These were all above the upper liniit of agecorrected normal values. Plasma glycerides were normal in all patients. The concentration of very low-density lipoproteins was normal in all cases; however, the 8-lipoprotein concentration was increased in 9 of the 10 patients with AIP. These increases ranged from 3 to 125 mg/100ml above age-corrected control values. Four of the patients whose total plasma cholesterol values were within normal limits nevertheless, had ,%lipoprotein concentrations that were elevated. N o correlation was found between the excretion of porphyrins and their precursors and the plasma cholesterol and the j3-lipoprotein concentrations. Taddeini et al. (1964) used an animal model in which the porphyriainducing chemicals allylisopropylacetamide and 3,5-dicarbethoxy- 1,4-dihydrocollidine were fed to rabbits; they observed marked elevation of serum cholesterol, total lipids, and phospholipids. The liver total lipids, cholesterol lipid phosphorus, and water concentrations remained unchanged, but the organs became greatly enlarged, reflecting active synthesis of new protoplasm. Taddeini et al. held that these compounds affect the lipoprotein transport of serum cholesterol in a manner inducing sequestration of the synthesized cholesterol in the liver cells-hence the activation of the hepatic feedback control for cholesterol synthesis. Another possibility was that allylisopropylacetamide and 3,5-dicarbethoxy- 1,4-dihydrocollidine enhance the production of one or more enzymes of cholesterol synthesis in a way analogous to what Granick and Urata (1963) demonstrated to occur for the 6-aminolevulinic acid synthetase of the porphyrin synthetic pathway. I n reviewing 51 patients with hepatic porphyria in whom serum cholesterol determinations were recorded, Taddeini et a!. found hypercholesteroiemia, although inconstant, to be a common occurrence in the acute, intermittent, and mixed forms of the disease.

G. MACROAMYLA~EMIA Hedger and Hardison (197 I ) reported transient macroamylasemia in AIP. Electrophoresis and gel filtration of the serum revealed the presence of normal amylase plus a large molecular complex (molecular weight greater than 100,000) with amylase activity. This substance disappeared after remission of symptoms. The studies regarding the amino acid excretion of

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porphyrics and the presence of macroamylasemia are intriguing and must be investigated. H. GROWTH HORMONE Perlroth et al. (1965) studied 7 patients with AIP between the ages of 21 and 54 and demonstrated an abnormal response to a glucose load in all of their patients. In contrast to normal patients who demonstrated a decrease in plasma hormone growth levels with rising blood glucose, the majority of their patients with AIP, some of whom were asymptomatic at the time, showed a rising growth hormone level. They suggested that this response of plasma growth hormone level to a glucose load could be a good criterion of activity of the disease when symptoms may be difficult to evaluate. XII. Therapy

The literature is replete with pathological descriptions of the findings noted during fatal attacks of acute intermittent porphyria in patients most of whom were given ACTH or steroids. Mason et al. (1933) indicated that the death rate in patients with neurological and psychiatric symptoms, reached as high as 90%. The use of ACTH or steriods will not therefore be detailed except to point out that they have been used with success only in acute porphyrics with abdominal colic alone whereas cases showing neurological and psychiatric symptoms are often made worse by these agents. Emphasis should be placed on the importance of good nursing care, the use of tracheotomy, and physical therapy during the acute attacks and in the recovery stages of AIP.

THERAPY A. CHELATION 1. Dimercaptopropanol and Edathamil In a series ofpublications, Peters (1954, 1956, 1961) and Peters et al. (1957, 1958) reported dramatic improvement in a large series of cases of AIP treated with metal-binding (chelating) agents, including dimercaptopropanol and Edathamil calcium disodium (EDTA). Painter and Morrow (1959) likewise reported a favorable response in three AIP patients given a full course of intravenous chelation therapy. Gulbrandsen and Wigmostad (1958) and also Luby et al. (1959) reported cases in which chelation therapy with EDTA seemed to have a favorable influence. Galambos and Peacock (1959) described 5 patients observed to have lead poisoning associated with the excretion of large amounts of urinary porphobilinogen and reported dramatic and prompt amelioration following intravenous administration of EDTA with cessation of abnormal pigment excretion. They concluded that they could not say to what extent the beneficial clinical results of EDTA therapy in their patients was due to the removal of metals or to a

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nonspecific effect of EDTA itself'. They also reported a patient, who after prolonged exposure to zinc and lead dust, developed AIP, but failed to respond to EDTA, his clinical course became more severe, necessitating discontinuation of EDTA therapy. Peters et al. (1957) described a case who failed to respond to dimercaptopropanol therapy, but later improved dramatically when ACTH therapy was instituted. This patient also had high blood levels of lead, and it is of interest that he responded to ACTH despite the fact that there were severe neurological and psychiatric symptoms. I t is possible that the pretreatment with dimercaptopropanol, prevented the expected worsening of the clinical course that is so often seen when steroids or ACTH are employed in the presence of neurological symptoms. Batchelor et af. (1964) noted that the intravenous administration of EDTA is followed by a 2- to 6-fold decrease in urinary potassium when compared with control values in the same patients. It is possible that potassium depletion might have figured in the failure of some AIP patients to improve with EDTA. Careful attention to electrolytes must again be stressed in the therapy of these patients. Galambos and Dowda (1959) speculated as to whether lead intoxication can actually induce AIP or the underlying abnormal porphyria metabolism makes these patients more susceptible to the effects of lead poisoning. Olsson and Ticktin (1963) alluded to 3 patients in whom EDTA had no demonstrable effect, although none of the details are given as to symptomatology, therapy, or the severity of the condition. Roman (1967a) also reported on a number of patients with AIP treated with intravenous EDTA therapy, in one of whom severe mental and other symptoms were alleviated only after intravenous chelation dosage was increased to 4 gm per day. Raedeli (1962) was impressed with the immediate relief of pain in 5 patients with AIP given repeated courses of intravenous sodium calcium edetate. Of interest was a case of AIP treated by Roman et al. (1969). The patient had received phenobarbital, diazepam, and pethidine for abdominal pain prior to the diagnosis. She became confused, mildly psychotic, developed mild hypertension, and had a tachycardia of 140 per minute, hypochloremia, hyponatremia, and mild fever. All drugs were stopped, and she was given orally 220 mg of zinc sulfate over 8 hours for 72 hours. Eight hours after the oral administration of zinc sulfate, the pain ceased, but her mental condition grew worse for several days. After a 3-day interval she was given 2 gm of calcium EDTA in 1 liter of normal saline twice a day, by intravenous infusion for 5 days. By the second day of therapy she was rational although her pyrexia and tachycardia persisted. She was discharged on the seventeenth day after taking calcium EDTA orally at a dosage of 8 gm per day for 5 days a week. She became asymptomatic and remained so for over two years. During the acute attack before institution of any treatment, absorption

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bands of free porphyin as well as zinc porphyrin were visible on spectroscopic study. After the oral administration of zinc, the free porphyrin bands disappeared, and all porphyrin was present as zinc porphyrin. After intravenous chelation was started, the zinc porphyrin disappeared and all porphyrin was present now as free porphyrin. Roman and his co-workers suggested that the abdominal pain may have been due to zinc deficiency, and this seemed to confirm their assumption that zinc ions were made available for lactic dehydrogenase of the muscle, making it possible thereby to complete the metabolism of accumulated lactic acid. Increased lactate had been found previously by Cowger et al. (1963) in HeLa cells grown in tissue culture under the influence of porphyria-inducing drugs. Ginsburg and Dowle (1963) had previously reported reduced lactate dehydrogenase activity in the liver of rats after porphyria was induced by drug administration. Peters (1956), however, reported a case of AIP in which the pain of a fracture dislocation of the shoulder was completely masked by the porphyria and the patient did not complain of pain until after she had improved in her porphyric symptoms, after the use of dimercaptopropanol therapy. This might suggest that in Roman’s patient zinc sulfate actually increased the porphyric disturbance, since mental symptoms definitely increased after administration of zinc sulfate and the abdominal pain may also have been masked by the porphyria. Wirtschafter et al. (1960) report a 16-year-old girl who developed focal seizures that were extremely difficult to control. Although she received intravenous Pentothal, dimercaptopropanol, primidone, Peganone, reserpine, and sodium Amytal during episodes of status epilepticus, this symptomatic treatment was incapable of controlling seizure activity due to the porphyria. Her clinical course was not helped during four courses of intravenous EDTA therapy. It should be noted that barbiturate anticonvulsant medication was maintained during this entire period, and little information was given as to electrolyte balance during her prolonged clinical course. This case was of special interest because of focal seizure activity. Peters also reported several cases of seizure activity including focal seizures, in which control came about with the start of chelation therapy and gradual withdrawal of all barbiturate anticonvulsants. One patient actually had an increase in seizure activity on institution of diphenylhydantoin therapy, but seizure activity was terminated as soon as dimercaptopropanol therapy was reinstituted. Acute intermittent porphyria is an unpredictable illness in that during acute severe episodes involving the nervous system, care of electrolytes and many other parameters must be evaluated before the effect of any therapeutic regimen may be determined. Yet, chelation therapy seems to have impressed many authors as most effective.

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In patients with chronic or PCT form of porphyria, there are other variables, such as alcoholic intake, iron storage, and often liver damage, that make the evaluation of treatment difficult, although to a lesser extent than in AIP. Schrumpf (1953) first described improvement in skin lesions associated with a fall in urinary coproporphyrin and uroporphyrin in one case of PCT chelated with dimercaptopropanol. Paul and Thyresson (1954) noted increased tolerance to sunlight after similar therapy in PCT. Woods et al. ( 1 958) reported a case of PCT successfully treated by chelation with Edathamil (EDTA) initially given intravenously for 4 days and followed by EDTA orally at 1.5 grn over a 2-year period. They later (1961) reported a 5-year remission of that same patient despite exposure to sunlight and the intake of beer. Redeker et al. (1959) reported a case of PCT effectively treated with intramuscular dimercaptopropanol. After chelation therapy, urinary and fecal porphyrins were markedly reduced and there was a return to normal liver function. Harmel-Tourneur and Dropsy ( 1 959) also report favorably on a case of PCT treated with dimercaptopropanol. Epstein et al. (1959) compared two cases of epidermolysis bullosa acquisita and PCT, both treated with oral EDTA; whereas chelation therapy had no effect on the clinical course of the former, the patient with PCT showed both subjective and objective improvement. Peters et al. (1966) reported treatment of eight unselectcd cases of hexachlorobenzene-induced PCT that were treated with EDTA. Four of these received initial intravenous series of injections, and all were given 1-2 gm of oral disodium EDTA for periods up to one year; all treated patients showed marked improvement characterized by loss of light-sensitive skin lesions, distinct reduction in hyperpigmentation and hirsutism, associated with weight gain and feeling of well-being. One patient relapsed slightly 10 months after oral therapy was discontinued and promptly responded on resumption of EDTA therapy. T h e urinary copper excretion was elevated in 2 of 4 cases studied prior to chelation therapy, marked diuresis of zinc, copper, and lead were noted as a result of intravenous and later of oral EDTA. T h e effect of EDTA on the abnormal porphyrin excretion included initial stimulation of porphyrin excretion followed by a decline to more normal limits after 9 months of oral EDTA. Donald et al. (1965, 1966) have reported 12 cases of PCT treated with sodium calcium EDTA without signs of toxicity in any patient and with a clinical response described as gratifying. Patients with a history of excessive alcoholic intake were the most difficult to treat, but patients without alcoholism responded well to treatment, and those patients showing in addition sclcrodermatous changes responded best of all. Donald et al. considered chelation therapy to be a safe and meful treatment in cutaneous porphyria and preferred the oral route of administration.

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The results of Peters et at. (1966) with hexachlorobenzene-induced porphyria likewise suggested that the results were favorable whether a n initial intravenous course of EDTA therapy was given followed by oral administration over a period of months, or whether only oral EDTA was utilized. The patients reported by Woods et al. (1958, 1961) is particularly impressive because the only variable in this nonalcoholic patient was in the introduction of a short intravenous course of EDTA followed by 1.5 gm of EDTA by mouth over a period of 2 years. Remission continued despite later beer intake and exposure to sun. All other variables seem to have been eliminated. Of great interest are the sclerodermatous changes in many of the Australian patients reported by Donald et al. (1965). They call to mind the observations of Price et al. (1957) and Rukavina et al. (1957) showing a favorable influence of chelation with intravenous EDTA in the treatment of scleroderma (acrosclerosis). I n the most refractory cases, Donald et al. utilized EDTA therapy in daily dosage as high as 8 gm of sodium calcium EDTA intravenously, the average intravenous dose in the less severe cases being 4 gm per day. The authors noted considerable zinc diuresis during chelation therapy and demonstrated that, during the course of intravenous therapy, coproporphyrin values a t times showed some initial increase. The same observation was made with the hexachlorobenzene-induced porphyria patients treated with EDTA intravenously by Peters et al. (1966), who described this as an unblocking effect on the pathway of porphyrin synthesis. Ippen (1961) said that polycythemia in PCT is one of the many mysteries associated with this disturbance. Donald et al. (1966) in a series of 12 cases of PCT showed 3 with a hemoglobin value exceeding 17.5 gm/100 ml and 6 with a hemoglobin value exceeding 16 gm.Their outstanding case was a 64-year-old Australian with a 12-month history of symptoms of PCT, who showed in addition to blistering of the light-exposed areas and fragility of the skin, sclerodermatous changes of head, neck, and upper trunk. He was excreting 1500 mg of coproporphyrin in 24 hours. Hemoglobin was 19.4 gm/lOO ml. After intravenous chelation therapy with sodium calcium edetate, begun October 1964, his condition improved steadily with the softening of all sclerodermatous areas. Oral administration of sodium calcium edetate, 2 gm per day, was continued, and his clinical improvement continued so that by the middle of 1965 all clinical symptoms had subsided. During the next 2 years the patient remained sign- and symptom-free without further treatment. Urinary porphyrin excretion reached normal values : hemoglobin had fallen from 19.4 gm/l00 ml to 16 mg/l00 ml. The authors were impressed with the return to normal hemoglobin values in patients treated with sodium calcium edetate and the close time relationship between treatment and improvement of sclerodermatous changes. Swerenton and Hurley (1971) studied the teratogenic effects of edathamil when administered orally to rats in dosage amounting to 2-3% of the body

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H. A. PETERS, D. J. CRIPPS, AND H. H. REESE

weight in a chronic dietary intake. They indicated that a teratogenic effect was seen in a high percentage of their animals. Hamilton (1971) was critical of the experiment because of the enormous dosage given, which was far in excess of those dosages of EDTA commonly employed and because EDTA is poorly absorbed from the gut. Tyler ( I 953) demonstrated that the addition of chelating agents such as EDTA, diethyldithiocarbamate, 8-hyroxyquinoline, a-benzoinoxime and various amino acids and peptides (glycine, alanine, valine, leucine, lysine, glutamic, histidine, phenylalanine, tryptophan, cysteine, glycylglycine, and glutathione) considerably extended the duration of motility and fertilizing capacity of sea urchin sperm diluted in sea water. Over 100-fold extension of life-span of dilute suspensions was obtained by these agents. Tyler thought that the increased survival of the sperm in the presence of amino acids, proteins, and chelating agents was due to the ability of these agents to bind heavy metals present in the dilution medium, and that copper and zinc were among the metals involved. Moskowitz (1960) demonstrated that EDTA could inhibit the toxicity of a lethal dose of Clostridium perfringens toxin. This inhibition was then reversed by the addition of zinc, cobalt, or manganese to the toxin. Foreman (1 960) demonstrated in studies on rats that parenteral disodium EDTA and disodium calcium EDTA will produce nephrosis when administered in large dosage. The specific nature of the lesion was severe hydropic degeneration of the proximal tubules. The lesion was found to be reversible since it showed clearing within a few days after cessation of the drug. O n the basis of these studies, Foreman suggested that the drug be used in doses of approximately 50 mg/kg per day, given for 5 days, followed by 2 days of rest before the treatment is repeated. He also suggested that if chronic renal disease was present one should be hesitant to use the drug. Peters et al. (1957) administered 6 gm per day for several days to a severely ill patient with tetraplegia and oliguria without development of kidney damage. Peters recommended that the dosage be in the range of 2 gm per day, for the most part, although higher doses have been employed with success by Roman and his co-workers.

2. Histidine Another excellent chelator of zinc is histidine. Neilson and Neilson (1957) described a nonpregnant AIP patient in critical condition in whom the administration of histidine brought about immediate improvement, but they also noted that remissions may be unpredictable. Page et al. (1954) noted that plasma levels of histidine are high in normal pregnancy and excessive amounts are excreted in the urine. Porteous (1963) describes a case of AIP complicated by three pregnan-

PORPHYRIA

333

cies, in the last two of which histidine was administered with good effect. Injection was in the form of histidine, 200 mg/5 ml intramuscularly twice daily for 3 days and then daily for a week. On occasion they administered guanethidine sulfate, 20 mg orally daily. D. P. Tschudy and R. Henkin (personal communication, 1973) also described very good therapeutic effect of histidine in AIP and called attention to its excellent role as a chelator of zinc, this is of special interest since it is a metabolic substance equal to the porphyrins as a chelator. Levine et al. (1973) in a preliminary abstract reported studies of 2 AIP cases and 1 PCT case on a constant diet. They measured urinary uroporphyrin and coproporphyrin in PCT, serum and urinary porphobilinogen and 6-aminolevulinic acid in AIP; and serum and urine zinc and copper and total body zinc during a sequential period involving a control period, an oral administration of 65Zn and zinc sulfate administration, a period involving 1-histidine, 8-32 gm/day, and a final control period. Zinc sulfate increased serum and urine zinc but had no consistent effect on copper, porphyrins, or porphyrin precursors. 1-Histidine (24-32 gm/day) significantly lowered urine and serum uroporphyrin and coproporphyrin in PCT and significantly reduced serum and urine porphobilinogen and 6-aminolevulinic acid in AIP ; there was concurrent decrease in whole body zinc as measured by decrease in biological TI,, of 65Zn, decrease in serum zinc without change in fecal 65Zn, and marked increases in urine zinc as compared to control periods of zinc sulfate feeding. Withdrawal of 1-histidine caused rapid increase in urine and serum 6-aminolevulinic acid and porphobilinogen in AIP. In PCT, withdrawal of 1-histidine did not result in increased uroporphyrin or coproporphyrin urine excretion levels. Levine et al. considered the action of 1-histidine to be related to a direct or indirect effect on porphyrin metabolism or to 1-histidine-mediated mobilization and excretion of a tightly bound tissue zinc pool. 3. Hematin Hematin has been shown to inhibit the action of 6-aminolevulinic acid synthetase, the limiting enzyme in heme biosynthesis according to Tschudy (1969). Bonkowsky et al. (1971) demonstrated in an AIP patient with renal failure a sharp decline in serum 6-aminolevulinic acid and porphobilinogen on administration of intravenous hematin, but this did not prevent a fatal outcome. Watson et al. (1973) were able to bring about remission in a case of AIP with tachycardia, abdominal pain, urine retention, and psychopathic behavior by the administration of hematin intravenously at 300 mg daily for 3 days. Levels of serum and urinary porphobilinogen and &aminolevulinic acid showed prompt lowering.

H. A. PETERS, D. J. CRIPPS, AND H. H. REESE

4 . Penicillam ine Hunter and Donald (1970) also described the effect of D-penicillamine as an alternative chelating agent in a PCT patient who had a clear-cut failure with sodium calcium edetate. He was given 120 mg of penicillamine orally 6 times per day for 5 days; clinical improvement was apparent within 3 weeks, and after such treatment in February, March and again in June, 1969, he was well and symptom-free, porphyrin excretion returning to normal by October, 1969. Another patient was treated for 5 days of every month at the same dosage level during October, November, and December; he, too, became symptom-free with a marked decrease in coproporphyrin and uroporphyrin levels. Hunter and Donald treated 4 PCT patients with D-penicillamine and did not see any toxic effects. I t would therefore appear that D-penicillamine may have merit as an alternative chelating agent. The toxic effects of D-penicillamine must be considered, including apparent induction of polymyositis as reported by Schraeder et al. (1972). As D-penicillamine chelates both copper and zinc, care must be taken not to create a deficiency in these essential trace metals. 5. Adenosine LClonophosphate( A M P ) Gajdos and Gajdos-Torok (1961) suggested the existence in AIP of a deficiency of phosphorylated derivatives of adenosine as also postulated in 1955 by Shemin and Talman. They chose to use AMP, 250 mg/day, in the treatment of porphyria, hoping that a deficiency in phosphorylated derivatives of adenosine might have been responsible for the clinical signs. They treated 10 AIP patients, the first of whom was a severe porphyric with abdominal, neurological, and psychiatric symptoms, and reported encouraging results with disappearance of symptoms after 5 days and disappearance of urinary uroporphyrin. Nine other patients were treated, with good response in 7 as judged by clinical and biochemical signs. In 2 patients, administration of AMP did not modify the course of the illness. They decided that their observations were too few to draw conclusions, especially in view of possible spontaneous remissions. In experimental porphyria, induced by injection of allylisopropylacetamide into the yoke sac of chicken and duck embryos, they also noted better embryonic development and diminished uroporphyrin excretion in the amniotic fluid, liver, and kidneys in AMP-treated embryos than in the controls. Inhibition of clinical and biochemical findings of porphyria induced by hexachlorobenzene intoxication in rats was also evident with AMP, whereas in rabbit AMP was not beneficial to this allylisopropylacetamideinduced porphyria. Oaks et al. (1 969) reported a case with clinical and laboratory findings characteristic of AIP in addition to which there were skin lesions following

PORPHYRIA

335

exposure to sun and trauma, suggesting the diagnosis of porphyria variegata. Two acute attacks were treated with AMP in doses of 250 mg/day intramuscularly. During the second attack, no improvement was noted until 8 days after the start of therapy. Calling attention to the report by Tschudy et al. (1 965), who noted the ability of glucose to repress 6-aminolevulinic acid synthetase, Oaks et al. postulated that Adenosine diphosphate acts through a metabolic pathway similar to that which gives rise to the glucose effect. According to Edman (1960), AMP like EDTA, is a chelating agent for zinc and thus have this mechanism of action in porphyria. 6. Cytochrome Proger and Dekaneas (1 947) gave injections of cytochrome c to 1 patient during attacks of AIP. Injection of a small dose (10 mg) of cytochrome c appeared to induce a slight aggravation of symptoms, whereas the injection of 50 mg was quickly followed by severe worsening of the condition. Riederer (1961), however, reported complete remission of symptoms of AIP in a 46year-old woman after daily injections of 15 mg of cytochrome c over a 3-week period. A later relapse of painful brachial plexus neuritis was again promptly terminated by the administration of cytochrome c daily after 2 days. No effect was noted in the urine, which remained strongly positive for porphobilinogen. Lang et al. (1968) reported 3 cases of AIP which improved clinically after the administration of cytochrome c. One of their patients had been treated previously with ACTH, chlorpromazine, dimercaptopropanol, EDTA, AMP, and testosterone without apparent benefit, although the dosage of chelating agents was not noted.

7 . Desferrioxamine Brehm and Holzmann (1964) made quantitative estimations of transferrin, haptoglobin, and serum iron levels in 10 cases of PCT. There was a statistically significant diminution of transferrin and elevation of iron in the serum of patients with PCT as compared with a group of healthy persons. Holzmann (1963) had treated four patients with PCT with desferrioxamine with improvement, although they noted that in contrast to hemachromatosis, they could not mobilize a level of 6 mg of iron in 24 hours. It should be recalled that Schrumpf (1953) first treated PCT with dimercaptopropanol because of a similarity to hemochromatosis. The infusions of desferrioxamine were accompanied by oliguria and drop in blood pressure as well as other symptoms in 2 of Holzmann’s 4 patients. Thivolet et al. (1968) used the iron-chelators 4 times, and disodic tetracemate, in a fifth case of PCT, with clinical results that were favorable. They noted a fast reduction in the rate of urinary uroporphyrin excretion and improvement in hepatic functional disturbances.

336

H. A. PETERS, D. J. CRIPPS, AND H. H. REESE

B. PHLEBOTOMY Ippen (1961) described marked improvement in PCT after repeated venesections and his findings were confirmed by Il’in (1963). Epstein and Redeker (1968) studied 20 PCT patients, from whom they removed 2.5-8.5 liters of blood over a period of 3-8.5 months, markedly reducing uroporphyrin excretion, thus bringing on remission of the clinical disorder in all the patients. Eighteen of their 20 patients showed persistence of clinical response associated with progressive reduction in porphyrin excretion levels ; 14 were observed for 1-32 years without a relapse, recurrences occurring in only 2 patients. Kalivas et al. (1969) alluded to experimental hexachlorobenzene-induced porphyria, in which they showed that superimposition of hepatic siderosis did not increase the severity of the porphyria. He concluded that hepatic siderosis may not play as crucial a role in the pathogenesis of PCT as had been previously surmised, and that the response to phlebotomy depends probably on a more complex mechanism than just simple elimination of iron. Hunter and Roman noted that with treatment with EDTA, iron storage likewise diminished toward normal, and it should be noted that other cations besides iron, such as zinc, are eliminated from the system by phlebotomy. H. A. Peters (1963, personal communication) administered daily oral hexachlorobenzene to rats and then after two weeks challenged them with ultraviolet light. The rats immediately developed hemorrhagic bullae over the ears, nose, and head. When the rats were fed hexachlorobenzene plus EDTA, they did not demonstrate any response to the ultraviolet light challenge. C. CHLOROQUINE The use of chloroquine phosphate in the treatment of PCT has been a debatable procedure. Linden et al. (1954) described acute porphyria that developed in a case of chronic discoid lupus erythematosus attributed to the administration of chloroquine. Davis and Vander Ploeg (1957) described 2 other cases in which acute porphyric symptoms were precipitated by chloroquine administration, one patient having chronic discoid lupus erythematosus and the other only light sensitivity. Marsden (1959) described another patient being treated for disseminated lupus who developed AIP symptoms on administration of chloroquine. The second case of Davies and Vander Ploeg showed chiefly cutanea tarda symptoms, but both patients gave a history of heavy consumption of alcohol with evidence of liver damage. The case of Linden had no evidence of liver damage and was classified as developing acute porphyria from chloroquine despite the absence of porphobilinogen in the urine. Woods ei al. (1958) described a case of PCT in which chloroquine, 250 mg, was prescribed every other day, and later daily, in an effort to combat

PORPHYRIA

337

photosensitivity. During the following year the patient continued to have cutaneous exacerbations and no decrease in symptomatology. Pain and shock sensations developed in the hand muscles but disappeared when chloroquine was discontinued. The laboratory findings, which at first included only coproporphyrin and uroporphyrin excretion, altered while on chloroquine therapy, and the patient began excreting 8-aminolevulinic acid, 7.8.-15 mg/liter, and porphobilinogen, 1.2 to 5.2 mglliter. Urinary zinc excretion was normal prior to therapy but rose to 1.7 mg/liter during chloroquine therapy. The patient was then placed on intravenous EDTA therapy, 3 gm per day for 3 days, then supplied 1 gm of oral disodium EDTA each day for the next 2 years. This chelation therapy brought about a 5-year complete remission, so that the patient was able to tolerate extreme exposure to sunlight and skin trauma without harm. Follow-up examination indicates that the patient remains asymptomatic, although still excreting abnormal levels of uroporphyrin and coproporphyrin in 1973. Cripps and Curtis (1962) also described a toxic effect of chloroquine in PCT. Donald’s group advised against the use of chloroquine in the treatment of PCT,but in a later communication,Hunter and Donald (1970), using one-half tablets (75 mg of chloroquine base) twice weekly, found the drug to be well tolerated in 5 cases with no ill effects and described the medication as being extremely effective. Chloroquine is not an innocuous drug. Whisnant et al. (1963) reviewed the side effects of chloroquine administration and included reports of corneal edema causing visual symptoms, bleaching of the hair, headache, weight loss, < c nervousness,” difficulty with accommodation to light, and Cambiaggi (1957) described macular degeneration, which was not reversed when administration of the drug was stopped. Sternberg and Laden (1959) as well as Hobbs et al. (1959) also described macular degeneration. Okun et al. (1963) reported 9 additional cases of chloroquine retinopathy and also described macular degeneration and the development of pericentral scotomas. Whisnant et al. (1963) described 4 cases in which weakness of the skeletal muscles developed during the administration of chloroquine; the weakness generally appeared after 500 mg/day of chloroquine had been taken for a year or longer. The clinical features and the electromyogram suggested neuropathic as well as myopathic components. The dosages used are much larger than those reported in the treatment of PCT, but nonetheless, careful observation for side effects is indicated with chloroquine therapy. London (1957) treated a case of PCT with chloroquine with relief of light sensitivity and no untoward symptoms. Colomb (1957) likewise reported 3 cases of PCT effectively treated with chloroquine. Felsher and Redeker (1966), Volger et al. (1970), and also Saltzer et al. (1968) and Arnold (1969) described good therapeutic effect in PCT without adverse side effects. Kowertz

33%

H. A. PETERS, D. J. CRIPPS, AND H. H. REESE

(1973) noted a n induced rapid remission of cutaneous symptoms in PCT in

2 patients with chloroquine. These two patients, in addition, showed improvement of hepatic function, which had been abnormal prior to treatment. I t would seem from the above reports that low doses of chloroquine are probably preferable to high levels in therapy and the possibility of developing symptoms of AIP should always be kept in mind.

D. CHOLESTYRAMINE A most interesting approach to the treatment of PCT was made by Stathers (1 964), who used cholestyramine, a strongly basic anion exchange resin containing quaternary ammonium functional groups which are attached to a styrene-divinylbenzene copolymer. This substance forms a n unabsorbable complex with bile salts in the gut of man and laboratory animals. They were able to demonstrate in uitro binding of uroporphyrin and coproporphyrin by cholestyramine which could be reversed by 10% HCI. Porphobilinogen in a patient with AIP, on the other hand, was not bound by the resin. Three patients with symptomatic PCT were treated o n the theory that the porphyrins would be bound by cholestyramine and this would deplete body storage by preventing a theoretical enterohepatic circulation. The resin was given in dosage of 12 gm daily in divided dosage with meals and brought about clinical improvement with decreased formation of blisters, cessation of septic involvement of blisters that did form, and associated decrease in skin fragility. One patient showed a loss of severe fatigability; another patient lost pruritus, which returned after discontinuation of the resin. There was no clear alteration in porphyrin excretion, but there was evidence that the resin did bind porphyrins in the gastrointestinal tract, with retained fluorescence in the fecal extract.

E. PYRIDOXINE Operating on the theory that pyridoxal 5-phosphate acts as a coenzyme for the decarboxylation process in porphyric metabolism and that a partial "enzyme block" in porphyrin synthesis exists in PCT, Petres et al. (1967) treated 4 PCT patients with intravenous doses of pyridoxal 5-phosphate or pyridoxine-HCI, 500 mg daily, and observed a fall in both porphyrin excretion and serum iron. Price et al. (1959) demonstrated a n abnormality in the tryptophan metabolism suggesting a functional pyridoxine deficiency. They were unable to show biochemical or clinical improvement as the result of oral pyridoxine supplementation. On the other hand, both clinical and biochemical improvement were often observed after treatment with chelating agents.

PORPHYRIA

339

Elder and Mengel (1966) also suggested that pyridoxine should be tried as a therapeutic agent in porphyria. They studied urinary xanthurenic acid excretion after an oral tryptophan load in an asymptomatic patient with known AIP. The abnormal response suggested pyridoxine deficiency, as already noted in porphyrics by Price et al. (1959). After the administration of deoxypyridoxine, there was further evidence of pyridoxine deficiency, and both S-aminolevulinic acid and porphobilinogen excretion decreased. The administration of pyridoxine had the opposite effect. Pyridoxal phosphate is the coenzyme for both production of S-aminolevulinic acid and succinylCoA from glycine and also for the further metabolism of xanthurenic acid precursors formed from tryptophan. This conforms with the observation that an antimetabolite of pyridoxine should reduce both S-aminolevulinic acid and succinyl-CoA. Elder and Mengel postulated that the increased 8-aminolevulinic acid synthetase activity in porphyria in some way makes pyridoxal phosphate unavailable to other enzymes, resulting in relative deficiency of pyridoxine. Zimmerman ( 1968) treated a 2 1-year-old gravid para 2, abortus 2 Negro female who had developed abdominal colic, psychosis, and severe peripheral neuropathy. Prior to the diagnosis of AIP the patient had been placed on barbiturates and other drugs. The addition of 400 mg of pyridoxine per day in divided'doses between March 24 and May 8 resulted in steady clinical improvement. Perhaps a large daily dose is needed.

F. TOCOPHEROLS Murty et al. (1969) reported a therapeutic effect of tocopherols in causing remission in the human type of porphyria, Nair et al. (1970) studied the induction of experimental porphyria in rats by allylisopropylacetamide characterized by elevation of hepatic 8-aminolevulinate synthetase and dehydratase activity. The administration of vitamin E 6 hours prior to initiation of the porphyrinogenic regimen prevented porphyria via control exercised on the activities of the two hepatic enzymes. The bone marrow of porphyric animals showed enhanced synthetase activity which was prevented by vitamin E. Nair et al. concluded that vitamin E is involved in the regulation of this inducible enzyme. When vitamin E was given alone to control animals, it induced a significant rise in the activity of both hepatic enzymes. In the experimental animals, allylisopropylacetamide administration produced a significant rise in bone marrow S-aminolevulinic acid synthetase, and this was prevented by prior administration of Vitamin E. With identical doses of allylisopropylacetamide, synthetase activities in vitamin E-deficient livers was approximately 5 times higher than in livers of rats fed vitamin E and also in the control livers. Nair et al. noted that addition in uitro of vitamin

340

H. A. PETERS, D. J. CRIPPS, AND H. H. REESE

E or liver homogenates of vitamin E-treated rats did not alter the activity of the induced enzyme in homogenates of porphyric rat liver, thus ruling out a direct inhibitory action of vitamin E on 6-aminolevulinate synthetase. On the other hand, Mustajoki (1972) studied the effect of vitamin E on the porphyrin excretion in 5 porphyric patients. During the evaluation, 1 patient had symptoms of manifest AIP and 1 patient had variegate porphyria. Four of the patients were classified as AIP, and 1 patient had variegate porphyria. Four of the patients were given dl-a-tocopherol, 100-200 mg per day, and one was given d-a-tocopherol, 400 mg per day. They noted during the 4-week observation period no significant alterations in the urinary excretion of 8-aminolevulinic acid, porphobilinogen, uroporphyrin, or coproporphyrin. The fecal excretion of coproporphyrin and protoporphyrin in the patient with variegate porphyria remained unchanged. The patient with symptomatic porphyria showed no clinical improvement. Since there are many tocopherols, in the light of the above observations, further investigation is needed.

G. CONTRACEPTIVE AGENTS Patients with AIP whose clinical attacks are associated with menses or gestation, may be benefited by contraceptive agents, according to Perlroth et al. (1965). In three patients, oral progestational agents were successful in preventing the appearance of symptoms, and the individual administration of estrogen, androgen, and progestin to one patient showed the same effect. The authors felt that inhibition of gonadotropin with stabilization of endogenous ovarian steroid production at a low level may be the effective mechanism. Hopmann (1968) described 3 females with ovulo-cyclic AIP, characterized by premenstrual exacerbation and described therapeutic effect of androgens as a counter hormone bringing about gonadotropin inhibition. XIII. Discussion

The reported beneficial effect of a number of different chelating agents as well as other techniques that lower the metal content of the body, such as phlebotomy, raise the question of which trace minerals might be responsible for the development of porphyria. It will be noted that EDTA, penicillamine, histidine, dimercaptopropanol, AMP, and even phlebotomy have considerable effect on zinc metabolism. Peters described increase in urine, zinc, andjor copper in AIP patients who are developing neurological and psychiatric symptoms. This excretion was not seen in the recovery phases of the disorder. Although zincuria was disputed by Olsson and Ticktin, Roman has confirmed the presence of zincuria during attacks of AIP and also demonstrated increased serum levels of zinc during the acute phase. Peters has noted that certain zinc-containing enzymes are inhibited by an excess

PORPHYRIA

34 1

amount of zinc, these include lactic dehydrogenase, insulin, equine gonadotropin, and ACTH. Metalloenzymes, such as carbonic anhydrase, uricase, catalase, peroxidase, carboxypeptidase, lactic dehydrogenase, and alcohol dehydrogenase, are all zinc-dependent enzymes. Uroporphyrin and coproporphyrin are excreted in AIP in both urine and feces as zinc metal complexes according to Watson and Schwartz (1941). Giinther (1922) recognized that urinary porphyrins were often combined with a metal. Watson and Larson (1947) called attention to the zinc complex being relatively larger in amount in a case of AIP during relapse. Peters postulated that the zincuria and cupruria in AIP patients could represent nature’s attempt to rid the body of toxic cations, the increased excretion of porphyrin metabolites representing a last-ditch effort to mobilize porphyrins for the needed purpose of chelation. The elevation of xanthurenic acid or kynurenine or both after a loading dose of tryptophan during acute stages ofacute, chronic, and mixed porphyria resembling the tryptophan metabolic pattern seen in patients with scleroderma or acrosclerosis by Rukavina et al. (1957) suggest that these tryptophan metabolites, which are also excellent metal-binding agents, are also participating in this function. It was felt that the body’s natural chelating ability had been exhausted, and the withdrawal of porphyrins from the central and peripheral nervous system, possibly for purposes of chelation, and their failure to be replenished because of a block in the metabolic progression toward protoporphyrin and coproporphyrin 111, might be the essential mechanism giving rise to porphyric symptoms. The demonstration of a block in uroporphyrinogen synthetase by Strand et al. (1972) could fit in with this hypothesis. Peters also demonstrated that in lead, arsenic, or mercury poisoning when a patient is becoming symptomatic, zinc and copper diuresis takes place. It is well known that one of the first depletions brought about by heavy metal intoxication is in glutathione levels. An attempt by Peters to estimate sulfhydryl groups in patients showing zinc and copper diuresis suggested that this sulfhydryl pool indeed was reduced. Lorber et al. (1964) determined serum sulfhydryl levels and found decreased levels in acute rheumatoid arthritis, the lowest levels being recorded in rheumatoid arthritis, complicated by necrotizing vasculitis, in active lupus, and polyarteritis nodosum. hluller et al. (1956) described a fatal case of acute hepatic porphyria associated with panarteritis and concluded that a screening of all cases of “arteritis” might demonstrate other cases of disturbed porphyrin metabolism. Peters reported a case of AIP associated with polyarteritis nodosum. He also recorded extremely high excretion levels of copper and zinc in polyarteritis nodosum as well as other conditions (see Tables 1-111). I t should be noted that Jaffe (1963, 1970) reported on the favorable influence of penicillamine in the treatment of rheumatoid arthritis and

342

H. A. PETERS, D. J. CRIPPS, AND H. H. REESE

TABLE I URINARY EXCRETION IN ALCOHOL ISM^ Clinical shape* 1

2 3

4

Total Total Total

I-ALA Average Sigma Count Average Sigma Count Average Sigma Count ilverage Sigma Count .i\verage Sigma Count

8.80 00 1 6.30 2.50 2 5.12 2.45 5 4.80 00 1 5.76 2.48 9

PBG

0.80 00 1 2.47 1.11 3 0.80 00 1 1.80 1.19 5

cu 0.0850 00 1 0.1450 0.0550 2 0.2151 0.1239 9 0.1200 0.0154 4 0.1774 0.1065 16

Zn

0.100 00 1 0.415 0.366 4 1.291 0.877 10 1.198 0.785 4 1.024 0.854 19

Pb

0.020 0.010

2 0.010 00 1 0.040 00 1 0.023 0.013 4

a Expressed in mg/liter. Normal copper = 0.03 mg/liter; normal zinc = 0.5 mg/liter. 3-ALA = 6-aminolevulinic acid ;PBG = porphobilinogen. Shape 1 represents clinical remission; shape 4, the most severe active stage of the disease process.

TABLE I1 URINARY EXCRETION IN PERIARTERIT~S NODOSUM Clinical shapeb

1

2

3

4

Total Total Total

3-ALA Average Sigma Count Average Sigma Count Average Sigma Count Average Sigma Count Average Sigma Count

2.40 00 1 -

3.00 0.60 2 2.80 0.57 3

PBG

0.80 00 1 10.00 00 1 2.37 1.87 3 3.58 3.57 5

cu

Zn

Pb

0.0950 00 1 0.0700 00 1 0.0950 00 1 3.570 00 2 1.4800 1.7065 5

0.360

0.020 00

00 1 3.00 00 1 2.450 2.090 2 6.380 3.638 4 4.223 3.588 8

1

0.020 00 1 0.028 0.011

4 0.025 0.010 6

a Expressed in mg/liter. Normal copper = 0.03 mg/liter; normal zinc = 0.5 mg/liter; I-ALA = 3-aminolevulinic acid ; PBG = porphobilinogen. Shape 1 represents clinical remission; shape 4, the most severe active stage of the disease process.

343

PORPHYRIA

TABLE I11 URINARY EXCRETION IN ACUTE HEPATIC PORPHYRIA Clinical shapeb 1

2

3

4

Total

Average Sigma Count Average Sigma Count Average Sigma Count Average Sigma Count Average Sigma Count

S-ALA

PBG

9.13 9.43 12 9.87 3.83 3 27.25 28.35 4 17.40 15.45 4 13.82 16.63 23

13.50 20.75 11 6.23 6.22 3 29.83 34.50 6 24.54 31.29 7 19.19 27.61 27

cu 0.0639 0.0251 9 0.0960 0.0933 5

0.1217 0.0753 3 0.1325 0.0325 2 0.0887 0.0654 19

Zn

Pb

0.623 0.460 15 0.743 0.339 6 1.896 1.624 7 5.248 6.891 5 1.615 3.236 33

0.022 0.015 6 0.020 0.010 2 0.025 0.015 2 0.070 0.010 2 0.030 0.022 12

Expressed in mg/liter. Normal copper = 0.03 mg/liter; normal zinc = 0.5 mg/liter; S-ALA = S-aminolevulinic acid; PBG = porphobilinogen. Shape 1 represents clinical remission; shape 4, the most severe active stage of the disease process. (I

necrotizing vasculitis. The value of penicillamine in rheumatoid arthritis seems to have been confirmed by a Multicenter Trial Group (1973)) despite a 30y0incidence of side effects. Thus a depleted sulfhydryl serum level may be an important gauge for the prediction of chelation effectiveness and should be further investigated in the porphyric patients. The remarkable tolerance exhibited by some of Peters’ AIP patients given huge doses of dimercaptopropanol which in the normal individual would quickly give rise to severe side effects, plus the frequent, prompt clinical improvement during chelation therapy, suggested that dimercaptopropanol andfor EDTA compounds are satisfying a real metabolic need. The normalization of tryptophan metabolism following recovery, at times induced by chelation therapy, in the porphyric group, as well as other patients, including several schizophrenics who showed this metabolic abnormality, also supported this view, In symptomatic lead poisoning, blood lead levels are noted to rise. Roman has noted elevations of serum zinc in porphyrics during exacerbation and this would suggest that zinc, too, might be behaving as lead in a toxic manner. Is there any evidence that zinc alone may act in a toxic manner? Tokuoka (1951) reported that caudal resection of the pancreas was effective

344

H.A. PETERS, D. J. CRIPPS, AND H. H. REESE

35

3.0 L; . t

't 2.5

E" 0

20

N

1.5

I

I .o

0.5 0.c

(m)

Fig. 1 . Urinary zinc in various disease states. Clinical shapes 1 represents clinical remission; and shape 4 the most severe active stage of the disease process. Normal zinc = 0.5 mg/liter.

(m),

in alleviating the epileptic seizures in 66.7% of 27 cases of refractory epilepsy and assumed that the disturbance of zinc metabolism caused by this procedure had some connection with the alleviating effect. Zinc content of the pancreas is many times the amount necessary to account for its insulin content and exists in the form of a zinc salt. Montgomery et al. (1943) have shown that injected zinc is eliminated by way of the external secretion of the pancreas. These observations were further explored by Tokuoka et al. (1967). Subtotal pancreatectomy in rats caused a marked disturbance of zinc metabolism in the early postoperative period with decrease of body retention of zinc, decrease in the distribution of zinc in the brain, and a decrease in the retention of zinc in the hippocampus and dentate gyrus. The changes in brain excitability and neurochemistry following this operation, were consistent with those in zinc-deficiency states. The animals demonstrated increased tolerance to electric shock, shortened response to maximum electric stimulation, decreased brain carbonic anhydrase, and increased brain calcium. Intracerebral injection of metal-chelating agents caused both a n increase in threshold to electric shock and diminution of zinc levels in the hippocampus and dentate gyrus. These observations fit in very well with the descriptions of Peters, in which seizure activity of porphyric patients were

345

PORPHYRIA

ailicol shpe 4.oy.O.la

lnical rhapr l,ovg.O.O48

(m)

Fig. 2. Urinary copper in various disease states. Clinical shape 1 represents clinical remission; and shape 4 the most severe active stage of the disease process. Normal copper = 0.03 mg/liter.

(w),

reduced and terminated and the electroencephalogramnormalized during and following chelation therapy, even though anticonvulsant drugs .were gradually eliminated. The development of AIP in patients with lead poisoning as described by Galambos and Peacock would seem more than fortuitous. I t is possible that the chelating potential of the body can be dissipated in a number of disease conditions, such as lead poisoning, mercury poisoning, arsenic poisoning, polyarteritis nodosum, and so forth, but that when the metabolic genetic defect is present, porphyria of the acute or mixed type, ensues. Batchelor et al. (1926) have noted that zinc workers absorb and excrete zinc in amounts considerably over the norm. Abnormal amounts of zinc may enter and leave the body for years without causing clinical symptoms of gastrointestinal, renal, or other organic pathology. Landouzy and Maumene (1850) and Sacher (1893) quoted cases, however, in which vomiting, intestinal colic, constipation, emaciation, and sometimes muscular paralysis with atrophy occurred. In this regard, cadmium could have been a contaminant of the zinc. Workers with lead, likewise, seem to have a variable susceptibility to lead poisoning, some workers on the same job, tolerating exposure for much longer periods than those that become toxic. Interestingly, Murphy (1970) reported an experience of a 16-year-old boy who ingested 4 and then 8 gm

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H. A. PETERS, D. J. CRIPPS, A N D H. H. REESE

of elemental zinc, mixed with peanut butter, having read that zinc will promote healing, and wishing to heal a minor laceration. The boy became inordinately sleepy, had difficulty waking after a full night’s sleep, developed light-headedness, slight staggering of gait, and difficulty in writing legibly. He denied gastrointestinal symptoms, headaches, or paresthesias. Increased serum lipase values were found, and a serum amylase value of 284 units was clearly elevated. Six hours after treatment with dimercaptopropanol, the patient became clear, and the following morning he awoke a t his normal time and was symptom free. Peters’ group described a case of AIP in which an exacerbation was associated with the use of zinc oxide ointment as a rectal lubricant. Vallee (1959) reported lethargy or decreased alertness when zinc was administered intravenously to dogs. When 4 mg of zinc gluconate was given per kilogram of body weight, lethargy, depressed tendon reflexes, and enteritis occurred. Orten (1966) stated that 85y0 of the blood zinc is found in the erythrocytes, 127, in the plasma, and 3y0 in leukocytes. According to Woodbury et al. (1968), after parenteral administration of a single dose of labeled zinc, the brain accumulates substantial amounts of the metal over the ensuing 7 days and subsequently releases it over an equal time. According to Montgomery et al. (1943), after its intravenous administration, zinc is excreted in large amounts in the duodenal juices and bile. According to Gubler (1956) and Vallee (1956), normal route of excretion for copper and zinc is almost entirely by the fecal stream, with an average daily urinary excretion of 0-70 mg of copper and 400 mg of zinc per 24 hours. Especially low serum zinc values have been noted in cirrhosis by Kallai et al. (1968). Vallee pointed out that there is a large zinc diuresis in cirrhosis and postulated zinc deficit as being responsible for many of the symptoms. Peters (1960, 1961) likewise called attention of zinc diuresis in alcoholics with delirium tremens, Wernicke’s encephalopathy, etc., and also noted that a copper diuresis is frequently present. Hunt et al. ( I 963) described the liver content of zinc to be reduced in cirrhosis and carcinoma of the liver, while the liver iron content in the cirrhotic patient suffering from hemochromatosis may be S t y times the normal value. Sullivan and Lankford (1965) noted abnormal excretion of urinary zinc in 42 of 124 chronic alcoholics. Age, duration of alcoholism, blood level of alcohol, and rate of alcohol metabolism could not be correlated with the abnormality in zinc excretion. The zincuria was transient and disappeared with 2 weeks in 8OoJ, of those studied, High urine zinc excretion was present in each of the 7 patients diagnosed as having delirium tremens. Abnormally low plasma zinc levels have been noted by Halstead and Smith (1970) in such conditions as active tuberculosis, indolent ulcers, uremia, before and after a single hemodialysis, myocardial infarct, nontuberculous pulmonary infection, Downs syndrome, cystic fibrosis,

PORPHYRIA

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growth retardation of patients in Iranian villages, and pregnancy in women taking oral contraceptives. Greaves and Boyde (1967) have demonstrated lowered plasma zinc concentrations in patients with cirrhosis, dermatoses, and venous leg ulcerations. MacMahon et al. (1968) have given supplementary zinc in the form of zinc sulfate in dosage of 100 mg in gelatin capsules, three times daily in the treatment of malabsorption. DeWys et al. (1967) reported inhibition of a Walker 256 carcinosarcoma growth by dietary zinc deficiency. Poswillo and Cohen (1971) also demonstrated inhibition of tumor formation with the zinc administration. According to Pfeiffer (1972), schizophrenics can be divided into a histapenic group with low blood histamine and a histadelic group with high histamine levels. The first group 'comprises about 50% of the schizophrenic population and is characterized clinically by paranoia and hallucinosis. The histadelic group comprises about 20oJ, of the schizophrenic population and is characterized by suicidal depression. Effective therapy restores normal histamine levels of 40-70 ng/ml. The two groups are further separated by their basophile count, the histapenic and histadelic groups being low and high, respectively. Copper is present in histaminase, and the histamine is stored in mast cells with the trace metal zinc. High serum copper levels have been reported in the schizophrenic by Nicolson (1966), and low zinc levels in the brain have been reported by Kimura and Kimura (1965), Pfeiffer (1972) has noted that schizophrenic patients excrete less copper and more zinc than normal controls. They further noted that the administration of zinc sulfate increased copper excretion and the combination of zinc sulfate plus manganous chloride greatly enhanced excretion (and also zinc excretion). Derrien and Benoit (1929) found an increased zinc excretion in urines of porphyric patients, who often are psychotic; this has been noted also by Peters (1960, 196l), Watson and Schwartz (1971), Nesbitt (1944), and Roman (1967a,b), as already mentioned. The chelatingactionofuroporphyrin has been given credit for this, although Price and Peters have pointed out that even the products of abnormal tryptophan metabolism are excellent chelators. Pfeiffer (1972) has administered zinc sulfate and manganous chloride to schizophrenics with good clinical response. Pfeiffer (personal communication, 1973) identified a patient with mauve factor and urinary cryptopyrrole excretion. Cryptopyrrole combines with zinc and pyridoxine and produces a deficiency of both. The schizophrenic patient showed white nail spots, failure to remember dreams, convulsions, sweetish breath odor, abdominal pain, and joint symptoms. She also had increased urine coproporphyrin, decreased blood histamine, increased serum iron and zinc levels. Administration of zinc sulfate and manganese orally, as well as very high doses of pyridoxine normalized the histamine level and brought about recovery from psychotic symptoms and cessation of seizures. Discontinuance

348

H. A. PETERS, D. J. CRIPPS, AND H. H. REESE

of medication brought relapse within 48 hours but recovery on reinstitution of treatment. Thus, zinc may be described as a friend or a foe, leading to the possible conclusion that it is the manner in which these cations are being handled by the body that determines their useful or pathological effect. The cation balance and metabolic studies described by Pfeiffer’s group with schizophrenics need to be extended and applied further to both the schizophrenic and the porphyric patients with psychosis. The early reports are impressive, and this approach could greatly further our knowledge of porphyria, cation metabolism, and the etiological factors in dissociative disorders. In summary, we have seen evidence that the natural porphyrin substances and precursors are not toxic in themselves, but that protoporphyrin and coproporphyrin naturally occur in the white matter of the central nervous system and in the peripheral nerves. Symptoms of acute intermittent porphyria can be explained on the basis of a deficiency of needed essential porphyrins that have (a) been withdrawn from the nervous system for purposes of chelation, (b) have failed to be replenished in the nervous system because of a n enzymatic block (uroporphyrinogen synthetase) in the synthetic pathway leading to protoporphyrin and coproporphyrin 111. T h e patient with AIP is vulnerable because this block exists, so that hepatotoxic agents that would not affect normal patients may be sufficient to seriously impair the supply of needed metabolites. The elevation of urinary zincland or copper in the acute stage of AIP when the patient is developing serious neurological and psychiatric symptoms is also seen in polyarteritis, delirium tremens, toxicity due to lead, mercury, or arsenic poisoning. This metal diuresis in those conditions may thus reflect an exhaustion of the body’s natural chelating ability. Altered tryptophan metabolites, depletion of SH levels (glutathione), and withdrawal of porphyrins may all be last-ditch efforts by the body to make up this deficit. Uncomplexed metals, e.g., zinc and copper, may be responsible for the enzymatic block in porphyrin synthesis as well as difficulties in other metabolic pathways. Chelation therapy may act by removing these enzymatic blocks. ACKNOWLEDGMENTS I wish to thank Ray Brown, Steve Kornguth, Michael Stein, and Laura Koplow for their kind assistance.

REFERENCES Arnold, H. L. (1969). Struub. Clin. Proc. 35, 115-117. Astley, C. E., and Williams, A. A. (1956). Lancet 1, 860. Bariety, M., Gajdos, A., Gajdos-Torok, M., Thibault, P., and Leymarios, J. (1960). Prcsse Med. 68, 825.

PORPHYRIA

349

Barnes, G. (1959). S. Afr. Med. J. 33, 274. Bashour, F. A. (1955). Bull. Minn. Hosp. Med. Found. 26, 423. Batchelor, R. P., Fehnel, J. W., Thomson, R. M., and Drinker, K. R. (1926). J. Znd. Hyg. Toxicol. 8, 322. Batchelor, T. M., McCall, M., and Mosher, R. M. (1964). J. Amer. Med. Ass. 187,305-306. Baumstark, E. (1874). Arch. Ges. Physiol. Menschen Tiere 9, 568. Becker, E. T. (1965). Arch. Dermatol. 92, 252-256. Becker, J. (1961). “Akute Porphyrie Und Periartereitis Nodosa In Der Neurologie.” Springer-Verlag, Berlin and New York. Bentz, H. E. A., and Bersohn, B. (1959). S. Afr. Med. J. 33, 939-944. Berger, H., and Goldberg, A. (1955). Brit. Med. J . 2, 85. Blanshard, T. P. (1953). Proc. SOC.Exfi. Biol. Med. 83, 512. Bleiberg, J. (1964). Arch. Dermatol. 89, 793-797. Bogorad, L. (1958). J . Biol. Chem. 233, 510. Bonkowsky, H. L., Tschudy, D. P., Collins, A. et al. (1971). Proc. Nut. Acad. Sci. US.68, 2725-2729. Booij, H. L., and Rimington, C. (1957). Biochem. J . 65, 4P. Bray, W. E. (1962). “Clinical Laboratory Methods,” 6th, ed., p. 47. Mosby, St. Louis, Missouri. Brehm, G., and Holzmann, H. (1964). Klin. Wochenschr. 42, 283-286. Brown, E. G. (1958). Nature (London) 182, 313. Brunsting, L. A., Mason, H. L., and Aldrich, R. A. (1951). 3. Amer. Med. Ass. 146, 1207. Calvy, G. C., and Dundon, C. C . (1952). Radiology 58, 204-208. Cam, C. (1957). Nester Vol. 1, No. 2. Cam, C. (1959). Dirum Vol. 34, p. 11. Cam, C., and Nigogosyan (1963). J . Amer. Med. Ass. 183, 88. Cambiaggi, A. (1957). A M A Arch. Ophthalmol. [N.S.] 57, 451-453. Cetingil, A. I., &en, M. 1. (1960). Blood 16, 1002. Colomb, D. (1957). Bull. SOC.Fr. Dermatol. Syphiligr. 64, 420-42 1. Cowger, M. L., LabbC, R. F., and Sweell, M. (1963). Arch. Biochem. Biophys. 101, 96. Cripps, D. J., and Curtis, A. C . (1962). Arch. Dermatol. 86, 575-581. Cripps, D. J., and Peters, H. A. (1970). Arch. Neurol. 23, ’79-84. Davies, M. J., and Vander Ploeg, D. E. (1957). Arch. Dermatol. 75, 796. Dean, G., and Barnes, H. D. (1958). Brit. Med. J . 1, 298-300. Dean, G., and Barnes, H. D. (1959). S. Afr. Med. J . 33, 246. Dean, S. (1971). “The Porphyrias-A Story of Inheritance and Environment,” 2nd ed. Pitman, London. Delangen, C. D., and Grotepass, W. (1941). Acta. Med. Scand. 106, 168-181. Denny-Brown, D., and Sciarra, D. (1945). Brain 68, 1. Derrien, E., and Benoit, C. (1929). Arch. SOC.Sci. Med. Biol. Montpellier 8, 456. DeWys, W., Pories, W. J., Richter, M. C., and Strain, W. H. (1967). Lancet 1, 121. Diamond, I., McDonagh, A. F., Wilson, C. B., Granelli, S. G., Nielsen, S., and Jaenicke, R. (1972). Lancet 1175-1 177. Dobriner, K. J. (1937). J . Biol. Chem. 120, 115. Dobrschansky, M. (1906). Wien. Med. Presse 47 2145. Dogramaci, I., Wray, J. E., Ergene, T., Sezer, V., and Muftu, Y. (1962). Turk. J . Pediut. 4, 138. Donald, G. F., Hunter, G. A., Roman, W., and Taylor, E. J. (1965). Aust. J . Dermatol. 8, 97-1 15. Donald, G. F., Hunter, G. A., Roman, W., Phil, D., and Taylor, A. E. J. (1966). Arch. Dermatol. 93, 392.

350

H. A. PETERS, D. J. CRIPPS, AND H. H. REESE

Dowle, E. B., Mustard, P., and Eales, L. (1967). S. tlfr. M e d . J . 41, 1093-1096. Drury, R. A. B. (1956). J . Pafhol. Bacferiol. 71, 211. Edman, K. A. P. (1960). Act4 Physiol. Scand. 49, 330. Elder, T., and Mengel, C. E. (1966). Amer. J. M e d . 41, 369. Ellinger, P., and Dojmi, L. (1935). J. Sod. Chem. Ind., London 3, 507. Epstein, J. H., and Redeker, A. G. (1968). iV.Engl. J. M e d . 279, 1301-1304. Epstein, J. H., Epstein, N. N., and Greenlee, M. (1959). -4MA Arch. Dermatol. 80, 713. Felsher, B. F., and Redeker, A. G. (1966). Medicine (Baltimore) 45, 575-583. Fenton, J. C. B. (1955). Proc. Roy. Sac. Ser. B 143, 279-280. Fikentscher, R. 2. (1935). Z. Geburtsh. Gynockol. 111, 210. Fischer, H., and Zerweck, W. (1924). Hoppe-Syler’s Z . Physiol. Chem. 132, 12. Foreman, H. (1960). “Metal Binding in Medicine” (M. .J. Seven, ed.), Lippincott, Philadelphia, Pennsylvania. Gajdos, A., and Gajdos-Torok, M. (1961). Lancet 2, 175-177. Galambos, J. T., and Dowda, F. W. (1959). Amer. J . M e d . 5 , 803-806. Galambos, J. T., and Peacock, L. B. (1959). .4nn. Intern. M e d . 50, 1056. Garrod, X. E. (1923). “Inborn Errors of Metabolism,” 2nd ed. Frowde, London. Gibson, J. B., and Goldberg, A. (1956). J. Pathol. Bnclpriol. 71, 495-509. Gibson. J. B., Parkes, W. C., and Brennan, C. F. (1957). J. Ir. M e d . Sci. 3, 137-138. Gibson, K. D. (1955). Porphyrin Biosyn. Metabol., Ciba Found. Symp., 1955 pp. 27-39. Gibson, K. D., Laver, Q. G., and Neuberger, A. (1958). Biochem. J. 70, 71-81. Gilardi, A,, Djavadi-Ohaniance, L., Labbt?, P., and Chaix, P. (1971). Biochem. Biophys. dcta 234, 446457. Ginsburg, A. D., and Dowle, N. B. (1963). S.Afr. J. Lab. Clin. M e d . 9, 206. Gitlin, N. (1969). Brit. M e d . J. 1, 9G98. Glenn, B. L., Glenn, G. G., and Omtvedt, I. T. (1968). Amer. J . Vet. Res. 29, 1653-1657. Goldberg, A. (1954). Biochem. J. 57, 55. Goldberg, A. (1959). Quart. J. M e d . [N.S.] 28, 183-209. Goldberg, A., and Rimington, C. (1955). Proc. Roy. Soc., Ser. B 143, 257-280. Goldberg, A., and Rimington, C. (1962). “Diseases of Porphyrin Metabolism.” Thomas, Springfield, Illinois. Goidberg, A., Paton, W. D. M., and Thompson, J. W. (1954). Brit. J. Pharmacol. 9, 91. Goldberg, A., Ashenbrucher, H., Cartwright, G. E., and Wintrobe, M. M. (1956). Blood 11, 821. Goldberg, A., Moore, M. R., Beattie, A. D., Hall, P. E., McCallum, J., and Grant, J. K. (1969). Lancet 115-118. Goldberg, A. M. (1955). Biochem. J. 59, 37-44. Goldstein, N. P., Martin, W. J., Brunsting, L. A., and Kirby, T. J. (1957). Proc. Staff Meet. iLfayo Clin. 32, 82-88. Granick, S., and Urata, G. (1963). J. B i d . Chem. 238, 821. Granick, S., and Vanden Schrieck, H. G. (1955). Proc. Soc. Exp. B i d . M e d . 88, 270. Greaves, M., and Boyde, T. R. C. (1967). Lance# 1019-1020. Greiner, A. C., and Nicholson, G. A. (1964). Can. itfed. Asso. J. 91, 627. Greiner, A. C., and Nicholson, G. A. (1965). Lancef 1165-1 167. Greiner, A. C., and Nicolson, G. A. (1966). Lancet 1, 344-347. Grinstein, M., Aldrich, R. A., Hawkinson, V., Lowry, P., and Watson, C. J. (1951). Blood 6,699-705. Grinstein, M., Bannerman, R. M., and Moore, C. (1959). Blood 14, 476. Gubler, C. J. (1956). J . Amer. Med. Ass. 161, 609. Gulbrandsen, R., and Wigmostad, K. R. (1958). Tidskr. Nor. Luegeforen. 9, 420. Giinther, H. (1911). Deuf. Arch. Klin. Med. 105, 89-146.

PORPHYRIA

35 1

Gunther, H. (1922). Ergeb. Allg. Pathol. Pathol. Anat. 20, 608. Halsted, J. A., and Smith, J. C. (1970). Lancet 322-324. Hamilton, R. D. (1971). Science 174, 172. Hansotia, P., Peters, H., Bennett, M., and Brown, R. (1969). Ann. Otol. Rhinol., Laryngol. 78, 388. Harley, V. (1890). Brit. Med. J . 2, 1169. Harmel-Tourneur, L., and Dropsy, G. (1959). SOC.Dermatol. Syph. 21, 545. Hedger, R. W., and Hardison, W. G. M. (1971). Gastroenterology 60, 903-907. Hellman, E. S., Tschudy, D. P., and Bartter, F. C. (1962). Amm. J. Med. 32, 734. Helmchen, H., Hippius, H., Hoffmann, I., and Selbach, H. (1967). Nnvenarzt 38,218-220. Hierons, R. (1957). Brain. 80, 176. Hiraoka, H. (1966). Bull. Yamaguchi Med. Sch. 13, 269-280. Hobbs, H. E., Sorsby, A., and Freedman, A. (1959). Lancet 2, 478-480. Holzmann, H. (1963). Med. Welt pp. 1078-1081. Hopmann, R. (1968). Deut. Med. Wochenschr. 93, 76-81. Hoppe-Seyler, F. ( I 871). Med.-Chem. Unters. ABT. 1-4-528. Hunt, A. H., Parr, R. M., Taylor, D. M., and Trott, N. G. (1963). Brit. Med. J. 2, 14981501. Hunter, G. A. and Donald, G. F. (1970). Brit. J . Dermatol. 82, 205. Huszak, I., and Durko, I. (1962). Psychiat. Neurol. 143, 407-415. Huszak, I., and Durko, 1. (1968). Proc. IV World Congr. Psychiat., pp. 2931-2933. Iber, F. L., Taxay, E., Nassau, K., and Plough, I. C. (1956). N. Engl. J. Med. 255, 86-88. Il’in, L. L. (1963). Vestn. Dermatol. Venerol. 37, 42-47. Ippen, H. (1961). Deut. Med. Wochenschr. 86, 127. Jaffe, I. A. (1963). Ann. Rheum. Dis. 22, 71-76. Jaffe, I. A. (1970). Arthritis Rheum. 13, 436-443. Kalivas, J. T., Pathak, M. A., and Fitzpatrick, T. B. (1969). Lancet 1184-1 187. Kallai, L., Keler-Bacoka, M., Marinkovic, M., Knezevic, S., Stojanovski, A., and Kosutic, Z. (1968). Schweiz. Med. Wochenschr. 98, 1007-1009. Kanig, K., and Breyer, U. (1969). Pharmakopsychiatrie 2, 190-201. Kappus, A., Song, S., Levere, R. D., and Granick, S. (1969). Med. Sci. 64, 557-564. Kaufman, L., and Marver, H. S. (1970). N. Engl. J. Med. 283, 954-958. Kebe, S. R. (1963). West. Med. 2, 46-48. Keilin, D. (1945). Science 101, 540. Kimura, K., and Kimura, J. (1965). Proc. Jap. Acad. 41, 943. Kluver, H. (1944). Science 99, 482. Kluver, H. (1954). Proc. 1st Internat. Neurochem. Symp., Magdalen College, Oxford, Academic Press, New York. Knudsen, K. B., Sparberg, M., and Lecocq, C. F. (1967). N. Engl. J . Med. 277, 350-351. Kowertz, M. J. (1973). J. Amer. Med. Ass. 233, 515-519. Kreimer-Birnbaum, M., Mosovich, L. L., and Bannerman, R. M. (1971). J . Med. 2, 149166. Krouch, R. B., and Hermann, G. R. (1955). Amer. Heart J. 49, 693-695. Labbt, R. F., Hanawa, Y., and Lottsfeldt, F. I. (1961). Arch. Biochem. Biophgs. 92, 373. Lageder, K. (1934). Arch. Verdau.-Kr. 56, 237. Landouzy and Maument (1850). Gar. Med. Paris 5, 409. Lang, P. A., Levine, H., Jones, C. C., and Mills, G. C. (1968). Tex. Rep. Biol. Med. 26, 525-534. Lees, R. S., Song, C. S., Levere, R. D., and Kappas, A. (1970). N. Engl. J . Med. 282, 432-433.

352

H. A. PETERS, D. J. CRIPPS, AND H. H. REESE

LeNobel, C. (1887). Arch. Ger. Phys. 40, 501. Levine, J. B., Aamodt, R., Tschudy, D. P., and Henkin, R. (1973). J . Clin. Invest. 52, 52a (Abstr. No. 188). Levine, W., Sernatinger, E., Jacobson, M., and Kuntzman, R. (1972). Science 176, 1341. Levit, E. J., Sodine, J. H., and Perloff, W. H. (1957). Amer. J. Med. 22, 831-833. Linden, S. H., Steffen, C. G., Newcomer, V. D., and Chapman, M. (1954). Calif. Med. 81, 235. Lockhead, A,, and Goldberg, A. (1961). Biochem. J. 78, 146. London, I. D. (1957). A.1414 Arch. Dermatol. 75, 801. Lorber, 4.,Pearson, C. M., Meredith, W. L., and Gantz-Mandell, L. E. (1964). Ann. Intern. Med. 61, 423-434. Luby, E. D., Ware, J. G., Senf, R., and Frohman, C. E. (1959). Psychosom. Med. 21, 34-39. Ludwig, G. D., and Goldberg, M. (1963). -4nn. N.Y. Acnd. Sci. 104, 710. McCabe, E. S. (1955). Arnn. Practitioner 6, 878. Machfahon, R. A., Parker, M.L., and McKinnon, M. C. (1968). Med. J. Aurt. 2,210-212. MackIunn, C. A. (1880). Proc. Roy. SOG.,Ser. B 31, 206. Magnus, I. A., Jarrrtt, A., Prankerd, T. A. J., and Rirnington, C. (1961). Lancet 2, 448. Marsden, G. W. (1959). Brit. J. Dermatol. 71, 219-222. Mason, V. R., Courvillc, C., and Ziskind, E. (1933). Medicine (Baltimore) 12, 355. Mauzerall, D., and Granick, S. J. (1956). J. Biol. Chenz. 219, 435. Mellinkoff, S. M., Halpern, R. M., Frankland, M., and Greipel, M. J. (1959). J . Lab. Clin. Med. 53, 35a363. Mentz, H. E. A., and Bersohn, I. (1959). S. Afr. Med. J. 33, 939-944. Messert, B., and Baker, N. H. (1967). J . NeuroE. 17, 6. Meyer, H., Nadler, S. B., and Bloch, T. (1954). Obstet. Gynecol. 3, 214. Meyer, U. A., and ‘Marver, H. S. (1971). Clin. Res. 19, 398. Meyer, U. R., Strand, J., Doss, M., Rees, A. C., and Marver, H. S. (1972). N . En& J. Xfed. 286, 1277-1282. hleyer-Betz, F. (1913). Deut. Arch. Klin. Med. 112, 476. Miyhei, K., and Watson, C. J. (1972). Blood 39, 1. Xlontgomery, &I. L., Shelinc, G. E., and Chaikoff, I. L. (1943). J. Exp. Med. 78, 151-159. Moskowitz (1960). In ‘‘Metal Binding in Medicine” (M. J. Seven, ed.). p. 190. Lippincott, Philadelphia, Pennsylvania. Mulder, G. J. (1844). J. Prakt. Chem. [l] 32, 186. Muller, S. E., Fravel, C. R., and Esmond, W. (1956). Ann. Intern. Med. 45, 288-298. Multicentre Trial Group. (1973). Lancet 1, 275-280. Murphy, J. \’. (1970). J . h e r . Med. Ass. 212, 2119-2120. Murty, H. S., Pinelli, A., Nair, P. P., and Mendeloff, A. I. (1969). Clin. Res. 17, 474. Mustajoki, P. (1972). J . Amer. Med. Ass. 211, 714-715. Nair, P. P., Murty, H. r\.> and Grossman, N. R. (1970). Biochim. Biophys. Actu 215, 112118. Nakao, K., Wada, O., Kitamura, T., Uono, K., and Urata, G. (1966). Nature (London) 210, 838. Weilson?D. K., and Neilson, R. P. (1957). Trans. Pa. Coast Obstet. Gynecol. SOC.25, 71-90. Nesbitt, S. (1943). Arch. Intern. Med. 71, 62. Nesbitt, S. (1944). J . Amer. Med. Ass. 124, 284-294. Nesbitt, S., and Snell, A. M. (1942). Arch. Intern. Med. 69, 573. Nicolson, G. A. (1966). Lancet 1, 344. Nielson, B., and Thorn, N. A. (1965). A m . J. Med. 38, 315.

PORPHYRIA

35 3

Oaks, W. W., Schultz, J., and Fleishmajer, R . (1969). Dermatologica 138, 10-18. Okun, E., Gouras, P., Bernstein, H., and von Sallman, L. (1963). Arch. Ophthalmol. 69, 59-7 1. Olson, E. S. (1971). Wis. Med. J. 70, 109-110. Olsson, R. A., and Ticktin, H. E. (1962). J . Lab. Clin. Invest. 60, 48-52. Orten, J. M. (1966). “Zinc Metabolism,” pp. 38-47. Thomas, Springfield, Illinois. Page, E. W., Glendening, M. B., Dignam, W., and Harper, H. A. (1954). Amer. J. Obstet. Gynecol. 68, 110. Painter, J. T., and Morrow, E. J. (1959). Tex. J . Med. 55, 811-818. Paul, K. G., and Thyresson, N. (1954). Acta Dermatol. Venereot. 34, 403. Perlroth, M. G., Marver, H. S., and Tschudy, D. P. (1965). J . Amer. Med. Ass. 194, 10371042. Perlroth, M. G., Tschudy, D. P., Waxman, A., and Odell, W. D. (1967). Metabolism 16, 87-90. Peters, H. A. (1954). Neurology 4, 477. Peters, H. A. (1956). Dis. Neru. Syst. 17, 2-8. Peters, H. A. (1960). In “Metal Binding in Medicine” (M. J. Seven, ed.), p. 190. Lippincott, Philadelphia, Pennsylvania. Peters, H. A. (1961). Fed. Proc., Fed. Amer. SOC.Exp. Biol. 20, 227-234. Peters, H. A., Woods, S., Eichman, P. L., and Keese, H. H. (1957). Ann. Intern. Med. 47, 889-899. Peters, H. A., Eichman, P. L., and Reese, H. H. (1958). Neurology 8, 621-632. Peters, H. Johnson, S. A. M., Cam, S., Oral, S., Muftu, Y.,and Ergene, T. (1966). Amer. J . Med. Sci. 251, 104-1 12. Petres, H., Schroder, K., Consbruch, U., and Ladner, H. A. (1967). Deut. Med. Wochenschr. 34, 1498-1502. Pfeiffer, C. C. (1972). Rev. Can. Biol. 31, Suppl., Printemps, 73-76. Porteous, C. R. (1963). J . Obstet. Gynecol. 70, 311-314. Posner, J. B., Ertel, N. H., Kossman, R. J., and Scheinberg, L. C. (1967). Arch. Neural. 17, 530-541. PoswiIlo, D. E., and Cohen, B. (1971). Nature (London) 231, 447-448. Price, J. M., Brown, R. R., Rukavina, J. G., Mendelson, C., and Johnson, S. A. M. (1957). J . Invest. Dermatol. 29, 289. Price, J. M., Brown, R. R., and Peters, H. A. (1959). Neurology 9, 456-468. Proger, S., and Dekaneas, D. (1947). Bull. N . Engl. Med. Cent. 9, 2 11. Prunty, F. T. G. (1946). A M A Arch. Intern. Med. 77, 623-642. Raedeli, A. (1962). Dermatologia 13, 17. Ranking, J. E., and Pardington, G. L. (1890). Lancet 2, 607. Redeker, A. G., Sterling, R. E., and Archer, B. (1959). A M A Arch. Intern. Med. 104, 779782. Redeker, A. G., Sterling, R. E., and Bronow, R. S. (1964). J. Amer. Med. Ass. 188,466-468. Riederer, J. (1961). Med. Klin. (Munich) 56, 96. Rimington, C. (1959). Proc. Roy. SOC.Med. 52, 963. Roman, W. (1967a). Enzymologia 31, 37-82. Roman, W. (1967b). Enzymologia 32, Fasc. 1. Roman, W. (1969). Amer. J . Clin. Nutr. 22, 1290-1363. Roman, W., Oon, R., West, R. F., and Reid, D. P., (1969). Med. J. Aust. 1, 633. Rukavina, J. G., Mendelson, C., Price, J. M., Brown, R. R., and Johnson, S. A. M. (1957). J. Inuest. Dermatol. 29, 273. Sacher, A, (1893). Arb. Pharmakol. Zwt. Dorpat, Stuttgart p. 88.

354

€3. A. PETERS, D.

J. CRIPPS, AND H. H . REESE

Saint, E. G., Curnow, D., Paton, R., and Stokes, J. B. (1954). Brit. Med. J. 1, 1182-1 184. Saltzer, E., Redrker, A. G., and Wilson, J. W. (1968). Arch. Dermatol. 98, 496-498. Saunders, S. .J. (1963). S. Afr. J. Lab. Clin. Xfed. 9, 277-283. Scherer, J. (1841). Ann. Chem. Pharm. 40, 1. Schirger, A., Ifartin, W. J., Goldstein, X. P., and Huizcnga, K. A. (1962). Proc. Staff .%feet. ,%fayoClin.37, 7-1 1. Schley, G., Bock, K. D., Debusmann, E. R., Hocevar, V., Paar, D., and Rausch-Stroomann, J. G. (1970). Klin. Wschr. 48, 616-623. Schmid, R. (1960).
PORPHYRIA

355

Turner, W. J. (1937). J . Biol. Chem. 118, 519. Tyler, A. (1953). B i d . Bull. 104., 224-239. Vail, T. J. (1967). J . Amer. Med. Ass. 201, 674. Vallee, B. L. (1956). J . Amer. Med. Ass. 162, 1053. Vallee, B. L. (1959). Physiol. Rev. 39, 443-490. Vannotti, A. (1954). “Porphyrins, Their Biological Importance.” Hilger & Watts, London. Volger, W. R., Galambos, J. T., and Olansky, S. (1970). Amer. J . Med. 49, 316-321. Waldenstrom, J. (1937). Acta Med. Scand. Suppl. 82. Waldenstrom, J. (1957). Amer. J . Med. 22, 753-773. Waldenstrom, J., and Vahlquist, B. (1939). Hoppe-Seyler’s 2.Physiol. Chem. 260, 189-209. Watson, C. J. (1935). J. Clin. Invest. 14, 116. Watson, C. J. (1946). Blood 1, 99. Watson, C. J. (1952). In “Diseases of Metabolism: Detailed Metlibds of diagnosis and Treatment: Text for the Practitioner.” (G. G. Duncan, ed.), 3rd ed., pp. 1079-1 101. Saunders, Philadelphia, Pennsylvania. Watson, C. J. (1954). Advan. Inter. Med. 6,235-299. Watson, C. J. (1960). N . Engl. J . Med. 263, 1205. Watson, C. J. (1963). In “Cecil-Loeb Textbook of Medicine” (R. Cecil and R. F. Loeb, eds.), pp. 1266-1271. Saunders, Philadelphia, Pennsylvania. Watson, C . J. (1973). Ann. Intern. Med. 79, 80-83. Watson, C. J., and Larson, E. A. (1947). Phy. Rev. 27, 478. Watson, C. J., and Schwartz, S. J. (1941). J. Clin. Invest. 20, 440. Watson, C. J., Schwartz, S., Schulze, W., Jacobson, L. O., and Zagaria, R. (1949). J . Clin. Invest. 28, 465. Watson, C. J., Lowry, P. T., Schmid, R., Hawkinson, V. E., and Schwartz, S. (1951a). Trans. Ass. Amer. Physicians 64, 345. Watson, C. J., Sutherland, D., and Hawkinson, V. (1951b). J . Lab. Clin. Med. 37, 8. Watson, C. J., Jeelani Dhar, G., Bossenmaier, I., Cardinal, R., and Petryka, Z . J. (1973). Ann Intern. Med. 79, 80-83. Waxman, A. D., Schalch, D. S., Odell, W. D., and Tschudy, D. P. (1967). J . Clin. Invest. 46, 1129. Waxman, A. D., Berk, P. D., Schalch, D., and Tschudy, D. P. (1969). Ann. Intern. Med. 70, 3 17-323. Wayne, E. J., Kontras, D. A., and Alexander, W. D. (1964). “Clinical Studies in Iodine Metabolism.” Blackwell, Oxford. Welland, F. (1964). Metabolism 13, No. 3. Whisnant, J. P., Espinosa, R. E., Kierland, R. R., and Lambert, E. H. (1963). Proc. Staff Meet. Maya Clinic 38, 501-514. Whittaker, S. R. F., and Whitehead, T. P. (1956). Lancet 1, 547. Wirtschafter, J. D., Turner, F. W., and Dow, R. S . (1960). Neurology 10, 787. With, T. K., and Peterson, H. C. A. (1954). Lancet 2, 1148-1157. Woodbury, J., Lyons, K., Carretta, R., Hahn, A., and Sullivan, J. F. (1968). Neurology 18,700-705. Woods, S., Peters, H. A., and Johnson, S. A. M. (1958). A M A Arch. Dermatol. 77, 559-567. Woods, S., Peters, H. A., and Johnson, S. A. M. (1961). Arch. Dermatol. 84, 920-927. Zeile, K., and Brugsch, J. T. (1934). Muenchen. Med. Wochenschr. 11, 2021. Zimmerman, E. A. (1968). Bull. Sloan Hosp. Women X I V , 14-21. Ziprkowski, L., Szeinberg, A., Crispin, M., Krakowski, A., and Zaidman, J. (1966). Arch. Dermatol. 93, 21-27.

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SUBJECT INDEX A

Acetylcholine (ACh) cardiovascular effects of, 67-68 effects on cerebral RNA metabolism, 217-218, 220,221 muscarinic receptors of, 67-68 nicotinic receptors of, 3-4, 67-68 bioeIectric measurements on, 17-32 Acute intermittent porphyria (AIP), 149ff, 305 symptoms and incidence of, 304-308 Adenoma, hepatic, porphyria from, 305,306, 308, 318, Adenosine monophosphate, in porphyria therapy, 334-335 Alcoholism amnesia in, 2 7 3-2 74 porphyria with, 177, 314-315 trace metal diuresis in, 344-346 urinary excretion in, 342 Amanita muscaria, psychomimetic agents of, 158 Amino acids, excretion of, in porphyria, 325 6 -Aminolevulinic acid, in porphyrin pathway, 319 6 -Aminolevulinic acid synthetase, overactivity of, in porphyria, 319-320, 323,333 Aminopentamide, as antiacetylcholine drug, 75 Amnesia, use to study memory, 273-2 7 6 CAMP effects on cerebrd RNA, 219 in membrane hyperpolarization, 32, 55 Antiacetylcholine drugs, 67-144 behavioral studies on, 116-125 chemical evolution of, 7 1-77 conformation features related to activity of, 97-100

crystallographic studies on, 100 derived from avtylcholine-like drugs, 73 factors influencing activity of, 105-111 metabolism of, 133-137 peripheral effects of, 68 pharmacoIogicaI methods used in studies of, 137-139 potencies of, in CNS and PNS, 111-116 structure-activity relationship in, 7 6-7 7 quantitative studies, 101-1 05 stereochemical aspects, 77-100 uses of, 69 Anticholinesterase, poisoning by, antiacetylcholine drug treatment of, 70, 125-133 Anticonvulsan t(s) kryptopyrrole as, 167 porphyria induced by, 314 Association cortex, evolution of, 244-250 Astrocytes, RNA synthesis in, 191 Atropine in anticholinesterase poisoning, 126, 130 behavioral studies on, 117-119 drugs derived from, 72 metabolism of, 134-135 pharmacology of, 7 1, 92 structure of, 72 Axons RNA in, 191-192 transport, 192-193

357

B Bantu porphyria, 305 symptoms of, 308 Barbiturates, porphyria induced by, 3 13

3.58

SUBJECT INDEX

Batrachotoxin. neurobiology of, 149, 172 structure of, 1 7 1 Belladonna, toxic psychosis from, 68 Behavior cerebral R X A effects on, 208-210 cortical function and, in primates, 2 60-289 in studies of brain anatomy, 237-238 Benapyrazine, as antiacetylcholine drug, 114-1 15 Benzetimide, as antiacetylcholine drug, 7 4 , 8 3 metabolism of, 136, 137 Benzhexol, as antiacetylcholine drug, 82,88 Benzilylcholine, as antiacetylcholine drug, 73 Benzodiazepines, neurobiology of, 158 Riogenic amine, effects on brain RNA, 217-219 Biological receptors, drug activity and, 78 Blood, pyrrole determination in, 159-1 60 Blood-brain barrier, pyrrole determination in, 160 Brain, (see nlso Cortex) anatomy of. 237-260 asymmetry of function of, 278-280 evolution of, 239-240 information exchange between hemispheres of, 280-281 information storage in, theoretical model, 222-228 of primates, cell density, 250-25 1 RNA metabolism in, 183-231 size evolution of, 939-240,243, 247 -248

C Cancer, tropical monopyrrole role in, 174 Caramiphen in anticholinesterase poisoning, 126, 127,130 structure of, 127

Carbachol, as antiacetylcholine drug, 112 Carbamylcholine, effects on cerebral RNA, 221 Cell density, in primate brains, 250-25 1 Central nervous system, antiacetylcholine drug effects on, 1 1 1-1 16 Cerebrospinal fluid, pyrrole determination in, 160 Chelating agents, in porphyria therapy, 309 Chelation therapy, of porphyria, 327-335 Chiari's syndrome, monopyrrole role in, 173 Chimpanzee, communication ability in, 284-287 Cholestyramine, in porphyria therapy, 338 Chlormethiazole psychoactivity of, 149 structure of, 150 p-Chlorophenylalanine, effects on cerebral RNA, 221 Chloroquine, in porphyria therapy, 336-338 Cirrhosis, Jamaican, monopyrrole role in, 1 7 4 Classification, in learning process, 266-269 Cobalamins, neurobiology of, 149 Coffee, pyrroles in, 175 Collagen diseases, monopyrrole role in, 173-175 Contraceptive agents, in porphyria therapy, 3.10 Copper, in porphyria etiology, 323, 340-348 Coproporphyria, hereditary, 305, 306 symptoms and incidence of, 307,321 Cortex anatomy of, 237-260 association area of, 244-250 Cortical functions, in primates, 233-299 anatomical aspects, 233-260 behavior, 260-289

SUBJECT INDEX

359

in anticholinesterase poisoning, 128, cross-modal abilities and, 253 130 lateralization of, 252-253 DNA, in neurobiology of pyrroles, 15 1 Corticosteroids, in induction of Drug potency, equation for, 101 pyrroloxygenase, 178 Dzhalangarsk encephalitis, monopyrCruetine, see Porphyrin role in, 173-175 Curare, effects on nicotinic effects of ACH, 28-29,30 E Cutaneous hepatic porphyria, types of, 305 Edathamil, in porphyria therapy, Cytochrome, in porphyria therapy, 335 32 7-332 Electric shock, effect on memory, 276-27 7 D Electrolytes, role in prophyria, 324-325 Deiters’ cells, RNA in, 190 Erythropoietic porphyria, 305, 306 Depolarization-hyperpolarizationcycle symptoms and incidence of, 307 acetylcholine and receptors in, 3-32 Erythropoietic protoporphyria, 305, current concepts of, 50-53 306 effect of various agents on, 30, symptoms and incidence of, 306 48-49 4-Ethylpyrazole hyperpolarization phase, 32-49 structure of, 150 cause of, 55-58 as tranquilizer, 149 membrane depolarization in, 5-6 Eye, kryptopyrrole effects on, membrane hyperpolarization in, 32 166-167 proposed model for, 2 Desferrioxime, in porphyria therapy, 335 F Diabetes, porphyria with, 314 Fasting, porphyria induction by, 315 Diazepam, kryptopyrrole compared to, 167 G Dibutyryl adenosine cyclic AMP, effects G3063, in anticholinesterase poisoning, on cerebral RNA, 221, 222 130 Dikryptopyrryl ether, in kryptopyrGanglion cells, RNA in, 189 role chromatography, 155-15 6 Glucose, in porphyria therapy, 1 7 7 Dimercaptopropanol, in porphyria therapy, 32 7 -332 H Diphosphoinositide (DPI), from triphosphoinositide dephosphory- Hansch equation, 102 Harmine, 1 7 6 lation, 58-59 Hematin, in porphyria therapy, 333 Diphosphoinositide kinase Hematoporphyrin as CAMPreceptor, 32-49 neurobiology of, 149 assay of, 35-36 toxicity of, 322 preparation of, 33-35 Hexachlorobenzene, porphyria inducDiscrimination, in learning process, 265-266 tion by, 308, 315 Histidine, in porphyria therapy, Ditran as antiacetylcholine drug, 119 332-333

3 60

SUBJECT INDEX

Hominids cranial capacities of, 257 fossil type, encephalometric indexes for. 248 Hoover sign, in porphyria, 309 Hormones, porphyria induced by, 3 12-3 13, 315-3 16 Horn cells, RN.1 in, 190 Hydrocortisone, effects on cerebral RNA, 221 Hyoscine as antiacetylcholine drug, 79, 92 behavioral studies, 118-124 stereochemistry, 9 6 in mtiacetylcholinesterase therapy, 130 metabolism of, 136 Hyoscyamine as antiacetylcholine drug, 78, 79 behavioral studies, 117-118 stereochemistry, 95-96 Hyperprolinemid, monopyrrole role in, 173-17.5 Hypoglossal cells, R K A in, 190 Hysterics, porphyrics as, 177

biochemistry of, 158-165 in blood, 159-160 chromatography of, 15 1-153 anomalies in, 153-156 excreted, biological sources of, 163-165 eye effects of, 166-167 lactams of propentdyoperits compared to, 156-157 molecular neurobiologv of, 145-182 neuroelectrical effects of, 167-1 68 oxidation products of, 153 biological activity, 157- 1 5 8 pharmacology of, 165-168 in porphyria, 160-1 62 in psychiatric disorders, 178 pyrrolooxygenases and, 162-163, 178 structure-activity studies on, 147-15 1 toxicity of, 165-166

L

Language brain structure and, 25 1-252 areas for, 254 I size factors, 254-257 Ibogaine, 176 importance of, 290-29 1 Indoles, as pyrroles, 171. 175-176 in nonhuman primates, 284-287 Information storage, in brain, Lateralization, of hemispheric functheoretical model of, 222-228 tion, 277-281 1,earning associative, 271-272 K cerebral RNA and, 21 0-2 1 7 KAO-264, as antiacetylcholine drug, evolution of, 242-25 1 115 neocorticalization and, 264-272 Kava, psychoactive components of, classification, 266-269 I58 discrimination performance, Kernicterus, bile pigments of, 149 265-2 66 Kryptopyrrole lateralization, 27 7-28 1 analytical techniques for, 158-159 Lipid metabolism, in porphyria, anticonvusant action of, 167 325-326 behavioral pharmacology of, 168-1 70 LSD discriminated avoidance studies, neurobiology of, 149 170 in studies of kryptopyrrole food reinforcement studies, 169 pharmacology, 159

361

SUBJECT INDEX

M Macroamylasemia, in porphyria, 32 6-32 7 Malvaria hypothesis, in pyrrole metabol. ism, 150-151 Man cortical functions in, 233-299 evolution of ancestors of, 234 Mandelyltropine, structure of, 72 Mauthner cell, RNA in, 189, 190, 192 “Mauve factor” identification of, 152 in psychiatric disorders, 148, 151 Memory brain information storage and, 222-228 evolution of, 251-252, 273-277 electrical stimulation studies, 276-277 lesion studies, 273-276 Methylcholine derivatives, as antiacetylcholine drugs, 88 Methyl-l,3dioxolans, derivatives of, as antiacetylcholine drugs, 73-74, 85 Methylethylmaleimide as kryptopyrrole oxidation product, 153 structure of, 154 Methylpiperidin-3-01, as antiacetylcholine drug, 99 Microglia, RNA synthesis in, 191 Molindone as antipsychotic agent, 149 structure of, 150 Monopyrroles in clinical psychiatry, 176-178 neurobiology of, 147 structure effects, 147-15 1 Motor functions evolution of, 241-242 neocorticalization and, 262-264 Mydriacyl, as antiacetylcholine drug, 75

N Neocortex, evolution of, 243

Neuronal perikarya, RNA in, 189 Neurons, RNA synthesis in, 19 1 Nitrosero tonin neurobiology of, 149 structure of, 150 Norepinephrine, effects on cerebral RNA, 219

0 Oligodendroglia, RNA synthesis in, 19 1 Ouabain, effects on cerebral RNA, 220 0 x 0tremorine as antiacetylcholine drug, 112-1 13 behavioral studies, 116-1 18 Oxykryptopyrrole lactams, neurobiology of, 149 P

PZS, in anticholinesterase poisoning, 129-13 1 pA,,definition of, 69-70 Paints, porphyria induction by, 315-316 Paravertebral sympathetic chain, isolation of, 18-19 Parkinson’s disease antiacetylcholine drug treatment of, 111 pathogenesis of, 117 Penicillamine, in porphyria therapy, 334 Peripheral nervous system, antiacetylcholine drug effects on, 111-116 Phlebotomy, in porphyria therapy, 336 Pheromones neurobiology of, 149 pyrrolic neurobiology of, 170-172 Piperidin-3-01 esters, as antiacetylcholine drugs, 98 Piperidin-4-0 1 esters, as antiacetylcholine drugs, 98-99 Platyphylline, as antiacetylcholine drug, 75

362

SllBJECT INDEX

PMCG, in anticholinesterase poisoning, 1'78, 130 Poiyarteritis riodostim porphyria and, 341 urinary excretion in, 342 Porphobilinoqen in psychiatric disease, 178 secretion of. in porphyria, 148, 15 1. 172-173 Porphubilinogrn-deaminase, neurobiology of, 162-163 Porph ?ria s) 3 0 1-3 5 5 amino acid excretion in. 325 biorhernipy of, 318-322 cardiorespiratory signs of, 3 10-3 1 1 clinical symptoms of, 308-309 dermatological signs of, 31 1 drug-induced, 3 13-3 14 elec-trolyterole in, 324-325 enLymt:s in, 31 9-322 gastrointestinal signs of, 310 genitourinary Tigns of, 310 q r o w t h hormone role in, 327 histc,ry of, 302-304 hornton~-indiiced,3 12-31 3 incidence of, 304-305 1ipid me tahol ism in, 3 2 5 -3 26 macroamylasemia in, 326-327 neuropathology of, 31 7 onset of, 308 pathogenesis o f , 322-327 pathology of. 316-3 I8 porphyrin pathways in, 31 9 precipitating factors in, 31 1-316 in pregnancy, 31 1-312 psychiatric symptoms of, 309 pyrrole neurobioIoLgy and, 147, 148, 139 - 1 i1, 1 6 0 - 1 62, 1 7 2- 17 3, 177-lT8 renal symptoms of, 3 16-3 17 therapy of. 327-340 by chelation. 327-335 trace metal role in, 323 urinary excretion in, 343 vasculitis in, 317-318 Porphyria cutanea tarda hereditaria. 305, 306

.

Porphyria cutanea tarda symptomatica, 305 symptoms and incidence of, 307 Porphyria variegata, 305, 306 symptoms and incidence of, 307 Porphyrints) disturbed metabolism of, classification, 305-308 excessive excretion of in nonporphyric porphyric disease, 308 role in porphyria, 304 Porphyrin-producing hepatic adenoma, 305,306, 308, 318 Potassium chloride, effects on cerebral RNA, 2 2 0 , 2 2 1 Pregnancy, porphyria in, 3 11-3 12 Primates, cortical functions in, 233-299 Procyclidine, as antiacetylcholine drug, 82.88 Propentdyopents, kryptopyrrole lactams compared to, 156-157 Protocoproporphyria, see Porphyria cutanea tarda hereditaria Pyridoxine, in porphyria therapy, 338-339 Pyrrole-2-carboxylic acid, biological activity of, 175 Pyrroles indoles as, 1 7 1 , 175-176 neuropharmacology of, 146, 170-176 pharmacology of, 165-1 68 Pyrrolizidine alkaloid(s) as antiacetylcholine drugs, 75 metabolites, as neurochemical disease agents, 174 neurobiology of, 149 Pyrrolonitrin, as nonindolic monopyrrole, 175 Pyrrolooxygenases, neurobiology and isolation of, 162-163 role in psychiatric disorder, 178

Q Quinuclidin-3-0 1, as antiacetylcholine drug, 99

363

SUBJECT INDEX

R Renshaw cell bioelectric processes of, 25-26 preparation of, 19 Reserpine, 176 RNA in brain, 183-231 age factors in, 199-203 anatomical and subcellular distribution in, 189 base composition, 187-189, 205 biogenic amine effects on, 21 7-2 19 content, 184-189 drug effects on, 2 19-222 during development, 1997203 function, 206-2 17 hybridization studies, 186-187 information storage and, 222-228 learning and, 2 10-2 1 7 metabolism of, 183-231 metabolism pools, 206 neuronal and glial metabolism, 204 nonlearning behavior and, 208-2 10 physiological and electrical stimulation of, 197-199 synthesis patterns, 204-205 turnover rates, 204 types, 204-206 in neurobiology of pyrroles, 15 1

S Salivation, antagonism of, as test for antiacetylcholine drug, 113 Schizophrenia porphyria and, 304, 309 pyrrole metabolism in, 147, 151, 1 7 7 trace metal metabolism in, 347-348 Sedatives, porphyria induced by, 313 Sense-modalities, information exchange between, 282-284 Sensory functions evolution of, 240-241 neocorticalization and, 260-262

Serotonin effects on brain RNA, 218,221 neurobiology of, 149 Skatole derivatives, neurobiology of, 149 Skin photosensitivity, in porphyria, 302 Solvents, porphyria induction by, 315-316 Spermidine, effects on cerebral RNA, 22 1 Steroids, as inducers of pyrrolooxygenase, 178 Sulfa drugs, prophyria induced by, 314 Superior cervical ganglion, isolation of, 18 T T-35, see 4-Ethylpyrazole TMB4, in anticholinesterase poisoning, 126 Tetrodotoxin, effects on cerebral RNA, 220,221 Thiphenamil, as antiacetylcholine drug, 75 Tobacco, pyrroles in, 175 Tocopherols, in porphyria therapy, 339-340 Trace metal, in porphyria etiology, 323 Tranquilizers, porphyria induction by, 313 Tranylcypromine, effects on cerebral RNA, 220 Tremorine, derivatives of, as antiacetylcholine drugs, 74 Trichodesma toxicosis, see Dzhalangarsk encephalitis Tricyclamol, as antiacetylcholine drug, 75, 82, 88 Trihexyphenidyl, as antiacetylcholine drug, 117 Triphosphoinositide phosphomonoesterase as ACh nicotinic receptor, 4 assay of, 10-12

3 64

SUBJECT INDEX

fractionation into subunits, 9-1 0 isolation of, 8-9 3-Tropanyl 2,3-diarylacrylates, quantitative studies on activity of, 103 Tryptamine derivatives, neurobiology of, 149 Tryptophan, abnormal metabolism of. in porphyria, 323-324 D-Tubocurarine, effects on cerebral RNA, 220 Turkish porphyria, 305 causes of, 308

Vestibular cells, R N A in, 190

W Waldenstrom’s protocoproporphyria, see Porphyria cutanea t a d a hereditaria

X X-porphyrin, in porphyria variegata, 306,307

U Uroporphyrinogen synthetase, in porphyria, 320-32 1

v Vascuiitis, in porphyria, 3 17-318 Venoociusive disease, monopyrrole role in, 173-175

Y Yohimbine. 176

z Zieve syndrome, porphyria in, 314 Zinc, in porphyria etiology, 323, 340-348

CONTENTS OF PREVIOUS VOLUMES Volume I

Recent studies of the Rhinencephalon in Relation to Temporal Lobe Epilepsy and Behavioral Disorders W. R . Adey Nature of Electrocortical Potentials and Synaptic Organizations in Cerebal and Cerebellar Cortex Dominick P . Purpura Chemical Agents of the Nervous System Catherine 0. Hebb Parasympathetic Neurohumors ; Possible Precursors and Effect on Behavior Carl C. Pfeayer Psychophysiology of Vision G. W. Granger Physiological and Biochemical Studies in Schizophrenia with Particular Emphasis on Mind-Brain Relationships Robert G . Heath Studies on the Role of Ceruloplasmin in Schizophrenia S. Mirtens, S. Vallbo, and B. Melander Investigations in Protein Metabolism in Nervous and Mental Diseases with Special Reference to the Metabolism of Amines F. Georgi, C. G . Honegger, D . Jordan, H. P . Rieder, and M . Rottenberg AUTHOR INDEX-SUBJECT INDEX

Volume 2

Regeneration of the Optic Nerve in Amphibia R . M. Gaze Experimentally Induced Changes in the Free Selection of Ethanol Jorge Mardones The Mechanism of Action of the Hemicholiniums F. W. Schueler 365

366

CONTENTS OF PREVIOUS VOLUMES

The ltoie of Phosphatidic Acid and Phosphoinositide in Transmembrane Transport Elicited by Acetylcholine and Other Hunioral Agents idorcell E. Hokin and Mabel R. Hokin

Brain Seurohormones and Cortical Epinephrine Pressor Responses as Affcctcd by Schizophrenic Serum Edward J . iI7alaszek The Role of Serotonin in Neurobiology Ermznio Costa Drugs and the Conditioned Avoidance Response Albert Hertz Lfetabolic and Neurophysiological Roles of y-Aminobutyric Acid Eqene Roberts and Eduardo Eidelberg Objective Psychological Tests and the Assessment of Drug Effects H . J . Eyseiick AUTHOR INDEX--SUBJECT INDEX

Volume 3

Subrriicr oscopic Morphology and Function of Glial Cells Edirardo De Robertis and H . LU.Gerschenfeld Microelectrode Studies of the Cerebral Cortex I'ahe E. dmassian Epilepsy Arthur A . il'ard, Jr. Functional Organization of Somatic Areas of the Cerebal Cortex Htroski .Vakahama Body Fluid Indoles in Mental Illness K Kodnight Some Aspects of Lipid Metabolism in Xervous Tissue G. R . Webster C:ori\ ulsive Effect of Hydrazides : Relationship to Pyridoxine Hariy I,. LVzlliams and James A . Bain

The Physiology of the Insect Nervous System D. '11. Vowles AUTHOR INDEX-SUBJECT INDEX

CONTENTS OF PREVIOUS VOLUMES

367

Volume 4

The Nature of Spreading Depression. in Neural Networks Sidney Ochs Organizational Aspects of Some Subcortical Motor Areas Werner' P . Koella Biochemical and Neurophysiological Development of the Brain in the Neonatal Period Williamina A . Himwich Substance P: A Polypeptide of Possible Physiological Significance, Especially within the Nervous System F. Lembeck and G. Zelter Anticholinergic Psychotomimetic Agents L . G. Abood and J . H . Biel Benzoquinolizine Derivatives : A New Class of Monamine Decreasing Drugs with Psychotropic Action A . Pletscher, A . Brossi, and K. F. Gey The Effect of Adrenochrome and Adrenolutin on the Behavior of Animals and the Psychology of Man A . Hofer AUTHOR INDEX-SUBJECT INDEX

Volume 5

The Behavior of Adult Mammalian Brain Cells in Culture Ruth S. Geiger The Electrical Activity of a Primary Sensory Cortex: Analysis of EEG Waves Walter J . Freeman Mechanisms for the Transfer of Information along the Visual Pathways Koiti Motokawa Ion Fluxes in the Central Nervous System F. J. Brinley, Jr. Interrelationships between the Endocrine System and Neuropsychiatry Richard P. Michael and James L. Gibbons Neurological Factors in the Control of the Appetite Andre' Soulairac

368

CONTENTS OF PREVIOUS VOLUMES

Some Biosynthetic Activities of Central Nervous Tissue R. V. Coxon Biological Aspects of Electroconvulsive Therapy Gunnar Holmberg AUTHOR INDEX-SUBJECT INDEX

Volume 6

Protein Metabolism of the Eervous System Abel Lajtha Patterns of Muscular Innervation in the Lower Chordates Quentin Bone The Keural Organization of the Visual Pathways in the Cat Thomas H . Meikle, Jr. and James M . Sprague Properties of Afferent Synapses and Sensory Neurons in the Lateral Geniculate Nucleus P. C. Bishop Regeneration in the Vertebrate Central Kervous System Carmine D. Clemente Neurobiology of Phencyclidine (Sernyl), a Drug with an Unusual Spectrum of Pharmacological Activity Edward F. Domino Free Behavior and Brain Stimulation Jose' M . R. Delgado AUTHOR INDEX-SUBJECT INDEX

Volume 7

Alteration and Pathology of Cerebral Protein Metabolism Abel Lajtha iMicro-Iontophoretic Studies on Cortical Neurons K . Krnjevit Responses from the Visual Cortex of Unanesthetized Monkeys John R. Hughes Recent Development of the Blood-Brain Barrier Concept Ricardo Edstrom

CONTENTS OF PREVIOUS VOLUMES

369

Monoamine Oxidase Inhibitors Gordon R. Pscheidt The Phenothiazine Tranquilizers : Biochemical and Biophysical Actions Paul S. Guth and Morris A . Spirtes Comments on the Selection and Use of Symptom Rating Scales for Research in Pharmacotherapy J . B. Wittenborn Multiple Molecular Forms of Brain Hydrolases Joseph Bernsohn and Kevin D. Barron AUTHOR INDEX-SUBJECT INDEX

Volume 8

A Morphologic Concept of the Limbic Lobe Lowell E. White, Jr. The Anatomophysical Basis of Somatosensory Discrimination David Bowsher, with the collaboration of Denise Albe-Fessard Drug Action on the Electrical Activity of the Hippocampus Ch. Stumpf Effects of Drugs on Learning and Memory James L. McGaugh and Lewis F. Petrinovich Biogenic Amines in Mental Illness Giinter G. Brune The Evolution of the Butyrophenones, Haloperidol and Trifluperidol, from Meperidine-like 4-Phenylpiperidines Paul A. J . Janssen Amplitude Analysis of the Electroencephalogram (Review of the Information Obtained with the Integrative Method) Leonide Goldstein and Raymond A. Beck AUTHOR INDEX-SUBJECT INDEX

Volume 9

Development of “Organotypic” Bioelectric Activities in Central Nervous Tissues during Maturation in Culture Stanley M. Crain The Unspecific Intralaminary Modulating System of the Thalamus P. Krupp and M. Monnier

370

CONTENTS OF PREVIOUS VOLUMES

The Pharmacology of Imipramime and Related Antidepressants Laszlo Gyermek Membrane Stabilization by Drugs: Tranquilizers, Steroids, and Anesthetics Philip A4. Seeman Interrelationships between Phosphates and Calcium in Bioelectric Phenomena L. G'. Abood The Periventricular Stratum of the Hypothalamus Jerome &tin Neural h4echanisms of Facial Sensation I. Darian-Smith AUTHOR

INDEX- SUBJECT

INDEX

Volume 10

A Critique of Iontophoretic Studies of Central Nervous System Neurons G. C. Salmoiraghi and C. N . Slefanis Extra-Blood-Brain-Barrier Brain Structures kb7erner P . Koella and Jerome &tin Cholinesterases of the Central Nervous System with Special Reference to the Cerebellum Ann Silver Xonprirnary Sensory Projections on the Cat Neocortex P. Buser and K. E. Bignall Drugs and Retrograde Amnesia Albert Weissman Neurobiological Action of Some Pyrimidine Analogs Harold Koenig

A Comparative Histochemical Mapping of the Distribution of Acetylcholinesterase and Nicotinamide Adenine Dinucleotide-Diaphorase Activities in the Human Brain ?: Ishii and R. L.Friede Behavioral Studies of Animal Vision and Drug Action Hugh Brown

CONTENTS OF PREVIOUS VOLUMES

37 1

The Biochemistry of Dyskinesias G. Curzon AUTHOR INDEX-SUBJECT INDEX

Volume I I

Synaptic Transmission in the Central Nervous System and Its Relevance for Drug Action Philip B. Bradley Exopeptidases of the Nervous System Neville Marks Biochemical Responses to Narcotic Drugs in the Nervous System and in Other Tissues Doris H. Clouet Periodic Psychoses in the Light of Biological Rhythm Research F. A . Jenner Endocrine and Neurochemical Aspects of Pineal Function B d a Mess The Biochemical Investigations of Schizophrenia in the USSR D . V. Lozovsky Results and Trends of Conditioning Studies in Schizophrenia J . Saarma Carbohydrate Metabolism in Schizophrenia Per S. Lingjaerde The Study of Autoimmune Processes in a Psychiatric Clinic S. F. Semenov Physiological Foundations of Mental Activity N . P . Bechtereva and V. B. Gretchin AUTHOR INDEX-SUBJECT INDEX

CUMULATIVE TOPICAL INDEXFOR VOLUMES 1-10 Volume 12

Drugs and Body Temperature Peter Lomax Pathobiology of Acute Triethyltin Intoxication R . Torack, J . Gordon, and J . Prokop

372

CONTENTS OF PREVIOUS VOLUMES

Ascending Control of Thalamic and Cortical Responsiveness ;M. Steriade Theories of Biological Etiology of Affective Disorders John .Lf. Davis Cerebral Protein Synthesis Inhibitors Block Long-Term Memory Samuel H. Barondes The Mechanism of Action of Hallucinogenic Drugs on a Possible Serotonin Receptor in the Brain J . R. Smythies, F. Benington, and R. D . Morin Simple Peptides in Brain Isanzu Sano The Activating Effect of Histamine on the Central Kervous System M. zllonnier, R. Sauer, and A. LV.Hatt Mode of Action of Psychomotor Stimulant Drugs Jacques .If. oan Rossum AUTHOR INDEX-SUBJECT INDEX

Volume 13

Of Pattern and Place in Dendrites -2ladge E. Scheibel and Arnold B. Scheibel The Fine Structural Localization of Biogenic Monoamines in Nervous T'issue Floyd E. Bloom Brain Lesions and Amine Metabolism Robert Y.Moore Morphological and Functional Aspects of Central Monoamine Neurons Kjell Fuxe, Tomas Hokfelt, and Urban Ungerstedt Uptake and Subcellular Localization of Neurotransmitters in the Brain Solomon H. Snyder, Michael J . Kuhar, Alan I. Green, Joseph T. Coyle, and Edward G. Shaskan Chemical Mechanisms of Transmitter-Receptor Interaction John T. Garland and Jack Durell The Chemical Nature of the Receptor Site-A of Synaptic Mechanisms J . K . Smythies

Study in the Stereochemistry

CONTENTS OF PREVIOUS VOLUMES

373

Molecular Mechanisms in Information Processing Georges Ungar The Effect of Increased Functional Activity on the Protein Metabolism of the Nervous System B. Jakoubek and B. Semiginovsky Protein Transport in Neurons Raymond J . Lasek Neurochemical Correlates of Behavior M. H. Ajrison and J. N . Hingtgen Some Guidelines from System Science for Studying Neural Information Processing Donald 0. Walter and Martin F. Gardiner AUTHOR INDEX-SUBJECT

INDEX

Volume 14

The Pharmacology of Thalamic and Geniculate Neurons J . W. Phillis The Axon Reaction: A Review of the Principal Features of Perikaryal Responses to Axon Inquiry A . R. Lieberman CO, Fixation in the Nervous Tissue Sze-Chuh Cheng

Reflections on the Role of Receptor Systems for Taste and Smell John G. Sinclair Central Cholinergic Mechanism and Behavior S. N . Pradhan and 5 '. N. Dutta The Chemical Anatomy of Synaptic Mechanisms : Receptors J . R . Smythies AUTHOR INDEX-SUBJECT

INDEX

Volume 15

Projection of Forelimb Group I Muscle Afferents to the Cat Cerebral Cortex Ingmar Rash Physiological Pathways through the Vestibular Nuclei Victor J . Wilson

374

CONTENTS OF PREVIOUS VOLUMES

Tetrodotoxin, Saxitoxin, and Related Substances: Their Applications in Neurobiology Martin H. Evans The Inhibitory Action of y-Aminobutyric Acid, A Probable Synaptic Transmitter Kunihiko Obafa Some Aspects of Protein Metabolism of the Neuron iMei Satake Chemistry and Biology of Two Proteins, S-100 and 14-3-2, Specific to the Nervous System Blake W. Moore The Genesis of the EEG Rafael Elul Mathematical Identification of Brain States Applied to Classification of Drugs E. R. John, P. Walker, D. Cawood, M . Rush, and J. Gehrmann AUTHOR INDEX-SUBJECT INDEX

A 4

6 5 C 6 D 7

E B F 9 G O

H 1 1 2 J 3