Russian Contributions to Invertebrate Behavior

Russian Contributions to Invertebrate Behavior

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Russian Contributions to Invertebrate Behavior Edited by

Charles I. Abramson Zhanna P. Shuranova Yuri M. Burmistrov

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Westport, Connecticut

London

Library of Congress Cataloging-in-Publication Data Russian contributions to invertebrate behavior / edited by Charles I. Abramson, Zhanna P. Shuranova, Yuri M. Burmistrov. p. cm. Includes bibliographical references and index. ISBN 0-275-94525-1 (alk. paper) 1. Invertebrates—Behavior. 2. Learning in animals. I. Abramson, Charles I. II. Shuranova, Zh. P. (Zhanna Petrovna) III. Burmistrov, IU. M. (IUrii Mikhailovich) QL364.2.R87 1996 592'.051—dc20 95-40578 British Library Cataloguing in Publication Data is available. Copyright © 1996 by Charles I. Abramson, Zhanna P. Shuranova, and Yuri M. Burmistrov All rights reserved. No portion of this book may be reproduced, by any process or technique, without the express written consent of the publisher. Library of Congress Catalog Card Number: 95-40578 ISBN: 0-275-94525-1 First published in 1996 Praeger Publishers, 88 Post Road West, Westport, CT 06881 An imprint of Greenwood Publishing Group, Inc. Printed in the United States of America

@r The paper used in this book complies with the Permanent Paper Standard issued by the National Information Standards Organization (Z39.48-1984). 10

9 8 7 6 5 4 3 2 1

Contents

Figures

vii

Preface

ix

1. Introduction Charles I. Abramson

1

2. History of Invertebrate Behavioral Studies in Russia Zhanna P. Shuranova

5

3. Memory and Morphogenesis in Planaria and Beetle Inna M. Sheiman and Kharlampi P. Tiras

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4. Innate and Acquired Behavior of Mollusks Pavel M. Balaban and Igor I. Stepanov

77

5. Individual Features in Invertebrate Behavior: Crustacea Yuri M. Burmistrov and Zhanna P. Shuranova

111

6. Learning, Memory, and Motivation in Ants Galina P. Udalova and Anna Ja. Karas

145

1. Local Orientation and Learning in Insects Vladimir M. Kartsev

177

VI

CONTENTS

Appendix A: Directory of Russian Scientists Engaged in Invertebrate Research

213

Appendix B: Bibliography of Invertebrate Articles Appearing in Neuroscience and Behavioral Physiology

217

Index

227

Addresses of the Editors and Contributors

229

Figures

Figure 2.1: V. A. Vagner (Courtesy of B. V. Lukin, Priroda, 1987, issue 1, p. 50).

11

Figure 2.2: N. N. Ladygina-Kots (From Ladygina-Kots 1923)

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Figure 2.3: L. A. Orbeli (Courtesy of Yu. Burmistrov).

17

Figure 2.4: L. V. Krushinsky (Courtesy of N. Krushinskaya).

19

Figure 3.1: Conditioning of planarians with light as the conditioned stimulus. For Day 9, only the conditioned stimulus was changed to vibration.

46

Figure 4.1: Experimental setup for behavioral experiments. The animal is fixed by its shell to a holder (H), but in such a manner that it can move freely on a plastic ball (B). I: indifferent carbon electrode, T: electrically driven tapper, S: electrode for manual application of electric shock to the skin.

81

Figure 4.2: Performance of snails in a food capture task. Curve 1 represents the time needed to capture a piece of cabbage. Curve 2 represents the time needed to capture a piece of carrot.

84

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FIGURES

Figure 4.3: Pneumostome closure responses in the snail during paired presentation of watermelon odor (filled dot) and moderately intense noxious stimulus (triangle, upper trace) across training trials. The upward shift in the trace represents pneumostome closure. The unfilled dot represents the presentation of carrot. Carrot odor was not paired with the noxious stimulus.

92

Figure 4.4: The dynamics of food avoidance learning in Helix fitted to the mathematical model. 1: conditioned stimulus—carrot, differentiated stimulus—cabbage; 2: conditioned stimulus—cabbage, no differentiated stimulus; 3: conditioned stimulus—carrot, no differentiated stimulus.

95

Figure 4.5: An application of the model to the instrumental conditioning of left tentacle withdrawal reflex in Helix after 1, 2, 3, or 4 training sessions.

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Figure 4.6: The influence of blood, sampled from donors after their first left tentacle withdrawal reflex learning session, on the learning of naive recipients in the snail Helix. Curve 1: the learning curve of the donors' first session. Curve 2: the learning curve of the recipients' first session. Curve 3: the learning curve of the recipients' second session.

99

Figure 4.7: The influence of blood, sampled from donors after their fourth left tentacle withdrawal reflex learning session, on the learning of naive recipients in the snail Helix. Curve 1: the learning curve of the donors' first session. Curve 2: the learning curve of the recipients' first session. Curve 3: the learning curve of the recipients' second session.

1000

Figure 4.8: Average frequency of bar contacts. Pooled results.

103

FIGURES

IX

Figure 6.1: The scheme of the experimental apparatus: I: the earthenware pot with ant nest. II: the tray. Ill: the screen. IV: the arena. V: the maze. 1: uprights. 2 entrance to nest. 3: the central bridge. 4: the side bridges. 5: the starting zone. 6: removable bridges. O: entrance to maze. A and G: target areas. B and V: symmetrical areas: T and P exits: K,L,E,C,D and Y,Q,J,U: other sections of right and left halves of maze, respectively.

155

Figure 7.1: Discriminative abilities in bees. 1: shape discrimination, 2: color discrimination, 3: shape of figures, 4: size of figures, 5: shape and size simultaneously.

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Figure 7.2: Performance of two groups of bees discriminating stimuli that lead to food (curve 1) or to the nest entrance (curve 2), over the course of 80 visits.

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Figure 7.3: Four possible trajectories around the perimeter of the experimental table containing four flower-like feeders; a and c were preferred flight paths, and b and d were rarely chosen.

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Preface

Analysis of associative and nonassociative behavior of invertebrates is one of the current approaches to understanding the neuronal and chemical bases of learning and memory. Associative and/or nonassociative learning has been demonstrated in all of the major classes of invertebrates, and underlying cellular substrates have been identified in several cases, largely in molluscs. The overwhelming majority of what American researchers know about invertebrate behavior, however, comes from Western laboratories. A large amount of the Russian invertebrate behavior literature remains untranslated and not readily available to the Western researcher. This book addresses and is a necessary step in correcting this imbalance. I first became interested in Russian studies of invertebrate behavior from reading the literature reviews of Gregory Razran (1901-1973). For nearly fifty years, Razran provided Western readers with surveys of the Russian literature on "higher nervous activity" or, as it is known in the West, learning. This work culminated in his 1971 book, Mind in Evolution: An East-West Synthesis (Boston: Houghton Mifflin), which reviewed over one thousand Russian studies. Razran's survey of the Russian work prepared me to find a vast and vibrant literature on invertebrate behavior. I was not disappointed. In a book describing, for example, the behavior of microorganisms, Tushmalova [1986, Functional Analysis of Acquired Behavior of Lower Invertebrates (in Russian), Moscow: Moscow University] cites 128 Russian studies and Sheiman [1984, The Regulators of Morphogenesis and their Adaptive Function (in Russian), Moskva: Nauka] cites 58 in her discussion of planarian and beetle behavior. In two books written by Shuranova and Burmistrov [1988, Neurophysiology of the Crayfish (in Russian), Moskva: Nauka; 1991, Progress in Crustacean Neurophysiology (in Russian), Moskva: Viniti], 123 Russian citations are presented on various aspects of crusta-

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PREFACE

ceans; and Balaban and Zakharov [1992, Learning and Development: Common Basis of Two Phenomena (in Russian), Moskva: Nauka] present 99 Russian citations relevant to the behavior of the snail. During my visits to Russia I had the opportunity to explore several research laboratories and speak with investigators engaged in invertebrate research, the work of some of whom is presented here. The continuation of invertebrate behavior work, however, is in jeopardy. It is obvious to any visitor to Russia that the growth of democracy is going to be a slow and painful process. The effect of this process is nowhere more evident than in the Russian scientific infrastructure. The current conditions, with an average salary of $30 a month, the cost of an airline ticket equivalent to at least two years salary, a shortage of even the most basic laboratory supplies, competition to obtain extramural funding with little or no grant writing skills, immigration of senior scientists to the West, a severe shortage of graduate students—for, after all, who but the most dedicated would risk spending a lifetime studying invertebrates for $30 a month—will in the short run confine the study of invertebrate behavior to a few relatively well funded laboratories studying only a few species. Without assistance from Western scientists engaged in the study of invertebrate behavior, the end of invertebrate research in Russia is rapidly approaching. In the course of conversations, Zhanna Shuranova, Yuri Burmistrov, and I came upon the idea that one way to provide such assistance is to make Russian work on invertebrate behavior known in the West. In this way collaborations in the form of, for instance, student and faculty exchanges, research and grant writing, and invitations to submit review articles and book chapters might be stimulated. Moreover, it would facilitate answers to fundamental questions in behavior by minimizing duplication and by fine-tuning research efforts. It is obvious from reading Western reviews of the invertebrate behavior literature that the Russian work is seldom cited. For example, out of a combined total of 700 references, only the 1927 work of Pavlov was mentioned in two major reviews of invertebrate behavior (Farley and Alkon 1985, Ann. Rev. Psy. 36: 419494; Carew and Sahley 1986, Ann. Rev. Neuros. 9: 435-487). In a recent review of invertebrate learning, no Russian work was cited in 257 references (Krasne and Glanzman 1995, Ann. Rev. Psy. 46: 585-624) even though a special issue of Neuroscience and Behavioral Physiology (1994, vol. 24) was dedicated to the neurobiology of the snail. The same situation is evident in books on invertebrate learning and behavior. For instance, in the thirteen chapters comprising Corning, Dyal, and Willows' three volume set on invertebrate learning there are no Russian contributors (Invertebrate Learning: Vol: 1-3, 1973-1975, New York: Plenum); and there is only 1 Russian contributor out of 103 in a recent encyclopedic treatment of learning and memory (Squire 1992, Encyclopedia of Learning and Memory, New York: Macmillan). One factor in the lack of Russian citations in Western journals and books is that the Russians themselves do not do a good job of making their invertebrate research known to Western readers. In a journal devoted to translations of selected Russian work a survey of Neuroscience and Behavioral Physiology between 1972 and

PREFACE

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1994, only 129 of 1661 papers pertained to invertebrates. The notable exception is a 1994 issue (vol. 24), which presents twenty papers dealing with snail behavior and physiology. As I mentioned above, this issue was not cited in a recent review of the literature. With this book the editors and contributors hope to increase the awareness of Russian contributions to invertebrate behavior. Russian Contributions to Invertebrate Behavior is an edited volume describing Russian experiments on invertebrate behavior, especially learning. Each chapter, written by a leading Russian scientist, provides a review of his or her subject area. Subject areas include a historical introduction to the role of vertebrate and invertebrate behavior in Russian science, the behavior of crustaceans, the orientation and learning of insects, the behavior and morphogenesis of invertebrates, and the role of motivation in learning and memory of ants. The audience for this book ranges from students and instructors of invertebrate behavior to any professional interested in knowing the contributions made by Russian scientists to the important field of invertebrate behavior. In addition to reviews of Russian work, additional features are appendices that provide the names and addresses of Russian scientists engaged in invertebrate work and a bibliography of invertebrate articles appearing in Neuroscience and Behavioral Physiology. It is hoped that students and professionals interested in invertebrate behavior can use the information to forge and to facilitate collaborative efforts between Russian and Western scientists. It is the sincere wish of the editors and authors that this book will serve the scientific community as a reference tool and, more important, will stimulate contacts between Russian scientists and Western researchers doing related work. We would like to thank Dolores A. Buckbee, Bryan Bruno, and Jim Hellwege for their help in the final stages of this project. Thanks are also due to Ann Newman, Liz Murphy, Linda Robinson, and Laura Clark from Praeger Publishing for making this book possible. Charles I. Abramson Departments of Psychology and Zoology Oklahoma State University

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Russian Contributions to Invertebrate Behavior

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Chapter One Introduction Charles I. Abramson

Among the many advances made possible by the study of invertebrates are an increase in knowledge of behavior, a greater appreciation of the plasticity inherent in stereotypic behavior, and the use of training methods to assist bees in the pollination of crops. In addition, researchers who have taken advantage of the invertebrate nervous system have made significant inroads in understanding the cellular mechanisms of behavior. As I have said elsewhere (Abramson 1994), for those of us who seek basic principles of behavior, invertebrates offer several important advantages. For example, the varieties of invertebrate nervous systems created in the course of evolution provide an excellent opportunity to begin to understand ourselves. When we understand the workings of the single cell of a protozoan, the regeneration of ganglia in a planarian, the retention of a memory in a beetle as it progresses from a larvae to an adult, the physiological basis of learning in a snail, or the individual behavior of a crayfish, we are that much closer to unravelling the mysteries of our own brains and behavior. In addition to what they can tell us about ourselves, invertebrates are useful to test the generality of both behavioral theories of learning and any proposed underlying physiological or biochemical mechanisms. Invertebrate nervous systems are, on the whole, more amenable to physiological and biochemical manipulations than vertebrate nervous systems. Moreover, because the behavior of invertebrates is generally less complex and more reflexive than our own, the genetic analysis of physiological mechanisms is readily amenable to experimental manipulations. The fruit fly, Drosophila, and the nematode Caenorhabditis elegans have become the invertebrates of choice in the effort to identify genetic components in behavioral traits. If we find, for instance, in a vertebrate that a particular type of learning depends on the presence of a cerebral cortex, a specific

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RUSSIAN CONTRIBUTIONS TO INVERTEBRATE BEHAVIOR

type of neural organization, or a certain level of cognitive development, an invertebrate can reveal whether such features are always required. We may find, for instance, that an invertebrate can learn the same task as a vertebrate but that the underlying mechanisms or solutions are different. Invertebrates are also used to answer questions such as, When does learning first appear in the animal kingdom? Is it possible to trace the evolutionary development of a particular type of behavior? Do invertebrates and vertebrates behave the same way, under the same conditions? Can invertebrates be used as a bioassay to detect pollutants? The intriguing nature of invertebrate behavior, the accessibility of the nervous system, and the use of genetic manipulations combine to make invertebrates an attractive group to answer questions about learning and memory. This attractiveness is multiplied by the sheer number and diversity of invertebrates, which make it easy to select behavior and physiology uniquely suited to a particular experimental design. For instance, if one is interested in the effect of regeneration of the nervous system on retention of a learned response, planarians make fine subjects. If one is interested in how conditions inside a cell influence learning, one approach is to use aneural single cells of such protozoans as Stentor. Russian Contributions to Invertebrate Behavior is an introduction to the invertebrate work performed by Russian scientists. The major emphasis is on studies of learning. In this book the editors and contributors have brought together contemporary Russian experimental data on the behavior of various invertebrates, including crustaceans, insects, and molluscs. The book should be useful for those interested in acquiring a working knowledge of the behavioral techniques, data, issues, and history of Russian studies of invertebrate behavior. It is also of interest to those studying the history of Russian behavioral science. The book is designed primarily to stimulate Western researchers in contacting and forming collaborations with Russian colleagues. It can stand alone, as a valuable resource of the Russian literature, or can be used as a supplemental text for courses in animal behavior and physiological psychology. The book consists of Seven chapters and Two appendices. Chapters Two through Seven begin with a history of Russian work in a particular area and highlights the investigators research. Each chapter contains a reference section citing related Russian articles. Much of the data discussed in the chapters was not previously available, or at least not readily available, to the Western researcher. Chapter Two provides a historical overview of invertebrate behavioral studies in Russian. Here you will see the contributions made by Russian scientists at the turn of the century and read about the downfall of Russian behavioral science due to the October Revolution, the resulting civil war, and the Communist leadership. You will learn about V. A. Vagner and the rise of the Russian ethological movement and about other Russian scientists, such as L. A. Orbeli and N. N. Ladygina-Kots, who contributed to the comparative analysis of behavior in Russia, often at great personal risk. Chapter Three presents a discussion of memory and morphogenesis using planarians and beetles as model animals. Experiments are presented that attempt to

INTRODUCTION

3

uncover the underlying mechanisms of the survival of memories following regeneration and metamorphosis. An experimental technique is presented for the training of planarians that eliminates the problems inherent in the conditioning procedures popular in the West during the 1950s and 1960s. Of interest also is a technique for quantifying the process of regeneration. Chapter Four is an excellent introduction to the type of simple systems work performed in Russia. The authors discuss a wide range of behavior in the snail Helix and discuss various training techniques and the influence of developmental stage on learning. A mathematical model is presented that accounts for performance under a range of training situations. A series of experiments that particularly captures the imagination comes from a transfer of training study in which the blood from a trained snail is injected into a naive snail. This work has much in common with that in Chapter Three on the transfer of learning in planarians following regeneration. Chapter Five provides a discussion of Russian studies of crustacean behavior. Of particular interest here is the value placed on the study of individual behavior when observed over a long period of time. The authors believe that long term observation under various conditions, coupled with physiological data, indicate that crayfish can be used to create an invertebrate model of the emotional process. A strong case is also made that the crayfish Procambarus cubensis, because of its size and ease of housing, makes an excellent subject for research. The behavior of this species is discussed in detail. Chapters Six and Seven outline several investigations on the learning and memory of ants and of bees and wasps, respectively. The learning of ants is studied under various levels of motivation; the data obtained in a complex maze situation indicate that the behavior of ants is more flexible than one might expect from Western data. The learning of bees and wasps is also studied under various conditions of motivation and under cognitive-like experiments based on problems of logic. The experiments with bees and wasps provide an excellent example of the comparative approach to the study of behavior and provide the surprising conclusion that bees and wasps can solve logical tasks; wasps are more flexible in their ability to do so than honey bees. The remaining sections of the book consist of two appendices. Appendix A presents the names and addresses of some Russian workers in the area of invertebrate learning to enable readers to contact scientists whose work excites them. Appendix B is a bibliography of every article using invertebrates (behavior, physiology, and/or a combination of both) that appeared in the journal Neuroscience and Behavioral Physiology until 1995. Other translated materials are also listed. SOME DIFFERENCES BETWEEN RUSSIAN AND WESTERN STUDIES OF INVERTEBRATE LEARNING AND BEHAVIOR In reading these chapters, one is struck by the similarities between Western and

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RUSSIAN CONTRIBUTIONS TO INVERTEBRATE BEHAVIOR

Russian studies of invertebrate behavior. As their Western counterparts, Russian scientists have exploited the invertebrate nervous system for what it can reveal about the underlying mechanisms of behavior; and, as in the West, invertebrates are recognized in Russia as important in research and teaching (Abramson 1990, 1994). There are also important differences. Russian studies of invertebrate learning, by and large, minimize the differences between instrumental/operant conditioning and classical conditioning. This stems from the Pavlovian tradition, which stressed instrumental conditioning as a special case of classical conditioning. In the West the distinction is stressed, although at the level of vertebrates the practice seems to be fading. A second difference is that the Russians employ an evolutionary perspective in their research much more than is done in the West. In the 1950s, for instance, both Schneirla and Beach noted the lack of an evolutionary perspective in comparative psychology. At least as it applies to invertebrates this problem still remains (Abramson 1994). As Chapter Two will show, there were a number of Russian theorists who routinely considered invertebrate data often at great personal risk. Related to the importance of having an evolutionary perspective is the need to conduct comparative analysis. In my opinion the West certainly lags behind the Russians. As Kartsev's chapter on learning in honey bees and wasps indicates he routinely compares the performance of several invertebrates, as do Sheiman and Tiras and others described in this book. The Sheiman and Tiras contribution is also interesting in that they continue to use the planarian in studies of learning and memory—an animal long ago forgotten in invertebrate learning research in the West. It should also be noted that there is a trend in the Russian invertebrate literature to consider cognitive capabilities in higher invertebrates such as Decapods and in insects such as ants and bees and wasps. Such research has been going on since the 1950s. I remember as a student thinking how silly it was to impart cognitive abilities to invertebrates. Yet now, in the West, for some, it seems silly not to. There also seems to be a greater Russian interest in the behavior of invertebrates particularly in the study of individual differences. As the chapters point out, a number of research institutions are devoted to the study of invertebrates and how they can be used as bioassays and, in the case of ants, "forest defense." In the West much invertebrate research focuses on their nervous systems. The above descriptions of Russian and Western perspectives come, of course, from a personal perspective. In the following pages, readers can determine for themselves the extent of the similarities and differences.

REFERENCES Abramson, C. I. 1990. Invertebrate Learning: A Laboratory Manual and Source Washington, D.C.: American Psychological Association. . 1994. A Primer of Invertebrate Learning: The Behavioral Perspective ington, D.C.: American Psychological Association.

Chapter Two History of Invertebrate Behavioral Studies in Russia Zhanna P. Shuranova

This chapter is dedicated to the memory of B. S. Kuzin, A. A. Lyubishchev, B. N. Veprintsev, and many other talented Soviet biologists who enjoyed doing research but could not do very much, for reasons that are so far from science.

Systematic investigations of invertebrate behavior in Russia began at the end of the nineteenth century, primarily because of the efforts of V. A. Vagner (Wagner) (Vagner 1896). It is well known that the twentieth century has been an extremely difficult time in Russian history. Therefore, it is impossible to discuss the status of any specialized scientific field (such as invertebrate behavioral studies) if we ignore the general picture of social life and the status of science and scientists in Russia (Aleksandrov 1992). The aim of this chapter is to analyze the main tendencies of invertebrate behavioral investigations in Russia from the beginning of this century. This time period can be divided arbitrarily into several subperiods. The first period continued from the end of the nineteenth century up to the beginning of World War I. Russian science during this period was a respectable member of the international scientific community, the equal of other highly developed Western countries. There was, for instance, an established tradition of sending young scientists abroad to further their education. Such famous Russian physiologists as V. M. Bekhterev, N. E. Vedensky, A. A. Ukhtomsky, I. S. Beritov, and L. A. Orbeli spent time working in laboratories abroad. I. P. Pavlov also spent time abroad as a student, spending two years (1884-1886) in Germany, where he worked in Heidenhain's and Ludwig's laboratories (Gureeva and Chebysheva 1969). Throughout his life Pavlov remembered these German physiologists and considered them among his teachers (see, for example, his speech after Heidenhain's death, October 23, 1897: Pavlov 1952, 6:108). As for Vagner, he had a rather long journey abroad sponsored by the St.

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RUSSIAN CONTRIBUTIONS TO INVERTEBRATE BEHAVIOR

Petersburg Society of Natural Sciences in 1889. He visited the zoological station in Naples and the Russian zoological station in Villafranca and spent time in Paris, Haage, Bruxelles, and Cairo (Lukin 1987). It is clear that both Vagner and Pavlov and other biologists of that time knew the chief European languages well. They could read scientific papers in German, French, and English; and they had no problem communicating with their foreign colleagues, often publishing their work in foreign journals (see, for example, both Pavlov's and Vagner's list of publications: Pavlov 1954; Vagner 1907). Thus, Russian scientists of the day were well informed about all scientific events, whether be by printed matter or from personal participation in international conferences (Yaroshevsky 1985). All this seems very simple and self-evident. However, this "normal" lifestyle began to deteriorate in 1914, when the world war resulted in the suspension of international scientific exchange. This situation was estimated at that time as "abnormal" and "temporary" (see, for instance, Ladygina-Kots 1923); but it lasted in Russia for many decades, fueled in part by the October Revolution in 1917. The period of the revolution itself and the resulting civil war was extremely hard on scientists, as it was for the average citizen. Life was a daily struggle; perhaps worse for many scientists, it interfered with their experimental and theoretical work. Though most naturalists had antimonarchist views and became loyal to the new power, often collaborating with it (see, for example, A. A. Ukhtomsky [Merkulov 1960; Pomper 1970; Shlupikova 1968], who held the title of "Prince" and whose ancestors were related to Rurik), the conditions for living were so hard that some scientists died from hunger (see short notes in the magazine Priroda in the years 1920-1921). In 1921, life began to be easier because of the "new economic policy" (NEP) of the Soviet government and Lenin. During the 1920s there was a short "Renaissance" in various areas of Russian cultural and scientific life (Shnol 1990). New scientific institutions were constructed, and there was a renewed interest in publishing scientific literature. There was unusual progress in printing books devoted to many biological problems. As the main reason was the intention to educate millions of nonliterate people, most of these printed materials were popular and inexpensive. Among them were books by Russian biologists and translations of interesting books by foreign writers (Sobol 1925). It should be noted that the books and magazines created during this period were to remain the primary reading material until the 1950s and 1960s. Some of these materials are still of interest today. The second time period spanned the beginning of the 1930s to about the 1950s. This period seemed to be harder on science and scientists than the preceding period. The main reason was that such scientific leaders as Bekhterev, Pavlov, Vagner, and Ukhtomsky died, with the leadership of Russian science being passed along to their pupils. Many of these new leaders were selected not according to scientific achievement but rather according to social and political activity, particularly as it related to the Communist party. Highly capable scientists with a deep interest in research found it difficult to maintain scientific careers. Some of

HISTORY OF INVERTEBRATE BEHAVIORAL STUDIES IN RUSSIA

7

these scientists were repressed by being confined to gulags. This was the fate of physiologists E. M. Kreps (Pavlov's pupil) and V. M. Merkulov (Ukhtomsky's pupil), the biochemist A. A. Baev (Engelgardt's pupil), and entomologist B. S. Kuzin. The head of Russian genetics, academician N. N. Vavilov; E. Bauer, the author of the brilliant book Theoretical Biology; the practical entomologist N. A. Alekseenko; and the talented scientific historian E. M. Vermel (among many other scientists) died in these camps and prisons. The great zoologist and geneticist N. K. Koltsov, who "fought" for many years against T. D. Lysenko, died at home in 1939. The main reason for his death was considered by many to be the frustration and sorrow of not being able to carry on his research. It is interesting to note that, though he was not repressed formally, books written by Koltsov were removed from libraries. His books still could not be obtained through official channels such as interlibrary loan as recently as the late 1970s. This time period was known as "the midnight of the century," (from the title of the book written in 1937 by the famous Russian-French writer V. Serge (V. Kibalchich). The period known as the midnight of the century was dominated by World War II ("the Great National War"). Many scientists died in the bloodshed, and scientific life all but stopped. There were attempts to move some scientific institutions away from the fighting. The physiological and medical institutions of Leningrad, for instance, were moved to Kazan and Middle Asia. Scientists who wished to stay in Leningrad (such as Pavlov's collaborator M. K. Petrova; the chief of the ornithological laboratory in Koltushi, A. N. Promptov; and the academician A. A. Ukhtomsky) tried to continue their scientific investigations and hold traditional scientific meetings. However, this heroic activity could hardly be successful in the face of the continued battle for survival. A. A. Ukhtomsky, for instance, died during the blockade of Leningrad, in the winter of 1942. Perhaps the most tragic event of this time as it relates to science is the so-called Vaskhnil Session (1948) of the Ail-Union Agricultural Academy, organized initially by T. D. Lysenko. The goal of this famous session appeared to be the destruction of true genetic investigations in Russia. The Vaskhnil Session spawned the "daughter" session of the "big" and "small" academies (Academy of Sciences and Medical Academy) devoted to Pavlovian "uchenije" (teaching). These daughter sessions were especially important for scientists studying behavior. The goal of these sessions appeared to be the same as that of the 1948 session: the destruction of true behavioral investigations in Russia. It is clear now that these two so-called scientific meetings were inspired directly by the Central Committee of the Communist party and personally by I. V. Stalin (Yaroshevsky 1991a, 1991b). After these Sessions (whose critical analysis has only just begun; see for example Leibson 1990; Grigorian and Roitbak 1991), L. A. Orbeli was relieved from the leadership of both the Biological Section of the USSR Academy of Science (in 1948) and the physiological institutes, which he had controlled after Pavlov's death. Some of Orbeli's collaborators lost their jobs in Leningrad; others had to change their topics. At this last session many scientists were criticized (I. S. Beritov, P. K. Anokhin, L. S. Stern, and A. D. Speransky, to name but a few), but

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the main coup was directed against Orbeli (Leibson 1990). Even now it is extremely difficult emotionally for many Russian scientists to reread the materials of that session. In fact, even in 1948 it was difficult to believe in the sincerity of many of those speakers. Thus it seems reasonable not to analyze seriously their arguments directed against their colleagues and teachers. This period of the "repressed science" ended in principle after Stalin's death in March 1953, when all life in the USSR began to change rather quickly. His death begins the third period, which continues to the present. However, the situation in science (at least in biology) seemed to remain unchanged for many subsequent years. It is important to note, though it seems unbelievable, especially for the Western reader, that the "Sessions" did not solve any scientific problems. The purpose of these sessions was to change the leadership in neurophysiology and related scientific fields. During many years and decades following these sessions the main scientific leadership positions were in the hands of those who "won the battle," inspired by nonscientific motives. For instance, the main "speakers" of these sessions attained high office: K. M. Bykov and A. Ivanov-Smolensky soon became the director and vice-director of the leading Institutes; L. G. Voronin and E. Sh. Ajrapetjants became the heads of the Departments of Higher Nervous Activity in Moscow and Leningrad Universities respectively. They held these positions mostly up to their deaths. Later their places were occupied by their pupils. It may be interesting to know that even in 1973 (twenty years after Stalin's death!) L. G. Leibson, who wrote a book about his teacher L. A. Orbeli, could not describe what actually happened at these sessions because the facts were unknown to ordinary scientists. The behind the scenes injustices of the Pavlovian sessions came to light only because of the influence of "perestrojka" in 1985 (Leibson 1990). However, Leibson's book is only the first bird signalling the spring. Critical analyses of these sessions on Russian science are now being performed (Krementsov 1991; Umrikhin 1991). Thus one may say that after Stalin's death the conditions for scientific work in Russia changed but the "repressive atmosphere" (at least in some biological sciences) remained for many years. The situation changed rather drastically only in the last five or so years, when scientists became much more free in their behavior. Ironically, few scientists can take advantage of this newfound freedom because of the worsening Russian economic situation. HISTORY OF BEHAVIORAL INVESTIGATIONS IN RUSSIA One may divide the behavioral studies performed in Russia during this century into at least three periods. The first period lasted from the beginning of the century up to the middle 1920s when the two main approaches to behavior, the psychological and the physiological, were equally respected. This sentiment is reflected well in the following quotation from a book by Ladygina-Kots: It [the book] is edited at the time of progressive development of the young science zoo-

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psychology when some scientists try to limit behavioral manifestations putting the animal in arbitrary, narrow experimental conditions (the American school), the others "talk" with animals by means of knocks and look for human capabilities in them (German school of Krallists"), the third glue the tag of "reflex" to all the psychical events (from the lowest to the highest) and look for the animal as for the automatic device (Russian reflexologists), the fourth (anatomo-physiologists) hope to understand the psychical life by means of skillful sophisticated surgical technique. (Ladygina-Kots 1923:9) During the second interval, lasting from the mid 1920s up to the end of the 1950s, there was a gradual and complete elimination of the psychological approach to the study of behavior. Moreover, at that time investigations that used physiological techniques differing from those favored by Pavlov and his school were eliminated. For example, at the end of the 1930s V. M. Borovsky (Borovsky 1936) had to stop his scientific work; and after the "daughter" Session in 1950 (the so-called Pavlovian Session) many research programs not fitting the traditional Pavlovian views were closed. In the third period, which began approximately at the end of the 1950s and the beginning of the 1960s, we see a gradual restoration of psychology and comparative psychology. During this period, which continues probably up to the present, we see a shift in the primary interest of Russian scientists to research and theoretical problems posed by Western science. Simultaneously, there appears to be a loss of interest in traditional questions of conditioned reflexes. This change is rather unexpected, considering the declarations of the Pavlovian Sessions. Another remarkable feature of our time is the revival of behavioral investigations using nonphysiological measures. Such research is based on ethological ideas that came to this country from abroad and not from the Russian investigations. It is a testament to the effectiveness of the Pavlovian Sessions and other Russian scientific catastrophes that the vast majority of Russian students have no collective recognition of the rich Russian tradition in ethology or "zoopsychology" started by Vagner. Let us look at these periods of behavioral research in more detail. "Objective Biopsychology" and the Theory of Conditioned Reflexes The development of "objective biopsychology" in Russia is connected entirely with V. A. Vagner (1849-1934). Today his name and his views are almost unknown to Russian behavioral scientists (Vagner 1900a, 1900b, 1910a, 1910b, 1913, 1914b, 1923). It seems that recently he is known better abroad than at home, because of the publications of Joravsky (1989) and especially of N. Krementsov (1992). Even here, however, the information about Vagner is incomplete. In this section I will present some information relating to Vagner's personality (Fabri 1969; Kim 1966; Roginsky 1940a; Strelchenko 1975). As a student Vagner entered both the Faculty of Law and the Faculty of Physics and Mathematics of Moscow University. Among his teachers were S. A. Usov,

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Ya. A. Borsenkov, and A. P. Bogdanov, who were pupils of the famous Russian zoologist K. F. Rulje. In his third year at the university he began to study seriously the anatomy and taxonomy of spiders. Figure 2.1 shows a picture of Vagner during this time period. While investigating the fauna of the Black Sea in 1882 he met with the famous biologists 1.1. Mechnikov and A. O. Kovalevsky. At this time he turned his attention toward the investigation of blood content in different sea invertebrates. Clearly, from the very beginning of his university education he had a deep interest in invertebrates. Vagner's thesis was devoted to an analysis of spider taxonomy in which he based his classification mainly on their sex organs (Vagner 1890). He proposed that behavioral features were good taxonomic indices, whose value was just as important as morphological indicators. His use of interspecies variability of behavioral features was heralded as a new method in comparative morphology. He also attempted to apply the scientific method to zoopsychology, which he called "objective biopsychology." For Vagner, biopsychology was the study of rules and tendencies in the evolution of behavior, using field observations and experiments under natural conditions. His main ideas, together with thorough, critical reviews of psychological and biological literature, are represented in his book "Biological Bases of Comparative Psychology: Biopsychology, printed in 1910 (vol. 1) and 1913 (vol. 2). The original data used in these books were obtained by Vagner earlier. Vagner's views concerning the evolution of behavior and the specificity of invertebrate behavior will be treated later in this chapter. One may state here, however, that Vagner provided a strong foundation for a naturalistic approach to behavior that is very similar to what is known in the West as ethology. The aims of biopsychology and its methods coincide with the program of "classical" ethological research (Krementsov 1992). It is obvious that his ideas were very popular at the beginning of this century. They influenced, for instance, such biologists as Kholodkovsky (1897), Shimkevich (1897), and Severtsev (1922). Vagner was well known, too, for his social activity. He lectured for many years on biology and comparative psychology at Moscow University. Later (from 1905) he was the director of the so-called Commercial College in St. Petersburg. In 1912 he realized his dream of creating a new popular magazine, Priroda. He discussed the idea of forming such a magazine with the writer A. P. Chekhov in the 1890s and became (with L. V. Pisarzhevsky) its first editor. It is interesting to note that the first issue of this magazine, which is still in circulation, has at the masthead a picture of the "assiduous bee" that Vagner displayed on his calling card. For ten years he was the editor of the famous series New ideas in biology; he also founded periodicals in the field of pedagogics. The physiological approach to behavioral research in this century was represented in Russia first of all by I. P. Pavlov (1849-1936) and V. M. Bekhterev (1857-1927). The names and works of both these famous scientists are well known in this country and abroad. Many papers and books have been written about their lives and scientific activity. Their works (especially those of Pavlov) were

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Figure 2.1 V. A. Vagner (Courtesy of B. V. Lukin, Priroda, 1987, issue 1, p. 50).

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translated into other languages and re-edited many times in Russian. Pavlov and Vagner were contemporaries. They worked for a time in the same city (St. Petersburg) and in the same scientific field. Both had common teachers and common views and were strong personalities. As so happens in science, despite sharing many characteristics, they were competitors rather then friends. They worked independently and moved in different circles. As is well known, Pavlov had both university and medical education. He had surgical skill, which he first applied to the study of the heart and the blood vessels. Later he used these skills to investigate the physiology of the digestive system. For this research he was awarded the Nobel Prize in 1904. However, at the end of the last century his interests shifted to the event called by him the "conditioned reflex." For the rest of his life Pavlov, together with many pupils, studied this wonderful phenomenon, mostly in dogs, by means of a technique he developed for this purpose (Pavlov 1923, 1951, 1954). V. M. Bekhterev, a psychiatrist and neurologist by education, worked in clinics and studied for many years the functional anatomy of the human central nervous system. Throughout his life Bekhterev maintained a deep interest in human psychology (Bekhterev 1907/1912). About the same time as Pavlov, Bekhterev also discovered the conditioned reflex, though Bekhterev called the phenomenon the "combination reflex." Rather than study the salivary reaction as Pavlov did, he preferred to study the movements of humans. He criticized Pavlov's technique but used much of Pavlov's data to confirm his own views. In the beginning of their research in this new field, Pavlov and Bekhterev believed that these phenomena belonged to the domain of psychology. However, later they changed their minds, realizing that all the "psychical" events can be studied by means of "conditioned" or "combination" reflexes. Thus there arose such terms as "higher nervous activity," introduced by Pavlov, and "reflexology," used by Bekhterev. Both of these "teachings" developed in the beginning of this century. Now they seem rather similar. At the turn of the century, however, they appeared contradictory, and their fate was different. "Victory" of Pavlovian Views: But at What Cost? I have tried to show that in the beginning of the century two big physiological schools in Russia were extremely active in the study of behavior. It seems that psychological views were not as popular even at that time. This probably reflects the general tendency of the development of natural sciences in the last century and the status of psychology itself at the end of the nineteenth century. However, as the Ladygina-Kots quote indicates, different approaches to the study of behavior and were available and given scientific expression in journals and other forums. This situation changed rather soon. In the 1920s the main way to study behavior became the method of conditioned reflexes (CR). Moreover, the theory of CR served as the foundation for not only physiology but psychology as well. Soviet

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psychologists viewed Pavlov's teaching as the basis for the establishment of a truly scientific approach to human psychical functions (Yaroshevsky 1985). As for Bekhterev, he was also extremely active during these years. Twelve institutions that he founded were eventually consolidated by Bekhterev into the State Psycho-Neurological Academy (Mojiseeva 1988). The academy had two scientific departments, clinical and experimental. It also included several additional institutions serving practical purposes, such as a school for retarded children. Bekhterev generated many ideas of great importance concerning human research in general and especially in children. He did not, however, have a systematic scientific program of research (Yaroshevsky 1985). He died suddenly in 1927 after visiting Stalin in his capacity as a psychiatrist. For many years there was a rumor that Bekhterev was poisoned (Guberman 1977). There are different opinions as to what happened to him. It is obvious that Bekhterev's name was forgotten soon after. Vagner, whose career was severely hurt by World War I and the civil war (Lukin 1987), was mainly occupied in the 1920s with pedagogical work (Ya. M. Pressman, personal communication) and with the organization of the Museum for the Evolution of the Nervous System and Comparative Psychology, which was based on the Anatomical Museum of the Bekhterev Institute of Brain Research. The exposition proposed by Vagner represented the correlation between nervous structure and function on one hand and behavioral and psychic activity on the other hand. The museum contained exhibitions of conditioned reflexes in fish and dogs, habits in ants, maze experiments with rats, and so on (Roginsky 1940b). Throughout these years, up to his death in 1934, Vagner wrote much but could publish rather little. His main book published during this time was The Essays in Comparative Psychology, which was based on the third volume of his Biopsychology (which was to be edited in 1915 but was "lost"). The essays were published in the form of ten thin booklets of low quality (especially if one compares them with the first two volumes) in 1925-1929. These volumes as well as other published work were never re-edited. Among his last published books is a very small and rare volume entitled Some Ways to Observe Animals (1926). His Comparative Psychology written at the end of his life, could not be published despite his and his wife's efforts (Lukin 1987). In Krementsov's opinion (1992), the main reason for the loss of Vagner's popularity after the October Revolution is the obvious discrepancy between his views and Marxist ideology. One may also add, the popularity and political power of Pavlov's views. There are no data about personal contacts between Pavlov and Vagner or about Pavlov's comments concerning Vagner's work. However, some of Pavlov's students (Yu. P. Frolov, G. P. Zeleny) "fought" with Vagner from the 1910s and for the next several years. These students of Pavlov denied the importance of behavioral research in itself and proclaimed the physiological approach to behavior as the only reasonable approach (Frolov 1925; Zeleny 1913). Vagner's reaction to this attack has been expressed several times (see, for instance, Vagner 1914a, 1914b). In Vagner's opinion, the physiological approach is insufficient, because

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higher mental capabilities are outside the methods used by the physiological school and because the reflex theory cannot explain the existence or formation of instincts. His conclusion was that the physiological school has major problems dealing with the facts of comparative psychology. The weakness of the physiological school becomes clear when they try to resolve what Vagner considered the main task of comparative psychology: to discover the rules governing the evolution of psychical events. Without this knowledge, according to Vagner, it is impossible to understand human psychology, just as it is impossible to understand human anatomy without an understanding of comparative anatomy. The criticisms that Vagner leveled at the physiological approach have been ignored since shortly after the October Revolution. At the end of the 1930s Vagner's contribution to Russian science was all but forgotten. He had few students; the best known were G. Z. Roginsky, who worked primarily with monkeys (Roginsky 1947), and B. I. Khotin, who studied imitation from an evolutionary perspective (Khotin 1990). Unfortunately Khotin could not work during the war (he was a soldier) and died soon after the war. With his death the comparative psychological work in the USSR also seemed to die. Around the period of the October Revolution, several famous scientists in addition to Vagner worked in the field of comparative psychology. D. N. Kashkarov, who is known as the founder of ecological zoology in the USSR, lectured on comparative psychology at Moscow University. The materials from these lectures were used for writing a book that remains useful even today (Kashkarov 1928). Another important figure was V. M. Borovsky, who was the head of the zoopsychological laboratory in the Moscow Psychological Institute. His topic was not far from Vagner's, but his approach was purely physiological (Borovsky 1936). He proposed, for instance, that instinctive actions are the result of unbalanced physiological rhythms in the organism. Moreover, he rejected the term "instinct" for its mystical and religious connotations. Although he had many more students than Vagner (for example, Akimov, Kobozjeva, Tikh, Skrjebitsky, Bibikova), like Vagner's, his contributions are all but forgotten. Another great personality of this time was N. N. Ladygina-Kots. She was the wife of the famous zoologist A. P. Kots, who worked for many years at the Moscow Zoo (Kots 1917) and was the founder of the wonderful Zoological Museum named for Darwin. In addition to her scientific training she was also a student of the famous Russian painter A. M. Vatagin, who is known most of all for his drawings of wild and domestic animals. She studied in detail the elaboration of new habits and sensory capabilities of monkeys, using methods she devised for the purpose. In Figure 2.2 she is seen holding one of her subjects. Ladygina-Kots proposed techniques for studying monkey behavior based on many hours of observation of the animal's natural habits under conditions of maximum freedom (Ladygina-Kots 1928, 1935, 1958, 1960). She criticized not only the purely physiological approach, by which some fragmentary reaction is studied, often under conditions of restraint, but also the approach of some psychologists of that time.

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Figure 2.2 N. N. Ladygina-Kots (From Ladygina-Kots 1923).

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In the author's opinion, one may force the silent animal to "open" his mouth only if the common language between the experimenter and the animal will be . . . specific for each animal (and only for it). If we stay face to face with Nature itself, before the live animal we should first of all not dictate to it our conditions and our demands but to try to sense it from the interior, to listen for it, to watch it and to find the best way to make contact with it; then it should "open" itself and "reveal" itself extremely easily and completely. (Ladygina-Kots 1923:9) Ladygina-Kots was certainly a unique scientist and not typical of Russian behavioral scientists of that time. The great bulk of young scientists who had an interest in animal behavior were involved by necessity with the views expressed by Pavlov and his school. After Pavlov's death in 1936, the main person responsible for the development of higher nervous activity became L. A. Orbeli (1882-1958). Orbeli was a student of Pavlov and a renowned scientist in theoretical and applied physiology. Orbeli is presented in Figure 2.3. His interests were rather broad. They included higher nervous activity, evolutionary physiology, trophic functions of the nervous system, kidney functions, and problems associated with pain (Leibson 1973; Svidersky, Veselkin, and Natochin 1988). Soon after he become the head of Pavlov's laboratories, he proposed a broad and innovative program for investigating "the teaching of conditioned reflexes." It is important to note that, for Orbeli, the application of the evolutionary, or comparative, approach to different functional systems, including problems associated with the central nervous system, was the correct approach. He wrote on many occasions that such an approach had been advocated by Pavlov himself at the end of his life. However, it now seems clear that it was mainly Orbeli's initiative to investigate the interaction between inborn and acquired behavior across taxons. In his behavioral experiments, he decided to use three lines of the evolutionary process: the insects, in whose behavior instinctive reactions predominate; the birds, which have a good representation of inborn and acquired behaviors; and the mammals, in which conditioned behavior attained its maximum expression. In his work with mammals the dog was often the animal of choice, but monkeys were also employed (Orbeli 1949). The comparative investigations of behavior took place primarily in Koltushi. Koltushi is a village not far from Leningrad where by special decision of the Soviet government, Pavlov founded one of his laboratories. Later it was reorganized into the Institute of Evolutionary Physiology and Pathology of the Higher Nervous Activity (see Zakharzhevsky and Andreeva 1984). Though most studies were performed at the institute on dogs, the ornithological laboratory and the laboratory of insect biology were also in operation. In some behavioral experiments Drosophila was used (Masing 1947). The major findings of the investigations performed in Koltushi during this time period are represented in a book devoted to the tenth anniversary of Pavlov's death but published some years later. In this book appear both a list of scientific publications during 1936-1946 and a list of theses defended in the institute. It offers an

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Figure 2.3 L. A. Orbeli (Courtesy of Yu. Burmistrov).

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interesting glimpse into scientific life during the war years. For the purposes of this chapter we will focus later on the work from the laboratory of insect biology headed by S. I. Malyshev (Malyshev 1949). As for general approaches to studying behavior, one should first mention A. N. Promptov (1898-1948). Promptov's views are the combination of the two main Russian approaches to behavior, the naturalistic (Vagner) and the physiological (Pavlov). Throughout his life, especially after leaving the Faculty of Physics and Mathematics of Moscow University, Promptov studied the behavior of birds (Promptov 1947). His main interest was in natural behavior. After 1940 he was invited by Orbeli to Koltushi, where he established a laboratory for the study of birds. One should note that during the war (and the famous blockade of Leningrad) he stayed behind in Koltushi so that he could protect his birds. Moreover, during this extremely hard time he had enough courage, strength, and will to continue his experiments (Lukina 1956). His main topic was the interaction of inborn and acquired behavior. For instance, he thoroughly investigated, using field and laboratory techniques, the orientation reaction to visual and auditory stimuli in such birds as skylarks, meadow pipits, northern nightingales and blue throats. In a book written during his last years (published in 1956, after his death) Promptov provided a description of the "biocomplex," a central theme of his research program. It may be described as "the set of reactions specific for every biological situation which has phylogenetically adaptive value and which is determined by the functional activity of the nervous and humoral systems" (Promptov 1956:110). It is similar to Vagner's idea of the "complex instinct." Rather close to Promptov's position was that of another scientist who was invited by Orbeli to Koltushi: L. V. Krushinsky (1911-1984). Throughout his life Krushinsky was associated with Moscow University (Semiokhina 1991). Like Promptov, he was a true naturalist who loved observing animals in their natural habitat. He was an expert in dog behavior and training, skills that were put to use during the war years. Later, using Promptov's idea about the role of excitability in the nervous system as a factor in the modification of animal behavior, he and his collaborators produced a rat that was extremely sensitive to sound. The line of such hyperexcitable rats (named KM, which means Krushinsky-Molodkina line) still remains unique in world practice and is used in many laboratories in this country and abroad. Figure 2.4 presents a picture of Krushinsky. When Krushinsky worked in Koltushi, his primary research was performing a behavioral-genetic analysis of learning in dogs (Krushinsky 1991). During thistime he wrote a very interesting paper entitled "Some integrative stages in theformation of behavior" (Krushinsky 1948). He argued that it was impossible to separate inborn from acquired behaviors and proposed the concept of "unitary responses." Such responses recognize the unity of both behavioral forms and the interrelations between them. He believed that these unitary responses combine to form a single adaptive action. He described the ways of forming these reactions and their possible involvement in behavioral evolution. Later, as a professor at the Department of Higher Nervous Activity in Moscow

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Figure 2.4 L. V. Krushinsky (Courtesy of N. Krushinskaya).

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University, Krushinsky investigated "elementary intellectual activity" in animals. He proposed techniques for studying this activity across developmental stages and across the phylogenetic scale. In his opinion, Intellectual activity is a kind of higher nervous activity; due to this, it is possible to solve— without some special learning—many tasks based on certain logical connections between the components. By means of intellectual activity one may detect the rules which connect the events and things in the environment. Certainly intellectual activity is a behavioral form, due to which the animal can react highly adaptively to rapidly changing environmental conditions. (Krushinsky 1977:207) Krushinsky and his collaborators investigated behavioral reactions of different animals when faced with a common set of problems. The results of these experiments indicated that monkeys were the best problem solvers, closely followed by dolphins. Following the dolphins were the carnivorous mammals, and much further behind were hares and rats. Good results were obtained also with birds, especially with the crow and its relatives. In contrast, "lower" vertebrates such as turtles and lizards could solve only the simplest kind of extrapolation task and fish could solve them only after extensive training. Thus Krushinsky showed that great differences are found between animals belonging to low and high stages of phylogenesis if one compares their capabilities to detect some simple natural rules and how to use them. It seems evident from the above-mentioned facts that the investigations headed by L. A. Orbeli, which took place in Koltushi during the 1940s had good prospects for continued success. The experiments showed the importance, indeed the necessity, of using a wide range of species in behavioral experiments and examining various types of behavior, not just higher nervous activity. Unfortunately this fruitful period was rather short, limited first of all by the Great National War and by the previously mentioned Pavlovian Sessions of 1948 and 1950. A different approach to Pavlov's study of higher nervous activity is connected with the name of the famous Georgian scientist I. S. Beritashvili (Beritov, in Russian transcription) (1885-1974). He was N. E. Vedensky's student at St. Petersburg University, where he specialized in neuromuscular physiology and was very skillful in electrophysiological techniques of that time. It is interesting to note that Pavlov did not approve of all this "wire" physiology. In 1915 Beritashvili obtained a job in the famous Novorosijsky University (Odessa), where he began to study conditioned reflexes in different animals. Though it was the time of World War I, which was followed by the October Revolution and civil war, the years spent in Odessa were some of the most fruitful in his life (Roitbak 1991). In 1919 he obtained work in the Georgian capital of Tbilisi, where he remained until his death. He began teaching in Tbilisi University and wrote books in various fields of neurophysiology. He had an encyclopedic knowledge of neurophysiology and was a popular person in scientific and social circles. Moreover, he was a brilliant experimenter who routinely developed new techniques. He also had the

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very rare ability of being able to generate novel scientific ideas. Being an expert in neuromorphology, he tried to connect structure and function. He was one of the first to approach a research problem from the physiological, morphological and biochemical perspectives (Roitbak and Bakuradze 1988). In addition to his work in neurophysiology Beritashvili simultaneously had a deep and lifelong interest in the study of animal behavior. He began replicating Pavlov's experiments with conditioned reflexes in dogs and soon understood their limitations. Like Vagner, Beritashvili believed that the behavior of the whole animal must be studied under conditions in which the animal is free to move about. He developed some specific techniques that allowed him to establish, among other things, the essential differences between reflexive and complex behavior, the ontogenetic and phylogenetic development of specific types of behavior, and the dependence of behavior on context. Though he was a "pure" physiologist and did much in the field of neurophysiology, his main approach to behavior was neuropsychological. He proposed the concept of "psycho-nervous" activity, which he considered to be a higher form of individual behavior than either reflexive or instinctive behavior. As opposed to conditioned reflexes, the reactions of the psycho-nervous type are formed rapidly. They arise due to remembering some events (that is, due to the psycho-nervous representation, or image, of the event) that the animal experienced only once (Beritov 1947). Later he investigated the mechanisms of spatial orientation of animals across species. At the end of his life he was completely occupied with the problem of memory. He divided the memory of vertebrates into three types: imaginative, emotional, and conditioned. He proposed that highly organized vertebrates have some elements of intellectual activity that permit goal-directed behavior. It had been noted previously that Beritov was one of the main figures condemned during the Pavlovian Session of 1950. In fact, the critique of him began some years earlier, after the publication of his small book entitled The Main Forms of Nervous and Psycho-Nervous Activity (Beritov 1947). It is necessary to pay attention to his extremely courageous behavior at that time. To gain some appreciation of this period in Russian scientific history, it is instructive to read a volume of his own material (most of which was completely unknown) published after his death (Beritashvili 1984). In this book are many papers written by him in response to accusations directed against him by some fellow neurophysiologists (for instance, Vatsuro and Voronin) and philosophers during the period before and after the Pavlovian Session of 1950. It was during this session that he was removed from the post of director of the Physiological Institute, Georgian Academy of Sciences. The Current Stage of Behavioral Investigations The beginning of this period seems to be at the end of the 1950s. As is known that one may see the great only from a certain distance, it is not easy to describe

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the most essential features of the time span to which we belong. From the historical point of view this period may be characterized as the time of "coming full circle." The pretensions of many neurophysiologists who believe that there is only one way of conducting behavioral research was expressed extremely well at the Pavlovian Session of 1950 and in the first few years after it. Such an attitude gradually disappeared as the Communist party loosened its grip on science and access to Western scientific literature improved. After approximately a thirty year absence, the teaching of comparative psychology was reinstated at the Psychological Faculty (opened at the beginning of the 1960s) in Moscow University. The restoration of the lectures on comparative psychology is the achievement of K. E. Fabri, who lectured at Moscow University for many years and wrote a book based upon these lectures (Fabri 1976). On the other hand, due mainly to the investigators in the field of vertebrate zoology (which had excellent traditions in this country), there was a revival of behavioral research based mostly on ethological concepts. Some results of their activity are represented in the proceedings of the All-Union Conferences on Animal Behavior organized by the Institute for Evolutionary Morphology and Animal Ecology and held at Moscow University in 1972, 1976, and 1984. In the preface to the proceedings of the last conference, one of its leaders, E. N. Panov, noted that there were big changes in the development of behavioral investigations during these twelve years. For instance, the number of participants increased from 146 to more than 600, and the topics of the reports became much broader. Reading the papers of these conferences one can clearly see the flowering of new approaches and new combinations of existing approaches to the study of behavior. It is obvious, however, that it is only the beginning in the development of this perspective. As for the achievements of the physiological school of Pavlov, in the narrow sense, it is not easy to estimate them. On the one hand, hundreds of papers have been written, especially in the 1950s, about the great victories of the teachings of Pavlov. The tradition of describing the investigations of conditioned reflexes exclusively in "pink light" is slowly fading (see, for example, Simonov 1991; Suvorov and Andreeva 1990). In my opinion Russians are not ready for a true critical analysis of the achievements and mistakes brought upon Russian behavioral science by the incidents reported here. It seems such an analysis is easier to conduct abroad (see, for example, Joravsky 1989). To conduct such an analysis properly, however, several generations of behavioral scientists should pass. This includes not only the old generation (whose life was more or less destroyed during these years of the "great jumps" and "big terror") but also people of young and middle age whose minds were damaged by the type of educational system that dominated in this country for so many decades. BEHAVIORAL STUDIES OF INVERTEBRATES The history of behavioral investigations of the main invertebrate groups will be

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presented in the chapters that follow. This section will offer some general considerations and provide a short review of studies omitted by other authors. My additional purpose is to illustrate (by examples taken from behavioral studies of invertebrates) the great influence of nonscientific factors on scientific work in this country. General Remarks It is well known that there were many great names in the last century in the field of invertebrate zoology in Russia (Strelkov 1967). First of all are the names of A. O. Kovalevsky and I. I. Mechnikov. Many brilliant investigations describing the natural history and morphological features of various invertebrates were performed at the end of the nineteenth and in the beginning of the twentieth century. These investigations were performed in universities throughout Russia, including those, for example, in St. Petersburg, Moscow, Kazan, Kijev, and Odessa. After the October Revolution there were a number of famous Russian invertebrate zoologists, including K. I. Skrjabin (the founder of helminthology in this country), E. N. Pavlovsky (who worked in many fields but is known primarily for his work with mosquitoes as disease vectors), V. A. Dogel (a specialist in ecological parasitology and comparative anatomy of invertebrates, whose manual Invertebrate Zoology first appeared in the 1920s and was re-edited until the 1980s, who for many years lectured at Leningrad University), V. N. Beklemishev (who lectured on comparative invertebrate anatomy at Moscow University), N. A. Livanov (who worked at Kazan University and had a special interest in the evolution of the nervous system of invertebrates), and L. A. Zenkevich (the specialist in sea hydrobiology who studied the evolution of movement in different invertebrate taxons), to name but a few. From just this short list it is easy to conclude that there was a rather high level of invertebrate research performed by Soviet zoologists. As for comparative physiological investigations of invertebrates, two main groups—one headed by L. A. Orbeli (in Leningrad) and the other headed by Kh. S. Koshtojants (in Moscow)—should be noted. Orbeli's students who were interested in evolutionary problems worked in the Institute of Evolutionary Physiology and Biochemistry, founded in 1956. Kh. S. Koshtojants (1900-1961), who was the head of the Department of Physiology at Moscow University, created the Moscow school of comparative physiology, which differed from Leningrad's mainly in its biological direction (Sakharov and Turpayev 1988). Koshtojants tried to incorporate physiology into the biological sciences. He had some personal experience in conducting invertebrate research from his work in G. Jordan's laboratory. He retained interest in these animals throughout his work at Moscow University and encouraged many of his students to use different invertebrates in their experimental research. The results of this research, together with an exhaustive review of the literature, were presented in his two-volume book, The Comparative Physiology (the second volume was devoted to the comparative physiology of the nervous system) (Koshtojants 1957). One

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may note that many students of Koshtojants retained an interest in evolutionary problems and invertebrate research, for example, T. M. Turpajev, who for many years was the director of the Institute for Developmental Biology; D. A. Sakharov, the great specialist in mollusc neurobiology; and J. Salanki, the director of the ADD Balaton Limnological Research Institute (Hungry), and president of the International Society for Invertebrate Neurobiology. However, the main interests of these scientists were rather far from behavior itself. As for behavioral research on invertebrates, it has been previously noted that for many years in this country there existed only one "acceptable" way of studying behavior, which, of course, was the method proposed by Pavlov. Unfortunately, he had no obvious interest in invertebrate behavioral studies. Though he once worked with the clam Anodonta, it was during his visit to Heidenhain's laboratory in 1884. The history of this work described by Pavlov himself is rather interesting (Pavlov 1885). He wrote in the introduction that he could not fulfill the work proposed by Heidenhain because not much was known about the organization of the nervous system as it related to the control of "leg" movements. Pavlov decided to investigate neural organization of motor control in the clam. This line of research was in fact related to his ongoing interest in the excitatory and inhibitory control of peripheral structures. Work on this problem was still of interest to Koshtojants' students (Koshtojants 1957). It seems, however, that it had no deep influence on Pavlov's investigations of behavior. It is well known that for Pavlov the conditioned reflex was the universal event in the animal world. However, this belief remained to be proved. It is obvious from the book of Majorov (Majorov 1954), which describes in chronological order all work performed in Pavlov's laboratories, that the first investigation of invertebrate behavior was made in 1924 by Kreps (Kreps 1925 and see below). Only then was the following problem considered: Is it possible to elaborate the conditioned reflex in animals without a cerebral cortex? (Majorov 1954). To judge the level of Pavlov's interest in invertebrates, it is helpful to look at his complete works including the "Pavlovskije sredy," three books of notes that were written during Pavlov's laboratory sessions held every Wednesday (Pavlov 1954). It was noted above that the evolutionary approach to studying behavior was generated by Orbeli. One of his students, L. G. Voronin (who was one of the "winners" at the Session of 1950) became the head of the new department founded at Moscow University after this session (the Department of the Physiology of the Higher Nervous Activity). Under his leadership, many investigations were done on the comparative physiology of conditioned reflexes, and some of them used invertebrates (see below). The main results of these studies and Voronin's view concerning this problem were represented in his lectures, which were edited several times (Voronin 1957, 1977, 1984). The comparative physiological approach to behavior and the study of the evolution of conditioned reflexes took place at the Rostov-on-Don University and were performed by A. B. Kogan. Kogan wrote a manual entitled The Bases of the Physiology of Higher Nervous Activity (first edition 1959; second edition 1988), in

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which he considered the problem of the evolution of higher nervous activity from the Protozoa on up. Moreover, his experimental course at the university was organized in such a way that students used various invertebrates for the study of behavior (Kogan and Shchitov 1954). The practical works in Kogan's course on physiology of the nervous system were divided into two groups: "lower" and "higher" nervous activity. In the first group, there was a description of such tasks as hydra reflexes, the reflectory movements of the isolated snail "leg," the reflectory coordination of worm movements, the role of the head ganglia in worm movement, the reflexive behavior of antennas in the cockroach, the reflexes in the isolated ant head, and circling movements of the crayfish after unilateral deletion of the cerebral ganglion. In the second part of this course there were experiments directed toward the study of the evolution of conditioned reflexes: conditioned orientation of searching movements in hydra (see below); conditioned reflexes to sound in Ascidia (see below); conditioned reflex to skin and muscle stimulation in worms; the extinction of natural defense reactions in the snail; the transition of the "negative" response to illumination into a "positive" one in the beetle Tenebrio molitor; and the elaboration of color discrimination in the cockroach. Considered below will be work done at Rostov University on the conditioned reflexes of Daphnia. Unfortunately, no data are available about other works in the field of invertebrate behavior that were made by this group of investigators. Following these general remarks is a description of some papers concerning behavioral investigations in invertebrates that were omitted in subsequent chapters and are important from a historical point of view. The first to be represented is a group of organisms considered by many not to be animals. There is, however, a long tradition of describing the nervous functions and behavioral organization in unicellular organisms, the Protozoa. Protozoa In the beginning of this century many countries, including Russia, expressed great interest in unicellular organisms. S. I. Metalnikov, who worked for many years with infusorians, published in the prestigious journal Izvestiya Imperatorskoj Akademii Nauk (Metalnikov 1915) a remarkable paper "Reflex as a creative act" which was later criticized by some scientists up until the 1950s (Koshtojants 1957; Lobashov 1951). In this paper, he discussed some general problems concerning reflexive reactions. In his opinion, even in infusorians the reflex reactions to different extrinsic influences are extremely variable. The variability of reflexes can easily be observed, "especially in unicellular organisms. In this case we can see every reaction in its entity, in all its appearances. The whole animal—the single cell—is before our eyes under the microscope, and we can discriminate all the peculiarities of every response" (Metalnikov 1915:1803). The variability observed in the reflexive behavior of protozoans suggested to Metalinikov the presence of individual behavior acquired through experience.

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Certainly this position of the author could not be accepted by many behavioral scientists of that time (and especially of the time that followed). The position expressed in that paper was criticized as an "idealistic" one. Different from this "idealistic" paper is another remarkable investigation on Protozoa which seems to be forgotten in Russian literature. This work was a "pioneer" in several respects. I refer to a paper of N. N. Plavilshchikov (18921962), who later became the famous entomologist who contributed much to the Zoological Museum at Moscow University. The paper, which was published in 1928 (Plavilstchikov 1928) in one of the journals arising at that time (see above), had the modest title, "Some observations on the irritability phenomena in infusorians." The purpose of the experiments was to elaborate the conditioned reflex in the colonial infusorians Carchesium lachmanni. Touching the colony produced a contraction, which was used as the unconditioned stimulus (US). The conditioned stimulus (CS) was a one minute illumination with red or blue light. Both the conditioned and the unconditioned response were estimated visually by means of a rating scale. After 150-200 CS-US pairings the infusorians began to contract in response to the CS alone. At this stage the CS was presented on test trials in the absence of the US. The conditioned reflex was retained for several days and could be extinguished. Plavilstchikov's experiment was one of the first on classical conditioning in Protozoa. A second purpose of his study was to see what will happen after the transplantation of trained organisms into a colony of untrained organisms. Part of the "learned" colony was transferred to a "naive" colony. The experiments began one or two days after the successful surgery. First, the effects of the CS and US on the "learned" organisms were tested. After 7-22 trials, the "learned" organisms were removed (together with a little part of a "host" colony), and the responses of the "host" colony to the same stimuli were compared with the responses of "controls" (colonies that were simply damaged). The main result was that the host colony had the same reactions to the light that were elaborated in the trained colony. "This second part of the experiment, in my opinion, is especially important because it indicates the possibility of a special kind of induction' of intracellular transmission of capability for a conditioned response" (Plavilstchikov 1928:24). Later, the author noted that he obtained similar results in experiments with worms. As for the mechanism of this phenomenon, he tried to explain it by means of the "irritation concept" of P. Lazarev and by the "biological rays" of A. Gourvich, which were popular at that time in Russia. It is evident that this work opened several lines of investigation. First of all it introduced a new technique for studying associative processes in Protozoa. Second, it indicated the possibility of memory transfer in these organisms (it is interesting to note that the problem of memory transfer seemed rather important in those years). Third, the author's explanation of his data indicated the high level of biological thought in this country during the 1920s; it was later destroyed when such biological branches as embryology, biophysics, and genetics were repressed. It would certainly be of interest to repeat these experiments today.

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Coelenterata A short paper of Zubkov and Polikarpov (1951), which was rather popular in its time, seems now to have interest only from a historical point of view. The main purpose of this work (as noted by the authors) was to discredit Beritov, who had suggested that animals with "primitive" nervous systems had only inborn or innate behavioral reactions. The experiments were performed on hydra. The animal was placed in a watch-glass filled with water. It was discovered that the incidents of the hydra's searching movements inside the glass depended on the animal's position relative to the previous water level. Changing the water level influenced these movements, but the movements remained for a time in the same form as before. The authors believed that this fact (keeping the movements characteristic of one situation an hour or more after a new water level was introduced) indicated the elaboration of a conditioned reflex in hydra. "This experimental fact gives the refutation of the metaphysical concept of the academician I. S. Beritov concerning the supposed lack of the acquired nervous activity at the early stages of nervous system development" (1951:302). The above mentioned paper seems strange in its purpose and in its results. A more detailed investigation made several years later (Chaylakhian 1957) did not support the conclusion of Zubkov and Polikarpov. He tried to form a classically conditioned reflex in hydras by means of combining illumination with electrical stimulation through the water. After special consideration of their responses to CS, US, and their combination, L. M. Chaylakhian concluded that "the phenomena which from the first sight seem to indicate the presence of contemporary connections in . . . Coelenterata after more detailed physiological analysis can be explained with more simple physiological processes" such as the rise of excitability, accommodation, and the like (Chaylakhian 1957:773). He noted too that the data obtained in Zubkov's and Polikarpov's experiments can also be interpreted this way. Ascidians As noted above, the first work on invertebrates done in Pavlov's laboratory belonged to E. M. Kreps, who is known as a prominent comparative physiologist and biochemist and as Director of the Institute of the Evolutionary Physiology and Biochemistry (1960-1975). At the time of that work, however, he was a student of the Military Medical Academy. He participated also in the work of Pavlov. In the summer of 1921 he visited the Biological Station near Murmansk, which was founded in the last century and belonged to the Society of Natural Scientists in Petrograd (the name of St. Petersburg in the first years after the October Revolution). Kreps convinced Pavlov to request this society to organize at the Murmansk Station, a laboratory for studying higher nervous activity of the primitive ("the lower") animals. This request was fulfilled, and Kreps began to investigate the behavioral reactions of animals abundant in the White Sea that

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could be kept easily in the laboratory. These included Ciona intestinalis, Pyura aurantia, Styela rustica, and Phallusia obliqua, who belong to a very strange taxon, Ascidia. Included in this taxon are very primitive animals with sedentary lives, which nevertheless are relatives, not of the invertebrates, but of Chordata. The results of this thorough investigation were reported at the meeting in Pavlov's laboratory ("Wednesday" meetings) and approved by Pavlov, who recommended them for publication (however, Pavlov never referred to them). Kreps began with a detailed study of the ascidian response to a single mechanical stimulus (a drop of water striking from above). The strength of the stimulation could be easily controlled by adjusting the height at which the drop was released. With this method it was possible to adjust the stimulation so that there was a contraction of only the syphon or, alternatively, a contraction of the whole body. Then he investigated these responses to repetitive mechanical stimuli in intact animals and in animals with ablated ganglion. He also studied the influence of environmental factors such as temperature and tried to elaborate the conditioned reflex to illumination and sound. It appeared that ascidians could produce only two kinds of movements: (1) contraction of the body or the syphon (defined as "defense reaction") caused by some extrinsic stimuli and (2) contraction of the syphon ("throw out reaction") in response to some intrinsic agents (such as chemicals introduced into the syphon). Weak artificial light was ineffective, and strong illumination resulted in a defensive reaction. Sound itself caused no reaction; after pairing with the mechanical stimuli, "it became the excitor of the defense reaction." It is evident that the author was very cautious in speaking about conditioning in ascidians. He was not sure that he could discriminate between the conditioned reflex in ascidians and the rise of excitability due to repetitive stimulation. "It is not easy to draw the distinct line between the conditioned response and the simple rise of the excitability in primitive animals with a very limited range of reactions" (Kreps 1925:226). It should be noted that Kreps maintained this point of view throughout the remainder of his life, though some authors tried to conclude on the basis of this work that even in ascidians the conditioned reflexes have been demonstrated unequivocally (Lobashov 1951). Thus the first work devoted to conditioned reflexes in invertebrates in Pavlov's laboratory was undoubtedly performed at the personal initiative of E. M. Kreps (Kreps 1989). He worked at the Murmansk Biological Station for ten years. However, it was his first and last work with invertebrate behavior. Very soon his interests shifted into comparative investigation of different functional systems, using biochemical techniques. It also seems that the behavioral investigation of these interesting animals has never been sustained in this country. Lower Crustaceans Work performed at the beginning of the 1950s at Rostov University concerning the hereditary fixation of the conditioned reflexes in lower animals (Kogan and Semenovykh, 1955) is representative of work performed at that particular time

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period in Russia and points nicely to the fact that the inheritability of conditioned reflexes was a popular research topic. The history of Pavlov's views on this problem and his experimental approach have been described many times before (see, for instance, Voronin 1957). Kogan and Semenovykh (1955) thought that the main factor hindering the experimental proof of the heredity of conditioned reflexes was the technical difficulty of producing so many generations of vertebrate material. They selected as their research animal a lower crustacean that is very common in small freshwater ponds: Daphnia. Daphnia needs only 2-3 weeks for parthenogenetic reproduction. In nature these animals prefer illuminated places where they search for food. In the laboratory, food was withheld during the daytime. Instead, they were fed only at night. Once a week the light-dark preference was estimated from the number of animals located in the illuminated and darkened area of the tank. The offspring were given a light-dark pretest to determine their preference. After this pretest, they experienced the same procedure as the "parents." By the seventh and eighth generations there was a significant increase in the "positive reaction to darkness," and by the fourteenth to seventeenth generations most of the animals preferred darkness. The authors concluded that "the conditioned feeding reflex to darkness which has been elaborated during many generations became the unconditioned one" (Kogan and Semenovykh 1955:111). This conclusion was criticized several times in the 1960s. As L. V. Krushinsky noted, It is absolutely clear that in this work there was no estimation of the individual survival and individual rate of reproduction as dependent on the time needed for the elaboration of the conditioned reflex to the darkness in a single animal. However, the individual differences in the adaptation to unusual conditions of feeding should be reflected obviously in the survival and the reproduction rate. Thus the selection process will be inevitable. (Krushinsky 1968:122) The authors proclaimed in their preface, however, that they paid special attention to eliminate the effect of natural selection. It is not easy to understand now why this opportunity to elaborate on hereditary conditioned reflexes seemed so pleasing to many scientists. Evidently the individual experience—even it would be hereditary—cannot influence the whole population and should be extinct. On the other hand, there are some questions concerning the above-described conditioned reflex in itself. Strictly speaking, it is difficult to compare it with the conditioned reflexes in mammalia, because of its schedule (first of all, there was no individual modification of behavior caused by the combination of CS and US) and to the incompleteness of such data in the paper. Insecta I would like to end this brief account of the works that have used rather "exotic"

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animals for behavioral research by referring to the investigation of the most "popular" invertebrates, which will be considered in the following chapters. The reason for this approach is twofold: First, these works have obvious interest from a historical point of view. Second, they are forgotten in this country and probably are not known abroad because of such nonscientific reasons as World War II and the period in which Soviet science was cut off from the West. It was noted previously that L. A. Orbeli built in Koltushi the laboratory of insect biology headed by S. I. Malyshev. There is no available evidence about the relationship between S. I. Malyshev and V. A. Vagner. It seems strange, however, that Malyshev's papers of that time make no references to Vagner (the same is true for his collaborators' papers). It is strange because this group of investigators used the same experimental animal as Vagner and used the same approach to the study of behavior as Vagner. Perhaps in the future the reason for this will be made clear. Before the war, Malyshev elaborated in detail the choice of experimental animals that can be used for the study of instinctive reactions (the paper devoted to this topic was sent to a journal in April 1941; it was published only after the war, in 1946). In his opinion, the best subjects for the investigation of instinct are insects, in which instinctive behavior is not complicated with behavior acquired through individual experience. In Malyshev's opinion, instinctive actions manifest themselves in the form of very complicated activities of the animal such as nest building, foraging, and the like. At the same time, they do not need individual experience for their expression. Thus, the main problem for him was studying the evolutionary changes in some instinctive activity in a group of related animals. His research program was based on several groups of Hymenoptera. He was, for example, able to trace the development of elementary instinctive reactions of the Hymentoptera ancestors of the wasp. The results of this work, which took many years to complete, were published in the Magazine of General Biology in 1949. It should be noted that this was a long and thoroughly edited paper containing a large list of references. This paper awaited publication for more than a year. In the same issue was a paper by Maksimova and Genkel (1949) on "The theory of stadial development and its importance for the plant physiology." The reason for including the latter paper is clear to Russian biologists, but it needs some explanation for the Western reader. The main purpose of this article was to promote the propaganda of Lysenko's views and to suppress the views of his opponents. It is obvious that the paper had been written especially for, and approved by, the leaders of the Communist party. I would like to note also a paper of Poljakov in the same magazine of that time entitled "The ideological meaning of the views about periodicity in mass birth of the field mice" (Poljakov 1949). This article was directed against the prominent Russian zoologists Formozov and Kalabukhov. The work of these zoologists was considered by some as "the propaganda of the mechanistic ideas of the foreign ecology"; the author appeared to struggle with these foreign ideas and with the native scientists who support such ideas. I apologize for this short distraction, the purpose of which was to remind you of the time when the Malyshev paper appeared. It is evident that almost

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nobody paid attention to it. As for the main content of Malyshev's paper, he described seven main stages of instinctive behavior necessary to supply larva with the food that is so important for its metamorphosis. The author could explain only a part of this long process. He wrote at the end of his paper: "At this point we are very close to the second part of our investigation: the evolution of instinctive activity of wasp-like insects" (Malyshev 1949:41). However, it was his last publication. There is some indication that Malyshev was forced to stop this work as it neared completion. In the same laboratory there were studies of instinctive behavior of other insects. First of all there is the beetle Deporaus betulae which lives on birch. It cuts a leaf, rolls it around the egg, glues the borders and gnaws through the leafstalk so that the leaf sinks to the ground, where the larva matures (Oksenov 1946). This paper was presented to the magazine in the spring of 1941; it was published only in 1946, with a mourning frame around the author's name. The main conclusion in this article was the presence of a high degree of plasticity of this very complicated behavior. Unfortunately, this work was never continued. Other work was performed on the larval behavior of different species of ant lion (Euroleon europaeus Mak. L., Myrmeleon formicarius L. and Formicaleon lineatus Fabr.). The investigation of their feeding behavior were carried out under natural conditions, with semiartificial arrangements in nature, and in the laboratory. It was noted that the larvae can easily be kept in the laboratory for 9-12 months. The main conclusion of this investigation indicated that there was considerable plasticity of behavior (Puzanova-Malysheva 1947). The work of Puzanova-Malysheva, like so much else in the laboratory of insect biology at Koltushi, was never extended. One may only guess what happened. It is probably not a coincidence that the laboratory disappeared soon after the Pavlovian Session of 1950. Moreover, it is interesting to note that the closing of the laboratory is not mentioned in a history of the Physiological Institute, part of which became the Institute in Koltushi after 1950 (see Zakharzhevsky and Andreeva 1984). Instead, there appeared the laboratory of behavioral genetics, which used insects as experimental animals. Its experimental approach, however, differed significantly from Malishev's. The head of this laboratory, M. E. Lobashov, who was a geneticist, did not approve of Malishev's and Vagner's interest in the evolution of instinctive activity. In his opinion, most insect behavior consisted of conditioned reflexes. He also believed that the heredity of conditioned reflexes can be discovered in invertebrates much more readily than in vertebrates (Lobashov 1951). MAJOR VIEWS CONCERNING BEHAVIORAL SPECIFICITY OF INVERTEBRATES It has been noted above that V. A. Vagner was the prominent specialist in invertebrate behavior. He also had, however, a deep interest in vertebrate behavior (see, for instance, his book about the evolution of nest-building activity in

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swallows). It is obvious that the problem of evolution of psychic (behavioral) activity (from amoeba to human beings) dominated his scientific interests throughout his life. His concepts and ideas concerning the evolution of behavior have been presented several times but most completely in his Essays in comparative psychology (1924-1929). Briefly, the essence of his views can be expressed as: "The excitability as the basement; the reflex as the consequence; the instinct as the hereditary knowledge; the emotions as the adaptation; the mind as the leader of the behavior; the progress as the inevitable result" (this quintessence of Vagner's concept was reproduced in each issue of the Essays). According to his scheme, which resembles a branch, the main stem represents the evolution of life. A branching "twig" begins with protozoa and ends with the infusorians. In multicellular organisms there are two main lines of evolution: the evolution of the excitability of the cells in body tissues responsible for the development of character and temper, and the evolution of the nervous system producing reflexive activity that develops into instinctive and cognitive behavior. Vagner believed that animals with a diffuse nerve net have only reflex reactions. That is, their behavior depends completely on extrinsic factors. He insisted on differentiating between reflexes and instincts (though in evolution the instinctive activity developed from reflexes). Based on some developmental data Vagner concluded that evolution of instinct had five main stages: in the first stage it may be confused with the reflex; in the second there are distinctive instinctive actions which appear due to mutations; in the third there are the instincts which developed from the above-mentioned stages by means of mutations or other events; in the fourth there are the instincts of the higher order, and in the fifth there is the modification of instincts due to environmental contingencies. Vagner insisted that there were important distinctions between the invertebrates and vertebrates and suggested that these distinctions were caused by the organization of their central nervous systems. Vertebrates having brains, especially those having cerebral cortexes, possessed higher psychic abilities, including complex instinctive and cognitive activity, good memory, true learning, a set of emotional reactions, and so on. Invertebrates that possess ganglionic nervous systems exhibit complex instinctive activity but lack emotional reactions, cannot learn "by means of individual experience or by imitation," and have a memory that is qualitatively different from vertebrate memory. It is worth noting that many years ago he observed the heart activity of spiders in different situations and concluded that frequency of heartbeats can vary easily. However, he concluded that "the change in the activity of the invertebrate vasomotor system has nothing in common with the changes which seem to be connected with emotions in higher vertebrates. Today Vagner's concepts and ideas seem rather rigid and mechanistic. They belong to the past. However, we must not forget that the published work of Vagner and of many other scientists of that time, was never reissued for a new generation of students or analyzed seriously. Critical appraisals of Vagner's views concerning invertebrate behavior are also absent. As I noted above, references to Vagner's

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spider work were missing even in Malishev's papers. It is difficult to believe such an omission was an oversight. One may suggest that Malyshev and his colleagues did not want to criticize Vagner and therefore preferred not to cite him at all. This seems possible because of the negative estimations of Vagner's contributions. Presumably any scientist publicly supporting Vagner's position would be subjected to the same criticisms and might be shunned (or worse) in academic circles. The lack of public interest in Vagner during the 1940s-1960s might not mean, however, the absence of private interest in his work. In researching this chapter, I came upon a copy of his major book Biopsychology (published only in 1910 and never reissued) which belonged, during the 1950s, to a prominent specialist in vision research, S. Kravkov. This particular copy contains many notes suggesting that Kravkov had a keen interest in Vagner. An objective evaluation of Vagner's work should be made in the future. Like Vagner, Krushinsky and Beritov also had an interest in the development of central nervous system function across the phylogenetic scale. Differences in the behavior of animals were correlated with the degree of organization in the central nervous system. The more complex the organization, the more complex the behavior. Both of these researchers, however, had only limited experience with invertebrates. Although Beritov did not have any personal experience in invertebrate research, he did develop a scheme describing behavior at all levels of the phylogenetic scale. "The lowest behavioral form is the inborn reflexes of the lower invertebrates. They have only one kind of behavior; that is, reflexive reactions in response to an extrinsic stimulus" (Beritov 1947:22). "With the development of the CNS arises the higher kind of inborn behavior—instinctive behavior which depends strongly on the intrinsic processes in the organism" (Beritov 1947:23). It should be noted that Beritov had a special interest in the spontaneous activity of nervous tissue and conducted electrophysiological experiments with a wide range of animals. As we move up Beritov's scale, we find that the next type of behavior is based on individual experience. He believed such behavior is typical of higher vertebrates and human beings. He also believed some higher invertebrates, for instance, crustaceans, insects, and cephalopods, also profit from individual experience. Beritov's views on the phylogenetic development of behavior are rather similar to those of Vagner. Beritov, however, did not see such a large gap between vertebrates and invertebrates as Vagner did. It is interesting to note that the idea that some invertebrates can profit from experience caused him to be demoted. In 1948 Beritov continued to maintain that some invertebrates can modify their behavior as the result of experience (Beritashvili 1984). After the Pavlovian Session of 1950, he modified his position and now, naturally, agreed with his critics that he was wrong: "I am convinced that my idea about the individual behavior ruled by the representation in the case of invertebrates such as crustaceans and cephalopods is doubtful. I myself did not perform any experiments with them, therefore I had no right to use them (Beritashvili 1984:603). In addition to the work of Krushinsky and Beritov, two other influential Russian

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scientists concerned with the evolutionary analysis of behavior were Fabri and Voronin. We will first briefly discuss the approach taken by Fabri and then conclude with a discussion of Voronin. Fabri's views have been based on the ideas of the prominent Russian psychologist A. N. Leontyev, who proposed two main stages in psychic development: (1) the "elementary sensory" stage and (2) the "perceptive" stage (Leontyev 1959). Leontyev defines elementary sensory psychic activity as that stage of reality representation in which organisms have only a single elementary "feeling." The perceptive psychic activity stage corresponds to the representation of the external world in the form of complex images. Fabri added to this general scheme by subdividing each stage into lower and higher levels. Basing his work on a review of the literature, he was able to superimpose his scheme of psychic development upon a picture of the phylogenetic tree (Fabri 1976). The attempt was not altogether successful. He believed that the primary difficulty was the fact that morphological indices used in constructing phylogenetic trees can not be used to accurately predict psychical development (Fabri 1976). Using a somewhat different approach to the evolutionary study of psychic activity was Voronin. It has been noted throughout this chapter that the dominant method of studying behavior in this country is not from the psychological perspective but rather by the physiological or, more strictly, by the conditioned reflex approach, founded by Pavlov. In later years the Pavlovian approach was broadened somewhat to include comparative analysis. Comparative research of learning phenomena from the Pavlovian perspective had a place for many years at the Department of the Physiology of Higher Nervous Activity, which was headed by Voronin at Moscow University. Using the technique of elaboration of conditioned reflexes similar to that used in Pavlovian experiments with vertebrates, comparative experiments were conducted with various species of worms, decapod crustaceans (Karas 1962, 1963; Burmistrov and Shuranova, this book), and social insects (see Udalova and Karas, this book). An important contribution has also been made by N. A. Tushmalova, who studied the capability to store individual experiences in protozoans and primitive worms (Tushmalova 1987). Based on the experiments of his coworkers, Voronin proposed that there are "levels in the evolution of higher nervous activity" (Voronin 1970, 1984). Combining this with the scheme of Tushmalova, the evolutionary process may be described in the following way. At the first level there are only "summation" responses. These responses are based on an increase of the excitability of the nervous system or (in Protozoa) of the intracellular structures caused by the summation of the traces of previous excitations with the existing state of excitation. Such reactions take place in Protozoa, multicellular animals with a diffuse nerve net, and in the spinal cord of vertebrates. At the second level we see the appearance of habituation. This relates, as does the previous stage, to the "nonsignal reactions of individual adaptation" common to many organisms, from the infusoria to the human being. At the third stage, there is the "non-retrieved (nonstable) conditioned reflex" described in planarians. At the fourth level "true" conditioned reflexes begin to appear. They probably arise first in annelids having

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mushroom bodylike structures in the cerebral ganglia. The insect, whose brain is extremely well developed, demonstrates positive and negative conditioned reflexes, chain reflexes, and instrumental conditioning. It has been shown that the rate of the elaboration of the conditioned reflexes does not depend on the position of the animal on the phylogenetic scale; thus, there is no difference between invertebrates and vertebrates in this sense. These "true" conditioned reflexes are rather stable; they may be stored many hours or days, and even years. The next levels (fifth and sixth) are characterized by much more complicated processes such as reasoning and extrapolation, and are typical only of higher vertebrates and man. CONCLUSION A dramatic and sometimes tragic situation characterized Russian science in the twentieth century. The effect of this situation is readily evidenced in all areas of Russian science, including behavioral research with invertebrates. Of course, there are universal problems confronting scientists and nonscientists alike in all countries. However, additional problems were faced by Russian scientists, specific to the Soviet period. These include ideological and administrative control of science and gradually isolation from the world scientific community, particularly from the 1930s into the 1950s. The time of "the repressed science" ended in essence in the 1950s. However, its influence is still felt even today. Looking back, as I have tried to do in this chapter, one sees clearly how many scientific movements, the reputations of excellent researchers, and the intellectual life of the average individual scientist were damaged or exterminated. Moreover, as time goes on it is doubtful that the scientific work, as reflected in books and research articles, of those who did not follow the "party line" will be found or republished for the next generation of students. It is difficult to predict what results in understanding invertebrate behavior would have been obtained in Russia if the various approaches to the study of behavior had been free to develop. I have tried to show that even under conditions of social and political pressure, some scientific investigation of animal behavior was in progress. In the chapters that follow this historical introduction, the experimental data come predominantly from the Pavlovian perspective (i.e., the position represented by Voronin). The reasons for this are the various social and political factors we have discussed. This "monastic" approach to the study of invertebrate behavior allows the reader to estimate personally the achievements and the mistakes brought about by factors not related to doing science. ACKNOWLEDGMENTS I would like to thank N. Yu. Alekseenko, Yu. M. Burmistrov, M. E. Ioffe, L. G. Leibson, Ya. M. Pressman, and M. E. Varga for reading this manuscript and encouragement. I am also grateful to N. L. Krushinskaya for supplying the photograph of her father.

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REFERENCES Aleksandrov, V. Ya. 1992. Trudniye gody sovetskoy biologii (Hard times in Soviet biology). Sankt-Petersburg: Nauka. Bekhterev, V. M. 1907/1912. Obyektivnaya psikhologiya (Objective psychology). In: Pamyatniki psikhologicheskoy mysli (Memorials of psychological ideas). Moskva: Nauka, 1991. Beritashvili, I. S. 1984. Trudy (Works: Problems of muscle physiology, neurophysiology and neuropsychology). Tbilisi: Metsniereba. Beritov, I. S. 1947. Ob osnovnykh formakh nervnoy i psikhonervnoy deyatelnosti (The main forms of nervous and psycho-nervous activity). Moskva, Leningrad: Izdatelstvo Akademii nauk SSSR. Borovsky, V. M. 1936. Psikhicheskaya deyatelnost zhivotnykh (Psychological activity of animals). Moskva, Leningrad: Biomedgiz. Chaylakhian, L. M. 1957. About the temporary connections in Protozoa and Coelenterata (In Russian). Zhurnal Vysshey Nervnoy Deyatelnosti 7:165-114. Fabri, K. E. 1969. V. A. Vagner and Modem Zoopsychology (In Russian). Voprosy Psikhologii 6:100-107. . 1976. Osnovy zoopsikhologii (Bases of zoopsychology). Moskva: Izdatelstvo Moskovskogo Universiteta. Frolov, Yu. P. 1925. Fiziologicheskaya priroda instinkta s tochki zreniya ucheniya ob uslovnykh i bezuslovnykh refleksakh (Physiological nature of instincts from the standpoint of the conditioned and unconditioned reflex theory). Leningrad: Vremya. Grigorian, N. A., and Roitbak, A. I. 1991. The bad years in the life of the academician I. S. Beritashvili (1947-1956) (In Russian). In: Repressirovannaya Nauka (The oppressed science), 297-304. Leningrad: Nauka. Guberman, I. 1977. Bekhterev: stranitsy zhizni (Bekhterev's life). Moskva: Znaniye. Gureeva, N. M., and Chebysheva, N. A. eds. 1969. Letopis zhizni i deyatelnosti akademika I.P.Pavlova. Tom 1:1849-1917 (I. P. Pavlov: Chronicle of his life and work. Vol. 1: 1849-1917). Leningrad: Nauka. Joravsky, D. 1989. Russian Psychology: A critical History. Cambridge Mass.: Basil Blackwell. Karas, A. J. 1962. Conditioned reflexes with food reinforcement in the Black Sea crab Carcinus maenas caused by visual, tactual and vibrational stimuli (in Russian). Zhurnal Vysshey Nervnoy Deyatelnosty 72:748-756. . 1963. New data concerning the conditional inhibition in the crab Carcinus maenas (in Russian). Nauchnye Doklady Visshey Shkoly, Seriya Biologicheskaya 13: 85-89. Kashkarov, D. N. 1928. Sovremenniye uspekhi zoopsikhologii (Progress in zoopsychology). Moskva, Leningrad: Gosudarstvennoye Izdatelstvo. Kholodkovsky, N. A. 1897. Instinkt i razum (Instinct and Mind). Nauchnoye Obozreniye 7:9-37. Khotin, B. I. 1990. Studying of the behavior of plovers and gulls in the bird colonies on Novaya Zemla, Bezymyanny cape (in Russian). In: Istoriya i nekotorye voprosy sovremennogo sostoyaniya eksperimentalnykh issledovaniy v otechestvennoy psikhologii (The history and some problems of experimental research in the native psychology) 266-294. Moskva: Institut Psikhologii an SSSR. Kim, S. D. 1966. Psikhologicheskiye i pedagogocheskiye vossreniya V. A. Vagnera (Psychological and pedagogical Views of V. A. Vagner). Moskva: Pedagogichesky

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Institut imeni V. I. Lenina. Kogan, A. B. 1959,1988. Osnovy fiziologii vysshey nervnoy deyatelnosti (The basis of the physiology of higher nervous activity). Moskva: Vysshaya Shkola. Kogan, A. B., and Semenovykh, A. P. 1955. About the hereditary fixation of the conditioned reflexes in lower animals (in Russian). Priroda, 9:110-111. Kogan, A. B., and Shchitov, S. I. 1954. Praktikum po sravnitelnoy fiziologii (Manual on comparative physiology). Moskva: Sovetskaya Nauka. Koshtoyants, Kh. S. 1957. Osnovy sravnitelnoy fiziologii. Tom 2: Sravnitelnaya fiziologiya nervnoy sistemy (Bases of comparative physiology. Vol. 2. Comparative physiology of the nervous system). Moskva: Izdatelstvo Akademii nauk SSSR. Kots, A. F. 1917. Zhizn zhivotnykh v fotografiyakh, risunkakh i bilogicheskikh nabroskakh iz Moskovskogo zoologicheskogo sada (Animal life: Photos, drawings and essays made in Moscow Zoo). Moskva: Izdatelstvo russkogo Obshchestva akklimatizatsii Zhivotnykh i Rasteniy. Krementsov, N. L. 1991. From agriculture the medicine (in Russian). In: Repressirovannoya Nauka (The oppressed science) 91-113. Leningrad: Nauka. . 1992. V. A. Wagner and the origin of Russian ethology. International Journal of Comparative Psychology 6:61-70. Kreps, Ye. M. 1925. Ascidian responses to the external stimuli (in Russian). Arkhiv Biologicheskikh Nauk 25:197-226. Kreps, Ye. M. 1989. O prozhitom i perezhitom (My life and experience). Moskva: Nauka. Krushinsky, L. V. 1948. Some stages of integration in the formation of behavior in animals (in Russian). Uspekhi Sovremennoj Biologii 26:131-152. . 1968. Are the conditioned reflexes inheritable? (in Russian). Priroda 7:120-123. . 1977. Biologicheskiye osnovy rassudochnoy deyatelnosti (Biological bases of the intellect). Moskva: Izdatelstvo Moskovskogo Universiteta. . 1991. Evolutsionno-Geneticheskiye Aspekty Povedeniya (Behavior: evolutionary and genetic principles). Moskva: Nauka. Ladygina-Kots, N. N. 1923. Issledovaniye Poznavatelnykh Sposobnostey Shimpanze (An investigation of chimpanzee abilities to reason). Moskva-Petrograd: Gosudarstvennoye Izdatelstvo. . 1928. Prisposobitelnye Motornye Navyki Makak v Usloviyakh Eksperimenta. K voprosu o "Trudovykh Protsessakh" Nizshikh Obezyan (Adaptive motor habits of the macaca rhesus under experimental conditions. A contribution to the problem of "labor processes" of monkeys). Moskva: Gosudarstvenny Darvinovsky Muzey. . 1935. Ditya Shimpanze i Ditya Cheloveka (A child of the chimpanzee and a human child). Moskva: Gosudarstvenny Darvinovsky Muzey. . 1958. Razvitiye Psikhiki v Protsesse Evolutsii Organismov (Psychical development in evolution). Moskva: Sovetskaya Nauka. . (1960). The Work of Soviet Scientists in Comparative Psychology (in Russian). In: Psikhologicheskaya Nauka v SSSR (Soviet psychology). Moskva: Izdatelstvo Akademii Pedagogicheskikh nauk RSFSR. Leibson, L. G. 1973. Leon Abgarovich Orbeli (in Russian). Leningrad: Nauka. . 1990. Akademik L. A. Orbeli. Neopublikovannye Glavy Biografii (The Academician L. A. Orbeli: Some unpublished chapters from his life). Leningrad: Nauka. Leontyev, A. N. 1959. Problemy Razvitiya Psikhiki (Problems in the development of psychical events). Moskva: Izdatelstvo Akademii nauk SSSR.

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Lobashov, M. E. 1951. The Principle of Temporary Connections in Invertebrate Behavior (in Russian). Uspekhi Sovremennoy Biologii 31:13-37. Lukin, B. V. 1987. V. A. Vagner: The first editor of the magazine Priroda (in Russian). Priroda 7:48-58. Lukina, E. 1956. Alexandr Nikolayevich Promptov (in Russian). In: A. N. Promptov ed., Ocherki po Probleme Biologicheskoy Adaptatsii Povedeniya Vorobyinykh Ptits 306310. (Essays on the biology of adaptive behavior in Passerine birds). Moskva, Leningrad: Izdatelstvo Akademii nauk SSSR. Majorov, F. P. 1954. Istoriya Ucheniya ob Uslovnykh Refleksakh (The history of the conditioned reflex theory). Moskva, Leningrad: Izdatelstvo Akademii nauk SSSR. Maksimova, N. A., and Genkel, P. A. 1949. The theory of "stadial development" and its importance for plant physiology (in Russian). Zhurnal Obshchey Biologii 70:1-13 Malyshev, S. I. 1946. The choice of objects for investigation of the instincts (in Russian). Izvestiya Akademii nauk SSSR, Seriya Biologicheskoya 7:97-104. . 1949. The ways and conditions for the evolution of the instincts in lower Hymenoptera (Symphyta and Terebrantia) in Russian. Zhurnal Obshchey Biologii 70:13-42. Masing, R. A. 1947. Variability and Heredity of Some Forms of Behavior in Drosophila melanogaster. 1. Variations of Photo- and Geotaxis. 2. Egg-laying Preference (in Russian). In: Trudy Instituta evolutsionnoy fiziologii i patologii vysshey nervnoy deyatelnosti imeni I. P. Pavlova, Tom 1. (Papers of the Institute of the Evolutionary Physiology and Pathology of the Higher Nervous Activity named by I. P. Pavlov, vol.1) 285-311. Leningrad: Izdatelstvo Akademii nauk SSSR. Merkulov, V. L. 1960. Alexey Alexeevich Ukhtomsky. Ocherk Zhizni i Nauchnoy Deyatelnosti (1875-1942) (A. A. Ukhtomsky: His life and scientific activity). Moskva, Leningrad: Izdatelstvo Akademii nauk SSSR. Metalnikov, S. I. 1915. Reflex as a creative act (in Russian). Izvestiya Imperatorskoy Akademii Nauk, Seriya 6, 1801-1819. Mojiseeva, N. I. 1988. The Peculiarities of V. M. Bekhterev Scientific School (in Russian). In: Bekhtereva, N. P. ed., Fisiologicheskiye Nauchniye Shkoly v SSSR Ocherki (Essays on the Physiological "Schools" in USSR) 85-93. Leningrad: Nauka. Oksenov, B. A. 1946. Behavior of the beetle Deporaus betulae L. in experimental conditions (in Russian). Izvestiya Akademii nauk SSSR, Seriya Biologicheskoya, 7:105-116. Orbeli, L. A. 1949. Voprosy Vysshey Nervnoy Deyatelnosti (Problems of the higher nervous activity). Moskva, Leningrad: Izdatelstvo Akademii nauk SSSR. Pavlov, I. P. (Joh. Pawlow). 1885. Wie der Muschel ihre Schaale offnet. Versuche und Fragen zur allgemeinen Muskelund Nervenphysiologie. Archiv fur die gesammte Physiologie des Menschen und der Thiere, Bd. 57:6-31. . 1923. Dvadtsatiletniy opyt Obyektivnogo Izucheniya Vysshey Nervnoy Deyatelnosti Zhivotnykh (Twenty years of the objective investigation of higher nervous activity in animals). In: Polnoye Sobraniye Sochineniy, torn 3, kniga 1 (Complete Works, vol. 3, book 1). Moskva, Leningrad: Izdatelstvo Akademii nauk SSSR. . 1951. Polnoye Sobraniye Sochineniy. Tom 1 (Complete works, vol. 1). Moskva, Leningrad: Izdatelstvo Akademii nauk SSSR. . 1952. In memory of R. Heidenhain (in Russian). Polnoye sobraniye sochineniy. Tom 6 (Complete works, vol. 6, 96-108). Moskva, Leningrad: Izdatelstvo Akademii

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nauk SSSR. . 1954. Bibliografiya Trudov I. P. Pavlova i Literatury o Nyem (Bibliography of Pavlov's works and of the materials about him). Moskva, Leningrad: Izdatelstvo Akademii nauk SSSR. Plavilstchikov, N. N. 1928. Observations sur l'excitabilite des infusoires. Russky Arkhiv Protistologii, 7:1-24. Poljakov, I. Ya. 1949. The ideological meaning of the views about periodicity in mass birth of field mice (in Russian). Zhurnal Obshchey Biologii 70:246-260. Pomper, P. 1970. The Russian Revolutionary Intelligentsia. New York: T. Y. Crowell Company. Promptov, A. N. 1947. On the evolutional biological peculiarities of the orienting response in some ecologically specialized bird species (in Russian). In: Trudy Instituta Evolutsionnoy Fiziologii i Patologii Vysshey Nervnoy Deyatelnosti imeni I. P. Pavlova, Tom 1 (Papers of the Institute of the Evolutionary Physiology and Pathology of the Higher Nervous Activity named by I. P. Pavlov, vol.1) 247-258. Leningrad: Izdatelstvo Akademii nauk SSSR. . 1956. Ocherki po Probleme Biologicheskoy Adaptatsii Povedeniya Vorobyinykh Ptits (Essays on the biology of the adaptive behavior in Passerine birds). Moskva, Leningrad: Izdatelstvo Akademii nauk SSSR. Puzanova-Malysheva, E. V. 1947. The Ant-lions and Their Funnel Traps (in Russian). In: Trudy Instituta evolutsionnoy fiziologii i patologii vysshey nervnoy deyatelnosti imeni I. P. Pavlova, Tom 1 (Papers of the Institute of the Evolutionary Physiology and Pathology of the Higher Nervous Activity named by I. P. Pavlov, vol. 1) 259284. Leningrad: Izdatelstvo Akademii nauk SSSR. Roginsky, G. 1940a. V. A. Vagner: K pyatiletiyu so dnya smerti, 1849-1934. (V. A. Vagner: Five years after his death). Sovetskaya Pedagogika, no. 7:96-100. . 1940b. Razvitiye Mozga i Psikhiki. Albom-Vystavka (Development of the brain and of the psychic life. The description of an exhibition). Leningrad: . 1947. Comparative psychology in the USSR (in Russian). Vestnik Leningradskogo Universiteta, 7:60-69. Roitbak, A. I. ed. 1991. Vospominaniya ob Ivane Solomonoviche Beritashvili (Memoirs about I. S. Beritashvili). Moskva: Nauka. Roitbak, A. I., and Bakuradze, A. N. 1988. I. S. Beritashvili: His Contribution into Physiology (in Russian). In: N. P. Bekhtereva, ed., Fisiologicheskiye Nauchniye Shkoly v USSR. Ocherki (Essays on the physiological "schools" in USSR) 157-170. Leningrad: Nauka. Sakharov, D. A., and Turpayev, T. M. 1988. Kh. S. Koshtoyants' School (in Russian). In: N. P. Bekhtereva, ed., Fisiologicheskiye Nauchniye Shkoly v SSSR Ocherki (Essays on the physiological "schools" in USSR), 241-246. Leningrad: Nauka. Semiokhina, A. F. 1991. L. V. Krushinsky. His Life and Work (in Russian). Zhurn. Vyssh. Nervn.Deyat. 47:618-621. Severtsev, A. N. 1922. Evolutsiya i Psikhika (Evolution and Psychology). Moskva: Izdadelstvo M. i S. Sabashnikovykh. Shimkevich, B. M. 1897. Consciouness, instinct and reflex (in Russian). Obrazovaniye 9:1-16. Shlupikova, A. V. 1968. AkademikA. A. Ukhtomsky (1875-1942) (The Academician A. A. Ukhtomsky). Yaroslavl: Verkhne-Volzhskoye Izdatelstvo. Simonov, P. V. 1991. Interaction of the scientific schools at the formation of the investigatory program (in Russian). Vestnik Akademii Nauk SSSR, 7:43-51.

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Sobol, S. L. 1925. Scientific and popular books in biology and zoology (in Russian). In: R. Goldsmit, ed. Ascarida: Obshchedostupnoye wedeniye v nauku o zhizni (Ascaris: Popular introduction in life science), Translated by S. L. Sobol 343-376. Moskva: Gosizdat. Strelchenko, V. I. 1975. V. A. Vagner as a zoopsychologist-darvinist (in Russian). Istoriya i teoriya evolutsionnogo ucheniya (History and theory of evolutionary teaching), 5:101-110. Strelkov, A. A. 1967. General account of the development of the invertebrate zoology (in Russian). In: Razyitiye biologii v SSSR (Development of Soviet biology), 175-189. Moskva: Nauka. Suvorov, N. F., and Andreeva, V. N. 1990. A problem of hereditability of conditioned reflexes in I. P. Pavlov scientific school (in Russian). Zhurnal Vysshey Nervnoy Deyatelnosty 40:3-14. Svidersky, V. L., Veselkin, N. P., and Natochin, Yu. V. 1988. Orbeli's school (in Russian). In: N. P.Bekhtereva, ed., Fisiologicheskiye nauchniye shkoly v SSSR Ocherki (Essays on the physiological "schools" in USSR), 94-106. Leningrad: Nauka. Tushmalova, N. A. 1987. The main regularities in the evolution of invertebrate behavior (in Russian). In: Fiziologiya Povedeniya: Neyrobiologicheskiye Zakonomernosti (The physiology of behavior: Neurobiological problems), 236-264. Leningrad: Nauka. Umrikhin, V. V. 1991. The "beginning of the end" of behavioral psychology in the USSR (in Russian). In: Repressirovannaya Nauka (The oppressed science), 136-145. Leningrad: Nauka. Vagner, V. A. 1890. Nabludeniya nad Araneina (Observations on Araneina). Trudy Sankt-Petersburgskogo obshchestva yestestvoispytateley, Vom 21. . 1896. Voprosy Zoopsikhologii (Problems in zoopsychology). St. Petersburg: Izdaniye L. F. Panteleeva. . 1900a. Vodyanoy pauk (Argyroneta Aquatica Cl), Ye go Industriya i Zhizn kak Material Sravnitelnoy psikhologii (Water spider Argyroneta aquatica CL: Its industry and life as a subject of comparative psychology). Moskva: Kushnerev. . 1900b. Gorodskaya lastochka Chelidon urbica, yeye industriya i zhizn kak material sravnitelnoy psikhologii (Urban swallow Chelidon urbica, its industry and life as a subject for comparative psychology). Zapiski Imperatorskoy Akademii nauk po Fiziko-Matematicheskomu Otdelu, Seriya VIII, 70:1-125. . 1907. Psycho-biologische Untersuchungen an Hummeln mit Bezugnahme auf die Frage der Geselligkeit im Tierreich. Zoologica 46:1-239. . 1910a. Biologicheskiye Osnovaniya Sravnitelnoy Psikhologii (Bio-Psikhologiya). Tom I. (Biological bases of comparative psychology: Bio-psychology, Vol.1). St. Petersburg, Moskva: Izdaniye tovarishchestva M. O. Volf. . 1910b. Biologicheskiye Teorii i Voprosy Zhizni (Biological ideas and life problems). St. Petersburg: Obrazovaniye. . 1913. Biologicheskiye Osnovaniya Sravnitelnoy Psikhologii (Bio-Psikhologiya). Tom II: Instinkt i Razum (Biological bases of comparative psychology: Bio-psychology. Vol. 2: Instinct and mind). St. Petersburg, Moskva: Izdaniye tovarishchestva M. O. Volf. . 1914a. Physiological and biological solution of psychological problems (in Russian). In: Novye Ideyi v Bilogii (New ideas in biology) 6:1-37. . 1914b. Segmentary psychology (in Russian). In: Novye Ideyi v Bilogii (New ideas in biology) 6:38-138.

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. 1923. Biopsikhologiya i Smezhniye Nauki (Biopsychology and related sciences). Petrograd: Obrazovaniye. . 1924-1929. Vozniknoveniye i Razvitiye Psikhicheskikh Sposobnostey (Origin and development of mental abilities). Issues 1-9. Leningrad: Nachatki Znaniy. . 1926. Metodika Nabludeniy nad Zhivotnymi (Posobiye dlya Prepodavateley) (Some ways to observe animals: A manual for the school-teacher). Leningrad: Gosudarstvennoye Izdatelstvo. Voronin, L. G. 1957. Sravnitelnaya Fiziologiya Vysshey nervnoy Deyatelnosti (Comparative physiology of the higher nervous activity). Moskva: Izdatelstvo Moskovskogo Universiteta. . 1970. Phylogenetic evolution of conditioned activity (in Russian). In: Rukovodstvo po Fiziologii. Chast 1 (Textbook of physiology. Part 1), 473-506. Moskva: Nauka. . 1977. Evolutsiya Vysschey Nervnoy Deyatelnosti Ocherki (Essays on the evolution of highernervous activity). Moskva: Nauka. . 1984. Kurs Lektsy po Vysshey Nervnoy Deyatelnosti (Lectures on higher nervous activity). Moskva: Izdatelstvo Moskovskogo Universiteta. Yaroshevsky, M. G. 1985. Istoriya Psikhologii (History of psychology). Moskva: Mysl'. ed. 1991a. Repressirovannaya Nauka (The oppressed science). Leningrad: Nauka. . 1991b. Stalinism and Soviet science (in Russian). In: Repressirovannaya Nauka (The Oppressed Science), 9-33. Leningrad: Nauka. Zakharzhevsky, V. V., and Andreeva, V. N. 1984. Ordena Trudovogo Krasnogo Znameni Institut fiziologii imeni I. P. Pavlova (proshloye i nastoyashchee instituta i yego laboratory) (I. P. Pavlov Institute of Physiology of the Academy of Sciences of the USSR: The past and the present of the institute and its laboratories). Leningrad: Nauka. Zeleny, G. P. 1913. Psychological reactions of the animals as the subject of natural science (in Russian). Priroda 70:1191-1207. Zubkov, A. A., and Polikarpov, G. G. 1951. Conditioned reflex in a coelenterate (in Russian). Uspekhi Sovremennoy Biologii 32:301-302.

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Chapter Three Memory and Morphogenesis in Planaria and Beetle Inna M. Sheiman and Kharlampi P. Tiras

This chapter is dedicated to the memory of James V. McConnell.

We will begin with a brief history of the study of memory and morphogenesis in invertebrates. During the 1950s and 1960s James V. McConnell excited the scientific world by his conditioning experiments with planarians. Infected by McConnell's results and enthusiasm, we began to study planarian regeneration, although our experiments have taken a different direction from his. The experiments performed by him and his many colleagues suggested the possibility that memories produced by conditioning survive regeneration following transection. With the interest in nucleic acids during the 1950s and 1960s, McConnell proposed that the memory trace was formed and stored in RNA molecules. These molecules are present in all planarian cells, including those that are critical for the process of planarian regeneration. The conclusions regarding the role of RNA in memory storage in regenerating planaria were not quite verified in our experiments. After our first results on planarian conditioning, the idea arose to study simultaneously planarian memory and morphogenesis. The results of these investigations are presented in this chapter. In our studies memory processes were analyzed by using the common mechanisms of morphogenesis, that is, the behavior of developing cells; their origin, growth, and differentiation; and, finally, how these cells are regulated. Two different models of invertebrate morphogenesis were used: planarian regeneration (regeneration as repeated development) and postembryonic development of insects, specifically the grain beetle. There is a very rich literature describing both planarian regeneration and insect metamorphosis and learning, but the relationship between them has not been studied. Establishing this connection has been the purpose of our investigations for many years, the results of which are reported in the following pages.

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In Russia, planarian investigations were first carried out at Kazan University. These investigations continue to the present (Porfiryeva and Diganova 1987). Planarian regeneration has been the object of investigation in Leningrad University (Krichinskaja 1972), and the study of conditioning and behavior in planaria was studied in Moscow University (Voronin and Tushmalova 1965). PLANARIAN MEMORY AND REGENERATION Planaria are typical flatworms, encased in a longitudinal body covered with ciliated epithelium. About 500 planarian species have been described. Most of them are freshwater animals, but there are also marine and terrestrial species. The length of planarians can vary from 1 mm to several centimeters. The majority are about 10-30 mm long. In Russia, the largest planarians live in Lake Baikal, where there are about eighty endemic planarian species. Planaria are beasts of prey and stalk their potential victims with well-developed chemical senses and photoreceptors. Planaria are hermaphrodites and can breed by sexual interactions. There are also asexual species that breed by fissioning. In this case, both parts of the animal develop into two new planaria. The ability to regenerate, morphogenetic plasticity, is one of the more remarkable features of planarians. In our work the following species of planaria were mainly used: Dugesia lugubris, Dugesia tigrina, Ijimia tennis, and Bdellocephala punctata. D. lugubris and /. tenuis were collected in lakes along the river Oka near the town of Pushchino. Sadly, over the course of thirty years we have witnessed the gradual disappearance of planarians in this lake because of pollution. This is why for the last few years the majority of our work has been performed on laboratory colonies of D. tigrina. In the laboratory our planarians live in large cups at room temperature and semidarkness. They are fed once a week with mosquito larvae. The water in their home containers is changed once every 3-6 months. The Nervous System and Planarian Conditioning Planaria are the first animal in evolution to possess a centralized nervous system. The central nervous system consists of two abdominal nerve cords and anterior paired ganglia. The nerve cords consist of nerve cell bodies and fibers. There are motor and associative neurons. The planarian ganglion represents the beginning of cephalization and consists of several thousand nerve cells surrounding the neuropil. The ganglion in different planarian species has the appearance of a butterfly or a horseshoe. Like other planarian cells, the neurons are characterized by their small size (about 10 pm) and large nucleus. There are multi-, bi-, and unipolar nerve cells. There are also various kinds of ganglion cells. Some of these are neurosecretory, and others resemble glial cells. Ganglion cells send their branches to neuropil and connect with the main sensory

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zones of the planarian body (Lentz 1968a). Planaria have various sensory receptors. The photoreceptors, for instance, enable the animal to detect weak light stimuli (about 1 lux) (Jones 1971); and the chemical receptors are sensitive to some proteins and amino acids: hemoglobin, lecithin, lysin, glutamine, and proline (Karpenko and Seravin, 1973). The various Pavlovian conditioning protocols for planarian research performed in Western laboratories are well known and will be discussed briefly here. Planaria can be conditioned to light or vibration conditioned stimuli when associated with electrical or a chemical-shock unconditioned stimuli. Planarians can also be conditioned in various instrumental situations employing mazes. As pointed out by Western researchers, the claim that planarians can be conditioned has engendered much controversy even today (Corning and Riccio 1970). We feel that the inconsistencies in the data and results can be traced to poor conditioning procedures and the difficulties inherent when one attempts an analysis by lumping together different planarian species. As to Russian studies of conditioning in planarians Krilov and Nasarian (1973) successfully trained planarians to make a correct choice in a cruciform maze. Tushmalova and Gromyko (1968) attempted to condition planaria to reverse their light/dark preferences. However, Voronin and Tushmalova (1965) concluded that planarian conditioned reflexes, when they could be formed, are primitive and probably based on sensitization. Our investigations in planarian behavior, which have spanned almost thirty years, allow us to make some conclusions about the origin of these contradictions and to analyze the properties of planarian conditioned reflexes (Sheiman and Tiras 1984). We have tried various combinations of training parameters, procedures, and species and are confident that we have found the optimal training variables and procedures for the elaboration of true conditioned reflexes in planarians. The majority of our experiments are performed with the species Ijimia tenuis (sexual race from the lakes near Pushchino) and Dugesia tigrina (asexual laboratory race). The planarians Dugesia lugubris and Rhymacephalus pulvinar (the latter is an endemic species from Lake Baikal) were also conditioned. In our first experiments we used the approach taken by McConnell in which subjects received massed training: 120 combinations of light and electrical shock presented in the course of a single training session (Cherkashin, Sheiman, and Sergeeva 1966a). We found essentially the same results as McConnell and observed, as others before us have, that all kinds of stimuli seem to evoked the same motor reaction: turning of the head part or longitudinal contraction of the planarian body. We also observed that the primary effect of the light and shock combinations was to inhibit the reaction to the light conditioned stimulus. This would, of course, underestimate the amount of conditioning and produce an unstable pattern of conditioned behavior (Cherkashin, Sheiman, and Sergeeva 1966b). From the results of our previous massed conditioning experiments, we thought conditioning would be better if a spaced trials procedure were used. We now conditioned animals with 25 trials per day for 10 days. In addition, the intensity of

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light used as a conditioned stimulus was decreased to 170 lux. This level of illumination was practically under threshold and did not evoke any motor responses from the planarian. As the experiment continued, the number of planarian reactions to the light gradually rose and became stable (17-20 reactions per 25 trials) by the fifth or sixth day. Similar results were obtained when vibration and electric shock were used as conditioned and unconditioned stimuli respectively. Figure 3.1 provides a summary of the results. The appropriate pseudoconditioning controls were employed in these experiments (Cherkashin and Sheiman 1967). The next modification of the experimental procedure permitted us to condition planaria in one day by using a mass conditioning situation. There were 40 combinations of light (70 lux) and electrical shock. Under these conditions the planarian Dugesia tigrina were well conditioned (Tiras and Aslanidy 1981). All of our data confirm the notion that the problem of planarian conditioning can be reduced to finding the right combination of species, training variables, and procedures and not to an inability of planarians to form true conditioned reflexes. Figure 3.1 Conditioning of planarians with light as the conditioned stimulus. For Day 9, only the conditioned stimulus was changed to vibration.

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McConnell, in his pioneering works, used a high-intensity light (about 2-3 thousand lux). He observed an increase in the number of planarian reactions to light (sensitization) but did not analyze this phenomenon (McConnell 1970; McConnell, Jacobson, and Kimble 1959; Thompson and McConnell 1955). As mentioned above, we could not form stable conditioned reflexes in planaria using McConnell's procedures (Cherkashin, Sheiman, and Sergeeva 1966a 1966b). Apparently, Tushmalova came across the same phenomena (i.e., unstable conditioned reflexes) when she used a 2000 lux light as a conditioned stimulus (Tushmalova 1973; Tushmalova and Gromyko 1968; Voronin and Tushmalova 1965). This no doubt contributed to her opinion that planarians cannot form true conditioned reflexes. However, as we mentioned earlier, if light intensity is decreased as in our experiments, true conditioned reflexes can be revealed in planarians (Cherkashin and Sheiman 1967). The importance of providing a "weak" CS intensity and using spaced trials in the study of invertebrate learning was mentioned earlier by E. Kreps in his work on ascidians (1925) and by Sokoloff in his work on sea stars (1961). Planarian conditioned reflexes are, however, obviously different from those found in vertebrates. Vertebrates can reveal the formation of conditioned reflexes with a wide range of behavior. Planarians, on the other hand, reply to any stimuli using the same set of behavioral reactions. Thus, conditioning in planaria must be expressed as an increase in the excitability to the conditioned stimulus. The common reaction to different kinds of stimuli promoted disagreements and contradictions in the planarian conditioning literature (Brown, Dustman, and Beck 1966; Cummings and Moreland 1959). Our data suggest several conclusions about the origin and particularities of planarian conditioned reflexes. Planaria have receptor systems that provide a distinct perception of various classes of stimuli such as light, vibration, and chemical. This is why there is an afferent differentiation of the conditioned stimuli, which leads to a specific increase of excitability in planaria. At the same time, however, their effector systems are not sufficiently developed. Thus, although their sensory systems can perceive specific classes of stimuli, their motor systems limit the type of locomotor behavior. Though planarian reflexes have much in common with summation or sensitization reflexes, they still preserve the basic character of classical Pavlovian reflexes. Planarian Regeneration Planarian regeneration is an excellent experimental model for studying morphogenesis (Brondsted 1969). As a classical object for regeneration research planaria have been used for 200 years. T. H. Morgan, for instance, before conducting his investigations in classical genetics published important work on planarian regeneration that is still cited (Morgan 1901). About 30 years ago McConnell was the first to use planarian regeneration for the study of memory. In our laboratory planarian regeneration was studied simultaneously with the investigation of planarian learning and memory.

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Planarian Regeneration: Morphological study The most investigated experimental model in our laboratory was the regeneration of the planarian head. Operations were performed after the animals were food deprived for one week. Morphological and functional investigations were carried out over the 10 day period following surgery. Morphometric Observation of Planarian Regeneration Much of the data was obtained by a combination of intravital computer morphometry and histological methods. The method of computer morphometry of planarian regeneration is based on the fact that intact planaria are covered by pigment cells. After decapitation, however, the regenerating blastema do not have such cells until several days have passed (Tiras and Khachko 1990). Therefore, the photocontrast between old (i.e., pigmented) and newborn (blastema) portions of regenerates can provide an accurate record of the time course of regeneration. The resulting photographs can be digitized for computer analysis. A computer program has been developed that constructs an image "skeleton" of the planarian and on the basis of this image computes the following primary traits: the area, length and width of the planaria and the area and length of the growing blastema. On the basis of these traits, a regeneration criteria can be computed. We used two regeneration criteria that reflect the restoration of the body proportions. The first reflects regeneration of the original proportions of the length of the head and the whole body. The second considers the area, including both linear indices (length and width). These criteria reflect the regeneration of the proportions of the areas of the head and the whole body (Tiras and Khachko 1990). The criterion of length, Cr(l), and criterion of area, Cr(a), were calculated according to the formulas:

where A(0) is the length of the intact planaria; C(0) is its area; A(i) and C(i) are the length and area of the regenerates at i- day after sectioning; B(0) and D(0) are the length and the area of the head part of the intact planarian body; B(i) and D(i) are the length and area of the blastema; m is the number of animals in the experimental groupand S is a constant. After the planarian was sectioned, an intensive reorganization of all the dimensions of the body was initiated. The length of the blastema increased during the experiment and reached a stable level after 20 days. Meanwhile, the total length of the body, which contracted after sectioning, gradually decreased over the whole period during which measurements were made. The width of the regenerated body

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increased immediately after sectioning because of the contraction of the body; it gradually decreased during regeneration down to 70% of the initial level. The length of the planarian body changes to a significantly lesser degree than its width, and the decrease in the total length of the body occurs during the increase in length of the regenerating head part of the body. The area of the anterior part of the body grew gradually during regeneration, but the total area of the regenerating body decreased. The area of the planarian profile decreased in exactly the same way. As the planaria were not fed during regeneration, obviously the observed changes are directly related to the morphogenetic reorganization of the regenerates. On the basis of the computed regeneration criteria collected during the 30 days of the experiment, Cr(l) reaches a level corresponding to 100% of the body proportions of intact animals by the 10th day of regeneration, but Cr(a) does so only by the 30th day. Thus, periodicity during regeneration is reflected in different criteria. The results permit an identification of the sequence of stages in the regeneration of the planarian body at a macromorphological level of organization. Stage I consists of the regeneration of the proportions of the length of the head and the whole body of the planarian. Stage II consists of the reestablishment of proportions related to area. The first stage takes approximately 10 days, and the second takes 30 days. Histological Investigations of Planarian Regeneration Histological studies of planarian regeneration were made in our laboratory using a modification designed to diminish preparation time (Bogorovskaya 1969). It was mentioned earlier that in a transected planaria the nervous system is the first to regenerate (Pedersen 1958). It takes about 5 days to form the frame of the new planarian ganglion. Twenty-four hours after the operation, the cut nerve cords lie free under the epithelial surface. The next day, the growth of thin nerve fibers were observed. On the 3rd day of regeneration, the number of these nerve fibers increased and their growth moved toward the median. On the 4th day, this tendency was clearer and the pioneer fibers established contacts. Finally, on the 5th day, these contacts developed into a strong junction, which then became thicker. The cellular composition of the new ganglion forms in parallel with the regeneration of the nerve cords. First of all, the small round or slightly extended cells concentrated around the remaining nerve cords. Usually, in planaria, such cells are called neoblasts. Two days after the operation small groups of such cells appeared under the wound surface. Over time, their numbers increased and they concentrated around the growing nerve fibers and formed the blastema. On the 5th day, the shape of the planarian ganglion, with the cell and fiber composition, was established. Thus, the planarian ganglion restores its structure in 4 or 5 days. The source of its formation is the growth of nerve fibers from the remaining nerve cords and neoblasts that are concentrated in the blastema.

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Cellular Destruction During Regeneration It has been previously mentioned that some planarian cells are destroyed during regeneration (Sheiman and Efimov 1973). The cell destruction is more pronounced in the remaining head area but not in the remaining tail area. The percentage of damaged cells (cell nucleus deformations) was calculated as a function of time since the operation. In the first hours following the operation, cell destruction in the remaining ganglion is pronounced. The maximum destruction (more than 50%) occurred 4 hours after the operation. As time continued, the number of intact cells increased and the number of destroyed cells decreased. After 12 hours postregeneration, the cell destruction increased again. Thus, this process has an oscillatory character. In the days following the operation, cell destruction in the ganglion slowly decreased. It must be mentioned that cell density in the ganglion during regeneration was not diminished but actually increased slightly. Although the whole structure of the anterior part of the regenerating planaria lost cells, cells from other parts of the body migrated to the ganglion region. Functional Changes in Regenerating Planaria It may be concluded on the basis of the morphological investigations that the primary ganglion structure formed on the 5th day after the operation. The functional differentiation of the planarian cells during regeneration will be illustrated by analyzing some cellular properties. Neurosecretion Neurosecretory cells have been described in planarian ganglion and nerve cords (Lender and Klein 1961, 1962; Sauzin-Monnot, Lender and Gabriel 1970). It is known that such cells play an important regulatory role in planarian regeneration (Grasso 1966; Lender 1964; Lindh 1959). N. Sakharova and R. Gordon (1974) from our laboratory described the neurosecretion in Dugesia tigrina using the Gabbe method. Apart from neurosecretory cells, some vacuoles filled with neurosecretion were also found. They were located at the periphery of the ganglion and also near the nerve cords. Two days after the anterior portion of a planarian was amputated, neurosecretion was found directly under the blastema and along the remaining nervous trucks. During the following 2-day period the supply of neurosecretion was exhausted and the vacuoles gradually disappeared. On the 5th day of regeneration, new neurosecretory cells appeared in the blastema. Simultaneously they were also found in the nerve trucks. Neurosecretory cells and vacuoles containing neurosecretion also appeared in both the parenchyma and along the nerve trucks. Apparently neurosecretion does play a role in planarian regeneration, as it is exhausted in the first days after the operation. The main conclusion of this work,

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however, was that new neurosecretory cells appeared in the new ganglion on the 5th day. This time period nicely corresponds to the morphological formation of the ganglion as described above. Acetylcholinesterase Activity Another histochemical characteristic of the new nervous structure in regenerating planarians is neuromediatory activity. Among other kinds of mediators, planaria of different species have acetylcholine and acetylcholinesterase (AChE) (Lentz 1968b; Tiras 1978; Tiras, Sheiman, and Sackharova 1975; Welsch 1946; Wolff 1962). The presence of AChE was revealed by the method of Koelle and Fridenvald, using the Gerebtzoff modification. The number of cells containing AChE particles in different regions of the regenerating planaria Dugesia lugubris and Planaria torva was calculated. In the ganglion region located in the head part of the operated on planaria the AChE cells increased in the first 2 days from 64% to 86%. In the remaining nerve trucks of the regenerating tail portion, few AChE cells were found. In the blastema such cells appeared only on the 4th day, and on the 5th day their quantity became considerable. At this time the normal picture of the ganglion AChE activity was established. The AChE and neurosecretion dynamics during the 1st day of regeneration indicate their important role in this process. Perhaps they are the elements regulating regeneration. Immediately after cutting, both of these systems are exhausted and are reestablished in the differentiating nerve elements. They indicate the functional maturation of the ganglion. Factors of Regeneration We would suggest that besides ACh and neurosecretion some specific factors of regeneration also take part in regulation. Such factors, as a special kind of embryonal inductor were described in many models of regeneration. For example, in hydra the specific neuropeptide activating the head regeneration was found (Schaller and Gierer 1973). Lender established that planarian ganglion cells excrete a factor that compels neoblasts to differentiate in "eyes" (Lender 1952, 1955), and Sengel has shown that the mechanisms of ganglion and pharynx regeneration are similar (Sengel 1959). Simultaneously, the pharynx region excretes the inductors for the pharynx itself (Sengel 1951, 1953). Lender also demonstrated the existence of both inhibitors and simulators of ganglion regeneration (Lender 1956a, 1956bb, 1960). The active factors stimulating and inhibiting morphogenesis are known as morphogens (or morphogenetically active substances) (Sheiman 1984). Their existence was known earlier from experiments in which planaria regenerated in homogenates (Lender 1960). There are also some data about the inhibitor of ganglion regeneration. It is glucoproteide with m.m. 2.4 * lOd (Steele and Lange 1977).

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The pharynx stimulator and inhibitor correspondingly have m.m. lower and higher than 15 kda (Wolff 1974). For revealing planarian morphogenetic factors, the homogenates of regenerating planaria were cut on different days and fed to intact recipients; 24 hours later, the recipients were sectioned, too. The regeneration of these recipient planaria were investigated using the computer morphometry method described earlier. The regenerates of the 1st to the 5th day were used as food and sources of regeneration factors (groups R1-R5) (Tiras and Sheiman 1984). The control animals were fed with intact planaria homogenates (group CI), with neutral food—mosquito larvae—(group CM), or not fed (group C). The results of the experiment indicate that in the control groups the restoration of the body proportions of the regenerates occurred as follows. Throughout almost the entire experiment, unfed animals (group C) regenerated the most slowly of any of the groups, while regenerates feed neutral food (group CM) developed more intensively. Planarians feed with intact planaria homogenates (group CI) outstripped both the preceding control groups and all groups of experimental animals. Planaria of the CI group significantly outstripped the animals of the C group from the 2nd to the 8th day, and the CM group on the 3rd, 4th, 7th, 8th and 11th days of the experiment. Regeneration in the CM group outstripped growth in the C group from the 3rd to 5th days following decapitation. Despite the fact that on individual days the differences between the control groups were insignificant, it may be concluded that feeding itself before decapitation stimulated regeneration. Feeding with homogenates from the intact planarians also stimulated regeneration. Moreover, feeding with intact planaria homogenates stimulated regeneration most effectively. It can be concluded that intact planaria Dugesia tigrina contains factors that stimulate regeneration. An essential factor in the course of regeneration of the head end of the planarian body is the formation and functioning of a new ganglion. This begins to occur on the 4th and 5th days following decapitation. Therefore, the experiments to be discussed will be analyzed in two stages: before and after the 5th day of regeneration. The animals of the Rl group regenerated more intensively than the animals of the CM group throughout the entire experiment. This difference was observed before and after the formation of a new ganglion in the recipients on the 5th day of regeneration. Significant differences in the value of the index Cr were observed on the 4th, 5th, 7th, and 11th days following decapitation. The most substantial difference in growth in comparison with the control planaria was on the 7th and 11th days (i.e., on the second stage of regeneration). The regeneration of planaria from group R2 in the first 5 days of the experiment was at the same level or below the level of group CM. In the second stage of regeneration, group R2 rapidly outstripped the CM group in development and significantly outstripped the CM group from the 7th to the 11th day. Planaria from group R3 regenerated similarly during the first 5 days of the experiment. However, a tendency was manifested here for a decrease in regen-

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eration rates, and on the 4 and 5 days after cutting, the Cr in this group was significantly lower than in the CM group. After the 6th day, planaria of group R3 overtook regenerates from the CM group. The development of animals in group R4 resembles the dynamics of restoration in group R3. The tendency for slow growth in this experimental group was enhanced. In the second phase of regeneration, from the 7th to the 11th days, the regeneration of planaria in group R4 was worse than that of the animals from all other experimental groups. The value of Cr in group R4 remained at the level of the control group CM. Planaria in group R5 outstripped the animals of the CM group in terms of development from the 2nd to the 5th day. This was especially noticeable at the 4th day. After the 5th day the rate of development of the animals of this group slowed down, but it was again intensified on the 8th to the 11th day of the experiment. On the basis of these data we can made the following conclusions. The regenerates of the 1 st day contained a factor that stimulates regeneration of planaria. The regenerates of the 2nd and 5th days also stimulate this regeneration. In regenerates of the 3rd and 4th days, a factor inhibiting regeneration was detected; this inhibition was more stable after feeding of 4th-day homogenates. On the 5th day after decapitation and in intact planaria, stimulators of regeneration were active, especially in the intact planaria. The stimulation of regeneration in the CI group was the least pronounced in comparison with all other experimental groups of animals. The main conclusions to be made regarding the existence of regeneration factors (RF) and of their dynamics during planarian restoration are the following. On the 1st and 2nd days following decapitation, an activator of regeneration turns on, thereby triggering the recovery process. On the 2nd day the effect of activation gradually decreases, and on the 3rd and 4th days the action of inhibitory factors predominates in the regenerates. Then, on the 5th day, after the formation of a new ganglion in the recipients, activity of the regeneration stimulators is again manifested. The intact planaria also contain a regeneration stimulator (Tiras and Sheiman 1984). The regeneration of planaria can be represented in the form of an active interaction of two types of regulators: stimulators and inhibitors, so that the real course of the process is determined by the preponderance of activity of the factor of one sign or the other. MEMORY TRACE IN REGENERATING PLANARIA Storage of Conditioned Reflexes After Planarian Regeneration McConnell was the first to find that conditioned reflexes in intact planaria were preserved after the animal was sectioned and regenerated (McConnell, Jacobson, and Kimble 1959; McConnell, Jacobson, and Maynard 1959; Murphy 1963). The conditioned reflexes to light were elaborated over several sessions. Then planaria

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were sectioned transversely into two parts and tested again 2 weeks later. Both regenerated parts reproduced the conditioned reflexes. The same results were obtained if the light was replaced with other conditioned stimuli. Similar results were obtained using conditioned stimuli other than light and in situations employing T mazes (Ernhart and Sherrick 1967; Nazarian 1973). McConnell believed that memory was stored in the neoblasts and was influenced by RNA (McConnell 1965). As mentioned previously, such an interpretation was popular in the 1950s and 1960s, when the idea that memory may be coded on the RNA as genetic information was suggested. Not enough control experiments were performed, however, to test this suggestion. Thus the phenomenon of memory storage during regeneration remained unexplained. An additional question concerns how the memory trace is dependent upon the planarian central nervous system. It was also of interest to know the relationship between the neoblasts, which play such an important role in regeneration, and memory formation in planarians. In an attempt to provide answers to these questions, experiments were conducted to analyze the storage of the conditioned reflexes in regenerated planaria (Cherkashin, Sheiman, and Bogorovskaya 1966). Fifty planaria Ijimia tenuis were conditioned to vibration combined with electrical shock. Then all the animals were sectioned across the midline. In the following days groups consisting of 10 animals (corresponding to an intital group of 5 intact planarians) were tested for the presence of conditioned reflexes. The results indicate that the level of conditioning in head regenerates did not fluctuate during regeneration. In the tail region (which regenerated a new ganglion), during the first 4 days of training the level of conditioned reflexes was significantly lower. This indicates that conditioned reflexes were not manifested. On the 5th day of regeneration, however, the number of reactions sharply increased and was stable for the remaining sessions of the experiment. Our results indicate that tail regenerates restore their conditioned reflexes on the 5th day after cutting. In the next series of experiments, different types of operations on the central nervous system were made to study the role of nervous system function in the storage of the memory trace. Planaria were conditioned to light, operated on, and checked on the 2nd and 14th days after the operation. The reults indicated that as in the previous experimental series, the conditioned reflexes were not manifested immediately after ganglion removing but were restored following a 2-week regeneration period. Following removal of the nerve trunks, the conditioned reflexes remained intact. After removal of both the ganglion and nerve cords (the lateral cords remained intact), conditioned reflexes were not restored. The final experiment from this series consisted of double sections. Intact planarians were conditioned to light and operated on in the usual manner. After 14 days the regenerated parts were removed again, and only a month after the first operation each of the 4 regenerated planaria (obtained from one intact animal) were tested. In this experiment 2 animals from every 4 consisted of the original planarian tissue, and the remaining 2 animals came from tissue regenerated at two

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stages. The results indicated that the number of conditioned reactions in the regenerated planarians was lower than for the intact planarians but reliably higher than in the corresponding control animals. These experiments confirmed the results of McConnell (McConnell, Jacobson, and Kimble 1959), who found that the memory of conditioned reflexes is present in regenerated animals. Moreover, our experiments indicate the dependence of such storage on central nervous system function. The memory of conditioned reflexes was not destroyed in the head parts that kept the ganglion. In the tail regenerates, memory of conditioned reflexes did not appear until the 5th day following the operation, that is, before the ganglion regenerated. Memory was not observed in regenerates who had only intact lateral nerve trunks. The memory trace was also found in planaria that had completely regenerated following a double section. This last result is evidence of the transfer of information during CNS regeneration and its fixation in new neural elements. Conditioned Reflex Formation During Ganglion Regeneration The previous series of experiments shows the possibility of memory storage for conditioned reflexes in planaria after cutting and regeneration. We will now present some data that indicate the possibility of forming new conditioned reflexes during regeneration (Cherkashin, Sheiman, and Bogorovskaya 1966). In these experiments planaria were cut across and conditioned for 5 days with a vibratory conditioned stimulus. These animals were tested for the memory of conditioned responses 2 weeks after the operation. The control animals were sectioned and then treated the same as experimentals, with the exception that they received only the conditioned stimulus. The results indicated that the number of responses to the conditioned stimulus did not change over 5 days of regeneration in both experimental and control groups. It was low. There was more activity, however, in the head regions than in the tail. However, in another experiment in which the presence of conditioned reflexes were tested following 14 days of regeneration, conditioned reflexes in trained animals, but not in controls, were revealed. The number of conditioned responses was similar to that found before amputation. The number of responses in the control group was constantly low throughout the entire experiment. The present experiments show that it is possible to condition planaria in the absence of the ganglion and during its regeneration. The results of such conditioning will appear only after ganglion regeneration. These results raise the question of whether conditioning depends on the maturity of the new nervous elements, the state of the remaining nervous structures, or perhaps some other factor. The following experiments were carried out to answer these questions. It was necessary to create a new experimental device and a new method of conditioning. To conduct this study, it was necessary to keep training times brief. Mass conditioning was therefore carried out intensively throughout a day. Forty

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combinations of light and electrical shock were given to a planaria restricted to a "cell." Learning was conducted in two daily sessions separated by a 2-hour interval. The basic illumination of the cell was 40 lux. The conditioned stimulus was increasing the light to 70 lux, and the unconditioned stimulus was electric impulses generated by electronic stimulator (current strength 10 uA; duration 10 ms). Progress in the formation of conditioned reflexes was monitored by an optical-electrical registration designed to record planarian movements automatically (Tiras and Aslanidi 1981). A light flux fell perpendicular to a sensor containing two photoresistors connected to different arms of a balanced bridge. Movements of the animal resulted in alterations of the illumination difference of photoresistors and, correspondingly, changed the signal of the bridge. Motor responses of planaria were registered in the course of the experiment as "bends" on a paper recorder. The ratio between the number of bends obtained under the action of a conditioned stimulus and the total number of bends for a combination of stimuli were calculated. The average value of 10 subsequent stimuli combinations was taken as a coefficient of conditioning (CR) and expressed as a percentage:

where A is the number of bends in the course of the action of a conditioned stimulus, B is the total number of bends for a stimuli combinations, and S is a constant. In the first set of experiments, planaria were trained after various numbers of days of regeneration (Sheiman and Tiras 1983; Tiras and Sheiman 1981). After sectioning of the head, experiments were carried out daily (up to the 5th day) in different group of animals. In 2 weeks, when the regeneration was practically completed, the memory trace preservation was tested using 10 conditioned stimuliunconditioned stimuli pairings. As controls, intact animals that had acquired the conditioned response 2 weeks previously were used, as was a second control group whose members were cut and subjected to the conditioning without receiving any preliminary conditioning. Each group contained 20-30 planaria Dugesia tigrina. As the activity of regenerated planaria was variable, it was impossible to compare the results of learning in different experimental groups. The results of conditioning were judged according to the a test session. The coefficients of conditioning in learned planaria on the 1st, 3rd and 5th days of regeneration are close to the level of responses of intact learned control planaria. Conditioning was very poor in planaria who received training on the 2nd and 4th days of regeneration: they were at the level of control planaria who also regenerated but were not trained. This set of experiments led to the following conclusions. The first group of control animals is the standard of learning, while the second one reflects those alterations in planaria motor activity and excitability that because of regeneration.

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Comparison between experimental and control groups showed that planaria subjected to the experiment on the 1st, 3rd and 5th days of regeneration appeared to be trained at the end of this process, while those trained on the 2nd and 4th days manifested motor activity characteristic of untrained regenerated planaria. As the deviations described are not connected with the linear character of ganglion development, it is natural to suppose that they are caused by some other factors operating in the course of regeneration. We believed that such a factor might be, in particular, the regenerating factors (RF). To test this supposition, the regenerative material was to be transferred to intact planarians prior to training. An adequate method of introducing this material was developed, and procedures were designed that made it possible to separate the source of RF and the object of action from the location where the formation, storage, and reproduction of the memory trace occurs. Planaria of different groups obtained the homogenates from planaria after different numbers of days (from 1st to 5th) of regeneration (groups H1-H5). The control groups were fed either with Tubifex worms or with homogenates from intact planaria (CT and CI correspondingly). On the next day after feeding, planaria-recipients were conditioned, and a week later they were tested for preservation of the memory trace. In all the experiments, the results of learning and reproduction of a memory trace were taken into account. The results indicate that the responses of planaria in different groups vary in the course of training. In all groups, deviation of the level of responses occurred in the same direction: In the second part of the experiments the response level was higher than in the first part. This indicated that motor activity increased in the course of training. The level of responses differed within experimental groups as well as between experimental and control groups. Motor activity of planaria fed with homogenates of the 1st and 3rd days of regeneration were higher than those in control group CT throughout the course of the experiment, although the differences were not statistically significant. The level of activity in animals obtaining homogenates of the 2nd and 5th days of regeneration was higher at the end of the experiment than in the control group CT. Finally, the group H4 obtained homogenates from the 4th day differed from all others (including the control groups) by a significantly lower level of responses throughout training. Hence, all experimental groups (except the H4 group) more or less expressed stimulation of learning compared to the control group. Only in one experimental group was an inhibitory effect revealed. To determine the preservation of the memory trace in the learned planaria of the previous experiments, these animals were tested one week after training. The response level in all groups of experimental animals were higher than those in the first experiment (after 40 training trials) and in control animals. Statistical analysis of the data allowed us to divide experimental and control groups as follows. The lowest level of motor activity was revealed in the control group CT; a higher level characterized the group of planaria fed with homogenates of intact planaria and regenerates of the 1st and 4th days. The highest level of activity was observed in

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planaria from the groups H2, H3, and H5. All experimental groups were reliably different from control group CT. There were no significant differences between the experimental groups H2, H3, H5 and HI, H4. The results show that in the interval between learning and testing the memory trace is enhanced. This is specific to experimental animals rather than to controls and is more pronounced in those obtaining homogenates of the 2nd, 3rd and 5th days and less in animals fed with those of the 1st and especially the 4th day. The results supported the hypothesis that in the course of regeneration there arise regenerating factors that stimulate or inhibit the formation of memory traces. An attempt was made to characterize these factors and reveal their effects on reproduction of memory traces. Intact planaria were trained by the standard method. Preservation of memory traces was tested a week later. Prior to testing, the trained planaria were divided into experimental groups; each group was fed with homogenates from the regenerating planaria (1-5 days) and Tubifex worms. The results indicate that in most experimental and control animals the response levels during the test differed only slightly. However, the group of planaria who obtained the regenerates of the 1st and 4th days manifested lower responses as compared with controls. In all groups except that of the recipients fed with homogenates from the 4th day, the level of responses in the test experiments were higher than in the course of training (Sheiman and Tiras 1983). Training of planaria with the amputated head containing ganglion showed the possibility of memory trace formation, but the reproduction of the memory trace requires the ganglion (Cherkashin, Sheiman, and Bogorovskaya 1966). The experiments on training regenerates at different days demonstrated the possibility of memory trace formation in decapitated planaria. The supposition was made that during days 1-3 of regeneration learning proceeds because residual nervous elements of the longitudinal nerve trunks and that on days 4-5 of regeneration, when the of base of the ganglion is practically formed, the function of memory trace formation should be carried out by a new ganglion. In such a case one could expect that, in the first set of experiments, the memory traces were preserved throughout the whole course of regeneration. The results of our experiments on memory trace reproduction after completing the process of regeneration indicated that the capacity of learning in the course of regeneration fluctuates and is not directly dependent on maturation of nervous structures. The memory trace can be formed in the absence of the ganglia (1st and 3rd days of regeneration) and may not arise even when the ganglion is developed (4th day of regeneration). These data support the idea that in the course of ganglion regeneration learning is affected by some additional factors (for example, RF) rather than by the substrate providing the fixation of a memory trace. We believe these factors to be directly related to the process of regeneration and its stages. The possibility of their transmission from one animal to another by means of corresponding homogenates indicates their chemical nature. Similar morphogenetically active factors that regulated regeneration of the ganglia were

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described, for example, in our own experiments (Tiras and Sheiman 1984). In the study described above, the activity of RF was observed under various conditions. This allows us to distinguish between their endogenous and exogenous effects and its influence on memory trace formation, its reproduction, and its time course. Experimental groups of animals were compared with control animals under all experimental conditions. The results revealed positive (stimulatory) and negative (inhibitory) and neutral effects of RF. Stimulatory effect of RF was most pronounced in the course of reproduction of the memory trace in intact planaria obtaining the homogenates of various groups prior to training (1-5 days of regeneration). This delayed effect, apparent after an interval, resulted from exogenous introduction of RF. Endogenous RF effects that were inhibitory were observed in experiments with regenerating planaria on the 2nd and 4th days of regeneration. Exogenous RF effects on the 4th day of regeneration was also observed to be inhibitory in intact planaria. Direct inhibitory RF effects on the 1st and 4th day of regeneration homogenates were observed under exogenous introduction and in the course of memory trace reproduction. The inhibitory RF effect was manifested mainly immediately. However, delayed effects were also observed: the total level of stimulation diminished in the intact planaria trained and tested a week after being fed with homogenates of the 1st and 4th days. The most distinct effects were those produced by RF in the last days: regenerates of the 3rd and 5th days had a stimulating effect, and those of the 4th day had an inhibitory one. As for RF of the 1 st and 2nd days of regeneration, the effects were not similar in different experiments. These results suggest that, at different days of regeneration, planaria contain active substances inducing different effects and that, moreover, different substances may exist together at the same stage of regeneration. Attention was drawn to the difference between the effects of RF of the early (1-2 days of regeneration) and the late (3-5 days of regeneration) stages. The latter group contains both stimulating and inhibiting agents and possesses some direct features relating to nervous system functioning. As these effects were revealed during the the process of differentiation and maturation of a ganglion, it was reasonable to suggest the existence of neurogenous factors specific to these stages of regeneration. At the same time, the indistinct character of the RF effect during the first 2 days of regeneration indicated their polyfunctionality and nonspecificity, at least as it relates to the function of the nervous system. At this stage of regeneration, such processes prevail as the healing of damaged surfaces and proliferation and migration of neoblasts, and it seems natural to expect that RF effects are directed toward the regulation of these processes (Sheiman and Tiras 1983). Earlier some indications were made that the factors arising in the process of regeneration of planarian ganglion have a morphogenetic effect (i.e., they act as embryonal inductors). They originate from the longitudinal nerve cords and the ganglion (Lender 1955; Wolff 1962), as well as from other parts of planarian body (Lender 1956a, 1956b). With respect to regeneration of homological structures they are inhibitors (Lender 1956a, 1956b).

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The results obtained here and the small amount of data relating to this question allow us to relate the regenerative factors we have described to the substances currently classified as neuropeptides and, on the other hand, as morphogens. This assumption received support from the recently revealed similarity between hydra head activator and the hypothalamus neuropeptide obtained from the rembryonal brain of a rat (Shaller 1975). To summarize our results thus far, the conditioned reflexes formed in planaria before cutting are stored during regeneration but are reproduced only after the formation of new ganglia. It is possible to condition planaria during regeneration, but the memory trace is not revealed until new ganglia are regenerated. Regenerator factors (RF) regulate the creation of the memory trace, its storage, and its reproduction. The most active RF occurs on the 4th day of regeneration. This RF was able to influence all stages of memory trace functioning. There are many different RFs, and their characterization requires additional biochemical investigations. INSECT MEMORY AND METAMORPHOSIS Part two of this chapter is concerned with a parallel analysis of development and memory on another preparation, insect metamorphosis. The complex multistage process of insect postembryonal development includes several larval stages separated by periodical molts, a pupa stage, and finally the appearance of the adult organism, the imago. All these forms differ sharply from each other in exterior structure and, more important, in inner structure. It is proposed that the insect genome consists of two programs. The first one is realized in the system of larval structure and behavior, and the other in the imagos inner organs and behavior. During metamorphosis one genetic system is regularly replaced by the other. The cyclic organization of insect development and metamorphosis is regulated by a system of hormones: activation hormones, molting hormone (ecdysone), and juvenile hormone. The data on insect development and metamorphosis are extensive, and we used these processes as a biological model for the study of memory. Accordingly, they initiated the following questions. First, how is a memory stored in the larval stage recalled after metamorphosis? This problem was solved in our laboratory for the first time. But it raised the following questions: (1) How does the periodicity in larval development influence the memory trace? (2) How do hormones produced during metamorphosis affect the insect memory trace? Unfortunately, it is not known exactly what happens to larval organisms during metamorphosis. It is also unclear if all larval cells are replaced or if some remain. The grain beetle Tenebrio molitor was used in our experiments (Cherkashin, Sheiman, and Stafekhina 1968). The animals were tested on a T-maze instrumental task. The stem and arms of the plexiglas maze were 50 mm in length. The animals crawled through the maze in a semicircular section of 10 mm diameter. The first trial of training was a preference test to determine if the right or left

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arm was preferred. In succeeding trials the animal was run against its preference. An incorrect choice was punished by a "light mechanical blow" delivered to the anterior portion of the animal. The correct response was rewarded with access to a cup of bran. A session consisted of 10 runs with an intertrial interval of 2-4 minutes. Such sessions were repeated 5 times for each insect, with a 1-hour interval. In some experiments animals received 10 sessions of training, in other experiments only 1 day of training. Sometimes insects showed prudence and preferred either to go back into the starting area or, simply, to stop. In such cases they were prevented from retreating or encouraged to continue (after 1 minute of inactivity) by a slight mechanical prodding. Metamorphosis Influence on the Storage of the Memory Trace More than 100 beetle larvae (about 25 mm long) were conditioned in a T-maze for 10 days. Some time after training they pupated and test experiments were conducted on the adult beetles 1 month after training. Another group of larvae about 10 mm long were trained similarly but tested as adult beetles 3 months after training. The results of this experiment indicate that the number of correct choices gradually increased both in larvae and in adult beetles and stabilized after 8 days of training. After the 1 month interval there were a large number of correct choices in all animals, and those animals in the experimental group performed better than controls. Small larvae learned as well as larger animals, although they moved more slowly. Moreover, the memory trace in small larvae was more stable than in adult beetles. After metamorphosis, trained animals retained the correct response (Cherkashin et al. 1968). The adult insect brain differs sharply from the larval brain. The adult brain is formed after the destruction of larval brain cells, and there is a proliferation, migration, and differentiation of new nerve cells. These processes are under genome control. Behavior of nerve cells in larvae and pupae depend on the stage of development (Pipa 1973). The mechanisms of postembryonal brain development are like those found in the embryonic stage. The number of cells in the insect nervous system significantly increased during the transformation into an imago. Neuroblasts and ganglion cells constantly divide (Panov 1963; Sbrenna 1971) the number of glial cells increase (Gymer and Edwards 1967), and the neuropil is enlarged (Afify 1960). The most straightfoward explanation of why the memory trace persists into the adult stage is that some nerve cells are preserved during the change from larva to adult. It is unclear, however, if the mechanisms of the memory trace is solidified during metamorphosis. Memory Trace Formation in Larvae and Cyclic Development To study this phenomenon one must investigate the dynamics of memory trace

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consolidation during metamorphosis in connection with larvae development. Larvae were used because the changes in their internal structures are not as pronounced as in pupae. This makes it possible to separate the metamorphosis processes into definite phases. The periodical larvae molts were used as a marker separating neighboring cycles. Memory traces were formed in different phases of one larvae instar (Sheiman, Khutzian, and Ignatovitch 1980). The white larvae of Tenebrio molitor (mealworms) obtained just after molting were selected for training, and this initial molt served as the zero-point for the developmental cycle. This insect is characterized by a great but variable number of molts, and tests were performed on the 3 last instars. The larvae were selected from the bulk of animals developing under constant conditions in bran. Mealworms, 20-22 mm long, were kept separately in cups containing bran at approximately 22° C. Under such conditions the intermolting period lasts about 20 days. Tests were conducted on 5 subgroups on day zero (i.e., the day of the molt), and on days 4, 8, 12, and 16 thereafter. Each developmental stage was represented by 10 larvae with the exception of the day 16 subgroup, which included 20 larvae. In order to reduce to a minimum the developmental changes of larvae within the time of an experiment, we used intensive training during carried out in single day. Because the behavior of untrained larvae at different stages of the intermolt period did not differ at all, we used as a control group 10 larvae selected randomly in regard to stage. They were studied in the same maze as the test animals but, in contrast, were not subjected to any reinforcement and were left to their own devices. The tested larvae that were not given training showed a preference for a definite direction, as was observed with the control group: 5 larvae of this group chose the right and 5 chose the left direction on their first run and during the rest of the test preferred their original direction with a probability greater than 0.95. The mean number of correct choices was 4.0 for the first session and 3.7 for the last session in this group. In all experimental subgroups the mean number of correct choices for the first session was somewhat more than that in the control group (group C), fluctuating from 4.5 to 5.3 in the different subgroups. In the second session the distinction became apparent between the mean number of correct choices for different subgroups: much greater for the day zero and day 16 subgroups than for the other subgroups, with the differences increasing in the subsequent sessions. In the last session the mean number of correct choices was approximately 7 for the day zero and day 16 subgroups but remained only slightly different from the original value for the other subgroups. Based on the obvious differences in the behavior of the experimental larvae at the various stages, two large experimental groups were formed. The day 0 and day 16 animals comprised the first such group (A); the second group (B) included all the others. Both Groups A and B differed from Group C by the larger numbers of correct responses beginning with the 1st session. A comparison of Group A with Group B revealed that the number of correct responses increased for Group A over the 5 sessions of the test but for Group B remained close to its first session value.

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The subsequent analysis by the bifactorial proportional complex scheme with the use of the modified Fisher's criterion confirmed the appropriateness of the division of the experimental larvae into 2 groups. In this scheme we considered the course of the test, namely, the number of a session as the 1st factor and the larvae's inclusion in either Group A or Group B as the 2nd factor. Thus, we derived a 5 X 2 design with 60 animals in all. We calculated the variances for each square, along each factor and for the whole design, and then compared them, taking into account the number of the degrees of freedom, of course. We used the numbers of correct choices during a session as the basic parameter because we found no reliable increase in number of correct responses within each session, either for each run taken separately or for the first 5 runs compared with the last 5. It was found that the stage of the instar period on the worm's aptitude for learning was significant. Thus, the training of larvae at different stages of their development revealed a clear distinction: learning was most efficient when approaching the molt and immediately after it. During most of the instar, learning was hampered. In order to determine more accurately the bound of the outlined phenomenon tests were conducted on 2 additional groups (day 2 and day 14) of larvae. The day 2 group showed hampered learning resembling that of the day 4 subgroup, and the day 14 group performed similarly to the day 16 larvae. Subsequent observations of the experimental larvae showed them to be mixed population in regard to the number of molts from pupation. Some belonged to the last instar and others to the second to last, but some were earlier instars. A comparison of larval behavior at different instars has shown that the periodicity in their aptitude for learning exist for all the investigated instars; no accurate data on instar influence on aptitude were obtained. The mealworms showed a different aptitude for learning depending on the stage of the instar period, a peculiarity inherent in different instars of larvae. Therefore, the dynamics of memory trace formation must exhibit a cyclic character. A large literature exists about the learning of adult insects, but little information is available regarding learning at the larval stage of insect development. Larvae possess such pronounced peculiarities, distinguishing this stage from both embryonic and adult stages, that one must consider it independently. The larval stage is a postembryonic stage of insect development and larvae can live, feed, and adapt themselves as well as adults do. The larvae are characterized by the active development of most of their organs and systems, in particular the nervous system, and by the cyclic nature of this development. A relationship may exist between the cyclic facilitation of learning in larvae and the sensitive periods in the development of vertebrate behavior. However, a comparison of the larval stage of insect development with the early period in vertebrate ontogenesis is extremely difficult to draw because the latter does not possess the genetic rigidity notable in insects. Only incidental information exists about the cyclic changes in the structure and function of the insect nervous system. An investigation by Tyshchenko (1969) of spontaneous electrical activity in nerve ganglia of caterpillars has shown that this

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activity abruptly decreases at the time of a molt and then increases gradually during the intermolting period. Motor activity also decreases at the time of the molt. Beetsma, de Ruiter, and Wilde (1962) described a change in the reaction to light between molts of the larvae of Smerinthus ocellata L. These animals show photopositive behavior at the beginning of the instar that is gradually supplanted by a photonegative response by the end. The knowledge about periodical changes in the structure of the nervous system of larvae is no more abundant. The process of reproduction of neuron bodies and their branches presumably goes on monotonously during the larval development, and only mitosis of the glial and neurolemmal cells possesses a cyclic character and occur at the time of a molt. At the same time the neuron population is replenished with continuous fission of neuroblasts and ganglion-mother cells (Nordlander and Edwards 1968, 1969; Panov 1960). Unfortunately, data on this matter are not numerous enough to explain the cyclic features in learning, and they need some explanation. The broadly manifested periodicity in the development of insect larvae is governed by a group of hormones related to metamorphosis. Interaction of the hormones affects the course of each cycle of larval development. By the end of each instar the molting hormones of metamorphosis, namely, ecdysones, may have an effect upon these processes. The experiments verifying the effect of hormonal extracts upon the cockroach's nervous system (Ozbas and Hodgson 1958) indicated that the hormones may have a direct influence upon the nervous system of insects. Ecdysone is responsible not only for molting but for a whole series of changes in the larval organism connected with a molt. However, there are no data about such a causal connection. There also are no data regarding the dependence of learning on ecdysone or other hormones. It follows from the results of our previous series of experiments that changes in the efficacy of memory trace formation may depend on the ecdysone content and its activity in the larvae organism. That is why the following special series of experiments were performed to investigate the direct effect of ecdysone on larval learning. Memory Trace and the Hormones of Metamorphosis Larvae 20 mm long were used immediately after molting. They were placed in cups containing bran at 25° C. The next molt at this temperature occurred 12-13 days later. The T-maze training was intensive. In accordance with the previous results of successive larvae training, this series of experiments was made 3, 6 or 9 days after molting. Two hours before the experiment larvae were injected with 0.6 ul mixture containing 2 ug of ecdysterone, dissolved in insect physiological solution. Control animals obtained only the physiological solution. Each experimental group contained 10 larvae (Sheiman and Ignatovich, 1977). The results of the experiment indicate that the control animals from the 9th day differ from the other animals both in the total number of correct choices and in the

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dynamics of the increase in correct choices as the experiment progressed. A quite other situation was seen in the experimental groups. In the 6th day, group training was the most effective. At the same time, the 3rd and 9th day groups did not significantly differ from the control groups. In a special series of experiments the effect of injecting large doses of hormone were tested. Larvae from the different groups obtained 4 and 8 ug of ecdysterone. The results showed that increasing the dose of hormone improved the training in the 3rd- and 9th-day groups (Sheiman and Ignatovitch 1977). This experiment with exogenous ecdysone confirmed the supposition about the dependence of the cyclic recurrence of larvae learning on the endogenous ecdysone content. This investigation was made on the larvae from different stages of instar (Sheiman 1976). It was found that degeneration of the protharacal gland (which secretes ecdysone in insects) was revealed just after molting. On the 3rd day, together with the destroyed cells, appeared many small cells that did not separate from the tracheal wall. On the 6th day mature gland cells were observed concentrating around the gland cavity. On the 9th day the gland was ready for secretion. Thus, in accordance with the changes in the gland secreting ecdysone, it may be concluded that on the 9th day of instar ecdysone is active in the larvae body. It is precisely at this time that the larvae reveal the best abilities to learn. Clearly, ecdysone facilitates conditioning in beetle larvae. In parallel with the gradual increasing of endogenous hormone titre, larvae conditioning sharply improves at the end of the instar. Injection of high doses of exogenous hormone improves learning in different phases of the instar. The larval reaction to hormone depends on its inner state, which changed during the intermolting period. Small doses of hormone effect only the middle phase of the instar. Perhaps at this time larvae tissue are ready to react to ecdysone coincidentally with prothoracal gland maturation. Probably the corresponding receptors become active in larvae tissue at that time. To summarize our results thus far, during complex morphogenesis, characteristic of postembryonal development in insects, the specific picture of memory trace functioning is shown. Grain beetle larvae may be conditioned to choose the correct direction in a T-maze, and this memory trace is preserved during metamorphosis and manifest in adult beetles. It was found that different abilities to learn were related to the different stages of one intermolting cycle. This ability does not manifested itself in the beginning and middle of the instar. However, larvae may easily learn in the end of the instar and immediately after it. The larvae ability to learn correlates well with the content in their body of the metamorphosis hormone ecdysone. REGULATORS OF MEMORY AND MORPHOGENESIS In two previous sections, the substances closely connected with regulation of development and also with memory trace functioning were revealed and outlined. These are planarian RF and the insect molting hormone ecdysone. As the main

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function of these substances is the regulation of morphogenesis, they belong to the morphogens. Naturally, the question is raised of whether such common effects on development and memory are characteristic of other morphogenetic regulators. To answer this question it is necessary to find morphogenetic properties among the active regulators. The most attractive were the neuropeptides. There are true regulators of morphogenesis, such as different growth factors and hormones, components of tissue-specific factors, invertebrate growth regulators (as hydra head activator, hydra morphogen, or insect activation hormones) (Sheiman, Balobanova, Martinovich, Polikarpova, and Slobodchikova 1984, 1990). All these substances may be directly classified as morphogens on the basis of their general function. The first task was to reveal morphogens among peptides, in particular, among neuropeptides. The second task was to reveal the effects of morphogens on memory processes. Neuropeptide Effects on Invertebrate Morphogenesis In our experiments, neuropeptides were obtained from the laboratories of the Russian Cardiological Center and in the Institute of Bioorganic Chemistry, Byelorussian Academy of Sciences. We used neuropeptides in concentrations ranging from 10(-7) to 10(-11) M. The peptides were added to the water containing the planaria. The peptides (1 ul) were injected into insects with a Hamilton microsyringe or were mixed in with food that was given to both larvae and adult beetles (Sheiman, Tiras, and Balobanova 1989). We use different neuropeptides: luliberin (LHRH), vasopressin, somatostatin (SRIF), dalargin (analog of leu-enkephalin), D-sleep peptide (DSIP), and hydra head activator (HA), specific for lower invertebrates. Among them, HA and SRIF have direct morphogenetic functions, LHRH only partially, and the remainder do not. The results indicate that neuropeptides may stimulate or inhibit development. There is a stimulating effect on planarian regeneration with hydra morphogen (HA), vasopressin, dalargin, and SRIF. There is an inhibitory efect with LHRH, no effect with Delta-sleep peptide (DSIP). Insect metamorphosis was stimulated with HA and LHRH. In the majority of cases, neuropeptides had stimulative effects. However, the dynamics of these effects differ considerably. Vasopressin and dalargin were active throughout the experiment, and the effect of dalargin even intensified by the end of the experiment. HA was reliably effective in the beginning and in the end of regeneration. SRIF also acted positively, but only in the second part of experiment. The data allowed us to conclude that the various morphogenetic mechanisms of neuropeptides have an effect on planarian regeneration (Sheiman, Tiras, and Balobanova 1989). Stimulation of Insect Development Only two peptides, HA and LHRH, were used in the experiments on insect

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development. Both of them proved to be stimulators of larvae development. If the neuropeptide is presented in the beginning of the instar, the molting in treated animals occurs earlier than in controls. However, this stimulating effect of neuropeptides is not always revealed. There were no differences in molting between experimental and control larvae after LHRH injection in the second instar (8th day after molting). Inhibiting Effect of Neuropeptides The effects of LHRH and DSIP on planarian regeneration differ from the effects mentioned above. LHRH had an inhibitory effect on planarian morphogenesis during the entire experiment. The next peptide, DSIP, had no effect on planarian regeneration. Thus, apart from stimulation, neuropeptides may also inhibit planarian morphogenesis. There is also no influence of neuropeptides on planarian regeneration. Effect of Neuropeptides on Development and Memory in Insects Two neuropeptides (HA and LHRH) were tested in experiments on development and memory of insects. Hydra Head Activator Effects on Development in Grain Beetles Experiments on development were performed using 300 larvae injected with hydra head activator (HA) following various days post molt (Sheiman and Bespalova 1988). There was an equal number of control animals. The larvae and pupae that molted were collected every day. Some of the larvae received HA in concentrations of 10 (-7) M and the others received 10(-9) M. The results indicate that molting and pupation were unsynchronized both in experimental and control groups; this is characteristic for grain beetles development. In the beginning of molting the number of larvae in experimental groups was larger than in controls. HA had the same effect on pupation: Treated larvae that molted to pupae developed more quickly than controls. Molting in larvae with injection on the 7th-9th day of instar was delayed in comparison with larvae with earlier injection. Both experimental groups differed from controls. The results of this experiment indicate that application of hydra head activator accelerates the molting and metamorphosis of mealworms to pupae. Hydra Head Activator Effects on Memory in Grain Beetles T-maze instrumental conditioning was carried out on adult beetles over the course of five training sessions (10 trials per session). Experimental (treated with HA) and control groups (untreated) contained 35 animals each. We predicted that

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animals treated with HA would perform better than controls over a series of training sessions. The results of the experiment supported this prediction (Sheiman and Bespalova 1988). The number of correct choices over the course of the experiment was significantly higher in treated animals than in untreated controls, although both groups improved. The data indicates that in treated beetles the number of correct choices within a session is also higher than in untreated controls (with the exception of the first and third training sessions). Improved performance of treated versus untreated animals was observed when 1 pi HA was injected in a concentration of 10(-9) M, but no effect was observed at concentrations of 10(-8) and 10(-10) M. The HA morphogenetic action on larvae observed in previous experiments is similar to the effect of ecdysone or prothoracotropical. It is possible that HA has a direct influence on molting or works indirectly on prothoracal glands, producing ecdysone. A third possibility is that HA exerts influence on ganglion secretory cells, excreting ecdysone (Sheiman and Bespalova 1988). HA was discovered and intensively studied in Professor H. C. Shaller's laboratory. HA is excreted by hydra neurosecretory cells and regulates regeneration and budding in hydra (Schaller 1976). HA morphogenetic function manifests itself in some cases in the activation of budding in hydra and in other cases in the acceleration of insect metamorphosis. The common result of administering HA is to increase the rate of development. HA effect on development can be seen, as we have done here, by making crossspecies comparisons. Another function of HA was revealed in our experiments on adult beetles, the stimulation of learning. We did not find a direct connection between rate of development and learning because development was studied in larvae but learning was investigated in adult animals. However, it was shown that the administration of ecdysone stimulated learning in larvae. This suggests the possibility that the action of HA on the memory of adult beetles and its effect on larval development in beetles is regulated by the same mechanism. Effects of Luliberin and its Fragments on Development in Grain Beetles and Larvae LHRH has a predominantly gonadotrophic effect in mammals. The purpose of this experiment is to test the effect of this peptide on grain beetles in the course of their normal development. Eggs were placed on fresh bran and, after they turned into larvae, were weighed every day, and the number of molts were counted. The peptide was administered to the larvae by mixing it in their food. The effects of LHRH and its fragments were studied. Fragments L(l-2) and L(9-10) were used. Every experimental group contained 100 larvae, and experiments were repeated 2-3 times. Most experiments were performed with L(9-10), the C-end fragment of LHRH. In 3 series of experiments, 5 mg of peptide was added to the animals' food. Larvae

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weight and weekly growth were calculated. The results indicated that the control larvae grew faster than treated larvae. The average number of molts before pupation in the experimental group were found to be lower than in untreated controls. This difference was considerable in the first period of development and then disappeared. After pupation, molting in both groups continued. By the end of the experiment, the average number of molts in the experimental group was 6.7 and in the control group was 7.4. This difference was due to the lack of stable instars in the mealworm population. The effect of neuropeptides in such unstable systems produced molts in experimental animals that were, in average, 1 molt less than in controls. The duration of the instar period during metamorphosis gradually increased in experimentals. We calculated only the average duration of one instar. This was found to be 9.1 for the control group and 10.7 for the experimental group. The first pupae appeared in both groups simultaneously. The pupation of the whole population took about a month. The dynamics of pupae appearance was calculated every 10 days. The onset of pupation in the experimental group was later than in control animals. The conclusion of the experiments is that L(9-10) delays growth and development in mealworms. This is reflected by a delayed growth in weight, an increase in both instar duration and time of pupation, and a decrease in the number of molts. These data were replicated in the next experiments. Just before the new experiments were performed, large larvae were selected and injected with peptide. Injection of peptide, rather than by administeration in food, appears to traumatize the animal. Despite this limitation, injection has the advantage that it allows us to know precisely the stage of development when the peptide was administered and its dosage. In these experiments the dose was 1 pi of 10 (-5) M. The injections were made at different stages of development, 2 or 8 days after molting (i.e., in the beginning or close to the end of instar). Accordingly, there were 2 experimental and 2 control groups of larvae in each set of experiments. The type of molt (to larvae or pupae) was a variable. Each group contained about 100 animals. The results indicated that the percentage of pupation in the first molt in both groups was similar. However, when the peptide was injected 8 days after molting, the number of larval molts were more than that observed in pupae molting. A molting criterion (Mc) was established to analyze these results, in which the ratio between the total number of animals molting into larvae and the total number of animals in the experimental population was calculated. In two control groups these values were 0.78 and 0.79. In the two experimental groups, however, the values were 0.9 (in the group injection on the 2nd day) and 1.34 (in the group injection on the 8th day). The results indicate that the peptide stimulated larvae to molt. The effect of L(9-10) was slight following the day 2 injection but was considerably stronger after the day 8 injection. Additional results indicated that pupation "intensity" following day 8 injection was a little lower than in the control group. However, in the group that received the injection on the 2nd day there were more pupae than in control group.

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The results indicate that L(9-10), whether injected or administered in the larvae's food during the last instar, inhibited development. Also of importance was the time of peptide action (i.e., the stage in the intermolting period). Peptide introduced in the end of instar was more effective than when introduced just after molting. Experiments with L(l-2) were carried out using the same methods just described for fragments L(9-10). The peptide introduced in the food (0.5mg/ml) increased larvae growth in experimentals relative to control animals. Increasing peptide concentration to 1 mg/ml led to a delay of larvae growth that was lower than in a control group. The rate of pupation did not differ in experimental and control animals. L(l-2) (1 pi, 10(-5) M) was also injected on day 2 and day 8 after the initial molt. In control groups half the animals molted into larvae, and the other half into pupae. In the experimental groups, however, an injection in the beginning of the instar period produced 75% larvae molts and 25% pupa molts. In the day 8 group, 36% produced larvae molts and 64% pupa molts. The molting criterion was 0.79 in control groups and 1.43 and 0.62 in experimental groups (2nd and 8th day injection). These results indicated that L(l-2) stimulates growth and development of larvae. LHRH was also given by injection in another series of experiments conducted at the same time as the previous one. There was a visible increase in the development of experimental larvae after injection on day 2. Intensive molting and pupation occurred in experimental groups. There was no effect after day 8 injection of LHRH. LHRH did not effect the molt and pupae ratio, which was equal in experimental and control groups. Effects of Luliberin and its Fragments on Memory in Beetles LHRH and its fragments were tested on groups containing 30 subjects each. LHRH injection improved learning compared to untreated controls early in training, but as training continued the controls reached the levels of experimentals. N-end peptide L(l-2) had a stimulating effect in the beginning of training that was not sustained as training continued. In fact, the number of correct choices decreased. The C-end fragment L(9-10) had the most expressive effect on conditioning. Experimental animals made significantly more correct choices than controls throughout the experiment. It should be noted that a high number of correct choices were made in the beginning of training. LHRH and L(9-10) had stimulative effect on the number of correct choices, and L(l-2) had an inhibitory, effect which was revealed as training continued. It is characteristic that by the end of the experiment (after 4-6 hours of training) the results of different peptides were similar. It seems that the effect of peptides on memory was transitory. Our results suggest that LHRH was able to influence insect behavior. This is one of the more intriguing examples of the effects of neuropeptides. To understand the morphogenetic effect of LHRH, it is important to analyze its

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influence and interactions with specific insect regulators of morphogenesis, (i.e., the hormones influencing metamorphosis such as ecdysone, juvenile, and activating hormones). The results of LHRH fragment injection at different stages of instar indicate that injection of LHRH did influence development when it was injected in the beginning of instar. It is possible that when LHRH did increase molting it limited the effects of juvenile hormone. Thus, the effect of L(l-2) injection on day 2 corresponds to some extent to the effects of L(9-10) injected on day 8 and vice versa. The first phenomenon—increasing the total number of molts and molts to larvae coupled with a decrease of pupations—probably is connected with the reinforcement of juvenile hormone action or weakening of ecdysone action. The second phenomenon expressed after L(l-2) injection on day 8 but weakly revealed after L(9-10) injection on day 2 demonstrates the limitation of juvenile hormone action and intensification of the action of ecdysone. LHRH and its fragments generally had a stimulative effect on grain beetle conditioning; only the N-end peptide had an inhibitory effect. The inhibitory action of this peptide, however, did have an initial stimulating effect on conditioning. The same biphasic influence of peptides was found for ACTH fragments (Sheiman, Ponomareva-Stepnaya, and Maksimova 1980). The positive effect of LHRH fragments on beetle training was revealed as an acceleration in learning but not in terms of the total number of correct choices. The end fragments of LHRH had different effects on adult beetle memory and larval development. However, based on these effects it can be concluded that the influence of LHRH is realized through the interrelations with its fragments. This may explain why the effect of LHRH administered alone is weak in comparison with the effects of its fragments. Finally, it should be mentioned that the possible mechanism of LHRH action on learning is either direct or perhaps indirect via the hormones that regulate metamorphosis. It is possible that this conclusion is applicable not only to larval development but also to adult beetles conditioning. For after all, adult beetles contain the hormones necessary for metamorphosis (Sheiman, Balobanova, and Martinovich 1984, 1990). CONCLUSIONS The comparative study of memory and morphogenesis in the simple biological models reported here and an analysis of their interrelations suggests the following conclusions. 1. The memory trace formed in developing animals is more stable than in intact (planaria) or adult (insects) animals. Retention of the memory of conditioning is preserved during the morphogenetic processes. 2. Memory of conditioning is retained despite considerable destruction of nerve cells during morphogenesis. It must be taken into account, however, that the destruction of these cells is gradual and occurs simultaneously with the develop-

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ment of new nerve cells. These new cells via neoblasts make contact with old nerve cells prior to the destruction of these old cells. The new cells obtain the specific properties of their precursors. The most remarkable data convey the contribution that these new cells make to the memory trace. The transmission of memory information from "old" cell to "new" cell resembles the process of embryonal induction as defined by Weiss (1961). In planarian regeneration, for example, the transmission of certain information takes place from cell to cell. 3. Regulatory factors are active in the planarian body during the course of regeneration. These factors as well as insect hormones regulating metamorphosis can stimulate or inhibit the unfolding of development sequences. These factors also influence the formation of a memory trace. 4. Planarian regeneration factors, like insect hormones, are not homogeneous. There are many regulators of regeneration. These can be classified as inhibitors or simulators. They can also be classified acording to their effects on such cellular behavior as proliferation, migration, destruction, and differentiation. During the various phases of morphogenesis, it appears that different regeneration factors can dominate. These factors are also active in the process of memory. 5. Some substances (neuropeptides) were found to regulate morphogenetic processes in much the same way as regeneration factors and those hormones related to metamorphosis. These substances may form a common group of regulators the morphogens that may be related to memory. ACKNOWLEDGMENTS We wish to thank our colleagues A. M. Cherkashin, N. Ju. Sakharova, G. I. Bogorovskaya, E. P. Sergeeva, I. A. Efimov, G. S. Ignatovich, R. Ya. Gordon, S. S. Khutzian, and E. Ph. Balobanova who took part in many of these experiments. REFERENCES Afify, A. M. 1960. Uber die postembryonale Entwicklung des Zentralnervensystems (ZNS) bei der Wanderheuschrecke Locusta migratoria migratorioides (R.u.F.) (Orthoptera, Acrididae). Zoologische Jahrbucher, Abt. fur Anatomie und Ontogenie der Tiere 75:1-38. Beetsma, J., de Ruiter, L., and Wilde, J. 1962. Possible influence of neonetine and ecdysone on the sign of phototaxis in the eyed hawk caterpillar (Smerinthus ocellata L.). J. Insect. Physiol. 5:251-256. Bogorovskaya, G. I. 1969. Regeneration of the planarian nervous system. Tsitologia 77:964-972. Brondsted, H. V. 1969. Planarian regeneration. London: Pergamon Press. Brown, H. M , Dustman, R. E., and Beck, E. C. 1966. Sensitization in planaria. Physiol. and Behav. 7:305-310. Cherkashin, A. N., and Sheiman, I. M. 1967. Peculiarities of conditioned reflexes in flatworms (in Russian). In: E. M. Kreps, ed., Evolyutsionnaya Nejrofiziologiya i Nejrokhimiya (Evolutionary neurophysiology and neurochemistry), 31-35.

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Leningrad: Nauka Cherkashin, A. N., Sheiman, I. M., and Bogorovskaya, G. I. 1966. Conditioned reflexes in planaria and regeneration experiments (in Russian). Zhurnal Visshey Nervnoy Deyatelnosti 76:1110-1112. Cherkashin, A. N., Sheiman, I. M., and Sergeeva, E. P. 1966a. Effect of combination of light and electrical shock on planaria (in Russian). Zhurnal Vysshey Nervnoy Deyatelnosti 76:266-273 . 1966b. Experimental reproduction of integration in planaria (in Russian). Zhurnal Vysshey Nervnoy Deyatelnosti 76:858-863. Cherkashin, A. N., Sheiman, I. M., and Stafekhina, V. S. 1968. Storage of conditioned reflexes during metamorphosis in insects (in Russian). In: E. M. Kreps, ed., (Fiziologiya i Biokhimiya Bezpozvonochnykh (Physiology and biochemistry of invertebrates), 117-120. Leningrad: Nauka. Coming, W. C, and Riccio, D. 1970. The planarian controversy. In: W. L. Byrne, ed., Molecular Approaches to Learning and Memory, 107-149. New York: Academic Press. Cummings, S. B., and Moreland, C C 1959. Sensitisation vs. conditioning in Planaria: Some methodological consideration. Amer. Psych. 14:591-600. Ernhart, E. N., and Sherrick, C, Jr. 1967. Retention of habit following regeneration in planaria (D. maculata) In: J. V. McConnell, ed. A Manual of Psychological Experimentation on Planarians, 85. Worm Runner's Digest: Ann Arbor: ML Grasso, M. 1966. Sui fenomeni di neurosecrezione durante la rigenerazione di dischetti isolati di Dugesia lugubris. Arch. Zool Ital. 51:321. Gymer, A., and Edwards, J. S. 1967. The development of the insect nervous system. 1. An analysis of postembryonic growth in the terminal ganglion of Acheta domesticus. J. of Morphology 725:191-197. Jones, F. R. 1971. The response of the planarian Dendrocellum lacteum to an increase in light intensity. Animal Behaviour 79:269-276. Karpenko, A. A., and Seravin, L. N. 1973. Role of chemical factors in feeding behavior of Dugesia tigrina (in Russian). Zoologichesky Zhurnal 52:1142-1148. Kreps, E. M. 1925. About the reaction of ascidians to external stimuli (in Russian). Arkhiv Biologicheskikh Nauk 25:191-221. Krichinskaya, E. B. 1972. The structure of nervous system in Turbellaria (in Russian). Archiv Anatomii, Gistologii i Embriologii 63:99-110. Krilov, O. A., and Nazarian, O. A. 1973. Effects of homogenates of regenerating planarians on their retention (in Russian). Zhurnal Vysshey Nervnoy Deyatelnosti 25:1303-1305. Lender, T. 1952. Le role inducteur du cerveau dans la regeneration des yeux d'une Planaire d'eau douce. Bull. Biol. Fr. etBelg. 56:140-215. . 1955. Sur l'inhibition de la regeneration du cerveau de la planaire Polycelis nigra. CR. Acad. Set, 247:1863-1865. . 1956a. Analyse des phenomenes d'induction et d'inhibition dans la regeneration des planaires. Ann. Biol. 52:473-485. . 1956b. L'inhibition de la regeneration du cerveau des Planaire Polycelis nigra et Dugesia lugubris en presence de broyats de tetes ou de queues. Bull. Soc. Zool. France 57:192-199. . 1960. L'inhibition specifique de la differenciation du cerveau des Planaires d'eau douce en regeneration. J. Embryol. Exp. Morphol. 5:291-301

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. 1964. Mise en evidence et role de la neurosecretion chez les Planaires d'eau douce en regeneration. Ann. Endocrinol. 25:61-70. Lender, T., and Klein, N. 1961. Mise en evidence de cellules dans le cerveau de la Planaire Polycelis nigra. Variation de leur nombre au cours de la regeneration posterieur. C.R.Acad. Sci. 253:331-333. . 1962. Les cellules neurosecretices de D. lacteum. Bull. Soc. Zool. France 57:380-384. Lentz, T. L. 1968a. Primitive Nervous Systems. New Haven: Yale University Press. . 1968b. Histochemical localization of acetylcholinesterase activity in planarian. Comp. Biochem. Physiol. 27:715-718. Lindh, N. O. 1959. Heteroplastic transplantation of transversal body sections in flatworms. Arkiv. Zool. 72:183-195. McConnell, J. V. 1965. A tape recorder theory of memory. Worm Runner's Digest 7:3-10. . ed. 1970. A Manual of Psychological Experimentation on Planarians. Worm Runner's Digest: Ann Arbor, MI. McConnell, J. V., Jacobson, A. L., and Kimble, D. P. 1959. The effects of regeneration upon retention of conditioned response in the planarian. J. Comp. Physiol. Psychol. 52:1-5. McConnell, J. V., Jacobson, A. L., and Maynard, D. M. 1959. Apparent retentioned response following total regeneration in the planarian. Amer. Psych. 74:410-417. Morgan, T. H. 1901. Regeneration. Chicago: University of Chicago Press. Murphy, J. 1963. Learning in the planarian. Worm Runner's Digest 5:37-41. Nazarian, O. A. 1973. Storage of traces about experience after regeneration in planaria (in Russian). Zhurnal Vysshey Nervnoy Deyatelnosti 25:120-125. Nordlander, R. H., and Edwards, J. S. 1968. Morphological cell death in the postembryonic development of the insect optic lobes. Nature 275:780-781. . 1969. Postembryonic brain development in the monarch butterfly, Danaus plexippus. 2. The optic lobes. Wilhelm Roux Archiv fur Entwiklungsmechanic der Organismen 762:197-217. Ozbas, S., and Hodgson, E. S. 1958. Action of insects neurosecretion upon central nervous system in vitro and upon behavior. Proc. Natl. Acad. Sci. (USA) 44:825-830. Panov, A. A. 1960. The character of reproduction of the neuroblasts, neurolemma and neuroglial cells in the brain of the Chinese oak silkworm larvae (in Russian). Doklady Akademii Nauk SSSR 752:689-692. . 1963. The origin and fate of neuroblasts, neurons and neuroglial cells in the central nervous system of the Chinese oak silkworm Antheraea pernyi Guer. (Lepidoptera, Attacidae) (in Russian). Entomologicheskoe Obozrenie 42:186-191. Pedersen, K. J. 1958. Morphogenetic activities during planarian regeneration as influenced by triethylene melamide. J. Embryol. Exp. Morphol. 6:308-334. Pipa, R. L. 1973. Proliferation, movement and regression of neurons during the postembryonic development of insects. In: D. Young, ed. Developmental Neurobiology of Arthropods, 105-130. London: Cambridge University Press. Porfiryeva, N. A., and Diganova, R. Ja. 1987. Planaria of the European part of the USSR (in Russian). Kazan: Izdatelstvo Kazanskogo Universiteta. Sakharova, N. Ju., and Gordon, R. Ja., 1974. Neurosecretory activity during planarian regeneration (in Russian). In: Pervaya Vsesoyuznaya konferentsiya po neyroendokrinologii (First All-Union Neuro-Endocrinological Conference), 152-154. Leningrad.

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Sauzin-Monnot, M. J., Lender, T., and Gabriel, A. 1970. Etude autoradiographique ultrastructurale des neoblastes dans le blasteme de regeneration de 24 h. et dans les tissus en arriere du blasteme, chez la Planaire Dugesia gonocephale (Turbellarie, Triclade). CR. Acad. Sci. 277:1892-1895. Sbrenna, G. 1971. Postembryonic growth of the ventral nerve cords in Schistocerca gregaria Forsk (Orthoptera, Acrididae). Boll. Zool. 38:49-74. Schaller, H. C 1975. A neurohormone from hydra is also present in the rat brain. J. Neurochem. 25:187-188. . 1976. Head regeneration in hydra is initiated release of head activator and inhibitor. Wilhelm Roux's Archives 750:287-295. Schaller, H. C, and Gierer, A. 1973. Distribution of the head-activating substance in hydra and its localization in membranous particles in nerve cells. J. Embryol. Exp. Morphol. 29:39-52. Sengel, P. 1951. Sur les conditions de la regeneration normale du pharynx chez la Planaire Dugesia (Euplanaria). lugubris O. Schm. CR. Soc. Biol. 745:1381-1384. . 1953. Sur Finduction d'une zone pharingienne chez la planaire d'eau douce Dugesia lugubris. Arch. Anat. Microsc. et Morphol. Exp. 42:57-66. . 1959. La region caudale d'une planaire est elle capable d'induire la regeneration d'un pharynx? J. Embryol. Exp. Morphol. 7:73-85. Sheiman, I. M. 1976. Memory in insects and neurometamorphosis (in Russian). In: Structural and Functional Basis of the Memory Mechanisms, 188-204. Moskva: Nauka. . 1984. Regulatory Morfogeneza i ikh Adaptivnaya Funktsiya (The regulators of morphogenesis and their adaptive function). Moskva: Nauka. Sheiman, I. M., Balobanova, E. F., Martinovich, V. P., Polikarpova, V. P., and Slobodchikova, L. K. 1984. The effect of LHRH and its fragments on memory of beetle Tenebrio molitor (in Russian). Zhurnal Evolutsionoy Biokhimii i Fiziologii 20:314-319. . 1990. Morphogenetical effect of LHRH and its fragments on the metamorphosis of grain beetle (in Russian). In: I. M. Sheiman, ed., Morfogeneticheski Aktivniye Veshchestva (Morphogenetical active substances), 145-154. Pushchino-na-Oke: Biological Research Center. Sheiman, I. M., and Bespalova, Zh. D. 1988. Effect of hydra morphogen on the development and learning of the grain beetle Tenebrio molitor (in Russian). Zhurnal Evolutsionnoy Biokhimii i Fiziologii 24: 420-425. Sheiman, I. M., and Efimov, I. A. 1973. The importance of cell destruction in planarian ganglion during regeneration (in Russian). Tsitologiya 75:1215-1221. Sheiman, I. M., and Ignatovitch, G. S. 1977. Moulting hormone effect on training of Tenebrio molitor larvae (in Russian). Zhurnal Obshchey Biologii 55:627-633. Sheiman, I. M., Khutzian, S. S., and Ignatovitch, G. S. 1980. Periodicity in the behaviour of grain beetle larvae. Developmental Psychobiology, 75:585-590. Sheiman, I. M., Ponomareva-Stepnaya, M. A., Maksimova, L. A, Nezovibatko, V. N., and Ashmarin, I. P. 1980. Effect of adrenocorticotropin fragments on memory in the grain beetle Tenebrio molitor (in Russian). Zhurnal Evolutsionnoy Biokhimii i Fiziologii 76:359-364. Sheiman, I. M., and Tiras, Kh. P. 1983. Influence of endogenous factors of regeneration on the memory of Planarians (in Russian). Zhurnal Obshchey Biologii 44:94-104. . 1984. Formation and properties of planarian conditioned reflexes (in Russian). In: V. F. Konovalov, ed. Nejrofiziologicheskiye Mechanismy Pamyati i Obucheniya

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(Neurophysiological mechanisms of memory and learning), 46-57. Sheiman, I. M., Tiras, Kh. P., and Balobanova, E. Ph. 1989. Morphogenetic effects of neuropeptides (in Russian). Fisiologichesky Zhurnal SSSR 75:619-626. Sokoloff, V. A. 1960. The conditioned reflex to light in Asterias rubens (in russian). Trudy Murmanskogo Morskogo Biologicheskogo Instituta 2:236-248. Steele, V. E., and Lange, C 1977. Characterization of an organ-specific differentiator substance in planarian Dugesia etrusca. J. Embryol. Exper. Morphol. 57:159-172. Thompson, R., and McConnell, J. V. 1955. Classical conditioning in the planarian Dugesia dorotocephala. J. Compar. Physiol. Psychol. 45:65-68. Tiras, Kh. P. 1978. Acetylcholinesterase activity in the planarian nervous system during regeneration (in Russian). Ontogenez 9:262-268. Tiras, Kh. P., and Aslanidy, K. B. 1981. Device for graphic recording of planaria behavior (in Russian). Zhurnal Vysshey Nervnoy Deyatelnosti 57:874-877. Tiras, Kh. P., and Khachko, V. I. 1990. Criteria and the stages of regeneration in planaria (in Russian). Ontogenez 27:620-624. Tiras, Kh. P., and Sheiman, I. M. 1981. Effect of regeneration on planarian conditioning (in Russian). Ontogenez 72:635-638. . 1984. Chemical factors of morphogenesis in planaria (in Russian). Ontogenez 75:374-379. Tiras, Kh. P., Sheiman, I. M., and Sakharova, N. Ju. 1975. Acetylcholinestarase activity in the nervous system of several triclads (class Turbellaria) (in Russian). Zhurnal Evolutsionnoy Biokhimii i Fiziologii 77:427-429. Tushmalova, N. A. 1973. The types of memory of some lower invertebrate (in Russian). In: M. N. Livanov, B. N. Veprintsev eds. Kletochnye Mekhanizmy Pamyati, 40-48. Pushchino-na-Oke: Biological Research Center. Tushmalova, N. A., and Gromyko, N. M. 1968. The conditioned reflexes on chemical stimuli in planarian Policelis nigra (in Russian). In: Fiziologiya i Biokhimiya Bespozyonochnykh (Physiology and biochemistry of invertebrates), 34-39. Leningrad: Nauka. Tyshchenko, V. P. 1969. The spontaneous electrical activity in insect nervous system (in Russian). Trudy Vsesoyuznogo Entomologicheskogo Obshchestva 55:148-181. Voronin, L. G., and Tushmalova, N. A. 1965. About the so-called conditioned reflexes in planarians (in Russian). Zhurnal Evolutsionnoy Biokhimii i Fiziologii 7:98-103. Weiss, P. 1961. Interactions between cells (in Russian). In: G. M. Frank, A. G. Pasynsky eds. Sovremennye Problemy Biofiziki, Tom. 2. (Modern problems in biophysics, vol 2), 176-184. Moskwa: Izdatelstvo Inostrannoy Literatury. Welsch, G. H. 1946. Evidence of a tropic action of acetylcholine in a planarian. Anat. Rec. 94:421-432. Wolff, E. 1962. Recent researches on regeneration of planaria. In: Regeneration, 53-84. New York:: Ronald Press. . 1974. Analysis of substances inhibiting regeneration of freshwater planarians. In: Biology of the Turbellaria, 446-459. New York: McGraw Hill.

Chapter Four Innate and Acquired Behavior of Mollusks Pavel M. Balaban and Igor I. Stepanov

NEUROBIOLOGY OF MOLLUSCANS: AN HISTORICAL OVERVIEW The study of the behavioral neurobiology of molluscans started in Russia with a classic paper of I. P. Pavlov (1885), in which he investigated the role of different ganglia in the reflex closure of valves in Anodonta. The investigation was carried out during his visit to the laboratory of Professor R. Heidenhain in the Physiological Institute in Breslau. Using two kinds of preparation with ablated cerebral and pedal or visceral ganglia, Pavlov demonstrated quite different effects of these ganglia on the muscles that close the valves. Results described in this work suggested the existence of motor fibers (originating from visceral ganglia) that elicit muscle contraction, and of inhibitory fibers originating from cerebral ganglia that cause muscle relaxation. Pavlov investigated reflex responses to adequate stimulation of mantle and gill and demonstrated the necessity of certain ganglia for behavioral responses. Investigating the role of environment, he changed the quality and temperature of the water in which the molluscans were maintained, used pharmacological blockers to alter the behavior (morphine), and described the dependence of behavior on environment. A "summation" phenomenon showing the effect of previous stimulation on subsequent responses was described. In fact, this work in invertebrates was a prelude to the investigation of the principles of learning and brain functioning that Pavlov carried out later, after a large interval of time devoted to the investigation of stomach functioning and brain regulation of visceral functions, for which he won a Nobel prize in 1904. It is interesting to note that this pioneering work has almost all the components of a contemporary neurobiological study: (a) investigation of behavior (b) ablation experiments (c) usage of pharmacological tools (d) modeling of brain functioning (e) application of external stimuli, and (f) change of the conditions of experiment

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(conditioning may have appeared as a concept in these experiments!). Surprisingly, another publication that had a great impact on invertebrate neurobiology in Russia appeared the same year: N. P. Vagner (1885), Invertebrates of the White Sea. This large book with perfect illustrations was simultaneously published in Russian in St. Petersburg and in German in Leipzig. Vagner was one of the founders of the first biological marine station on the Solovetzky Islands in the White Sea where, between studies of other animals, he thoroughly investigated the pteropod mollusc, Clione limacina. The level of investigation of ecology, behavior, and morphology was so high that without new technologies there is nothing to add to this description. He described individual giant neurons and traced some of their branches and spoke about how it is possible to provide a complete investigation of the nervous system in this animal using the simplicity and clarity of its structure. Investigation of molluscan neurobiology on a large scale was started in Russia by Kh. S. Koshtojants, who himself was a student of a famous Dutch comparative physiologist, Hermann J. Jordan. In the laboratory of Koshtojants in Moscow University, the first studies concerning the influence of a "nerve factor" on snail muscles and behavior were carried out. Now we know the nerve factor under the name of neurotransmitter. Findings in invertebrate neurobiology were summarized in the extensive volume Comparative Physiology of the Nervous System (Koshtojants 1957), which was and still remains a brilliant insight into the comparative physiological approach. Investigation of transmitter functioning traditionally has remained one of the main themes in Russian neurobiology of invertebrates. Two students of Koshtojants (K. Rosza and J. Salanki), established a well-known laboratory in Hungary, while D. Sakharov continued to study the role of transmitters in invertebrate brain organization and behavior using electrophysiological, cytochemical, morphological, and phylogenetical approaches. Comparative investigation of structure and function of nervous system in pteropod Clione (Sakharov 1960) and later in different species of nudibranch mollusks (Sakharov 1962; Veprintsev, Krasts, and Sakharov 1964) provided evidence of the existence in two quite different species of identifiable cells with completely similar properties including specificity in biochemistry. This notion shifted the scientific interest of D. Sakharov to considering the problem of the genesis of multiplicity of transmitters in the brain. The questions he posed were: Why do we have a strikingly similar set of transmitters in phylogenetically very different animals? How does a similar set of transmitters appear in different animals, by analogy or homology? His findings and a hypothesis considering homology as a principle underlying the appearance of numerous transmitters in different animals are summarized in the book Genealogy of neurons (Sakharov 1974), which became a rarity the next day after its appearance, not only because of the presence in it of a transmitter poligenesis theory but implicitly because of the author's logical style and intelligible and expressive language. With the onset of an era of intracellular physiology, the quantity of laboratories

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working in molluscans flourished. Mostly to obtain an understanding of biophysical properties, giant nerve cells were chosen as the focus of many experiments. Among the first investigators was B. N. Veprintzev, who worked on giant neurons of a pond snail and succeeded in the investigation of ionic channels in isolated neurons; in his laboratory, isolated snail neurons were kept in culture (Kostenko, Geletyuk, and Veprintsev 1974). In the Ukraine, the membranes of snail neurons were used for investigation of ionic channels; the method of dialyzed neurons was introduced by P. G. Kostyuk and his collaborators (Kostyuk, Krishtal, and Doroshenko 1975). Behavioral and electrophysiological investigation of simple forms of plasticity in pond and terrestrial snails was started at the end of the 1960s in a newly formed Department of Psychophysiology (Moscow University). This group of researchers was directed by E. N. Sokolov, who switched from studies of orienting reflex and plasticity in humans and vertebrate animals to cellular mechanisms (Sokolov 1969). The main goal of this group of researchers was to provide a connection between simple networks investigated on an intracellular level and behavior. Habituation of neuronal membrane to repeated electric stimuli was one of the models used to investigate cellular and subcellular mechanisms of plasticity (Sokolov and Jarmizina 1970). A group of devoted snail researchers (to which P. Balaban belonged) in the 1970s moved from Moscow University to the Institute of Higher Nervous Activity headed by a collaborator of Pavlov, E. A. Asratjan, to investigate neural mechanisms of learning in invertebrates. It is important to note that, at the end of the 1970s in Leningrad, a group in the Institute of Experimental Medicine (I. Stepanov among them) started investigating factors influencing learning in terrestrial snails. Their laboratory was located in the famous "Tower of Silence" constructed specifically for the behavioral experiments of Pavlov at the beginning of the century. Thus, we can watch how a loop of a giant helix of scientific knowledge started by Pavlov turned a 100-year wheel. Principles of analysis of behavior and investigation of underlying brain structures have not changed too much but are completed by new technological tools like immunochemistry and electrophysiology. In the present chapter we will try to describe the approaches used to investigate behavior and its modifications in terrestrial snails Helix. BEHAVIORAL REPERTOIRE OF HELIX In the behavior of snails one can find all the major forms of behavior characteristic of higher vertebrates, including humans. They are feeding, escape (avoidance), exploratory, and sexual behavior. We will describe briefly all these behavioral forms before beginning an analysis of behavioral plasticity. Defensive Behavior Terrestrial snails demonstrate withdrawal reactions accompanied by mucus re-

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lease to all kinds of noxious stimuli. Defensive and feeding behavior of terrestrial snails (Helix pomatia, Achatina fulica) is easy to investigate if an animal's shell is fixed with plasticine to a holder as shown in Figure 4.1. A snail can freely crawl upon the surface of a polyethylene ball floating in water, and the behavioral responses can be recorded. A tactile stimulus, irritating chemical stimulus, or electric shock initiates defensive withdrawal responses. It includes a contraction of optic tentacles (ommatophores), small tentacles (rhinophores), a closure of pneumostome opening, and a withdrawal of a foot into the shell. Some mucus is secreted if noxious stimulus intensity overcomes the threshold. A normal snail exerts well-coordinated defensive behavior. Chemical substances injected into the body cavity can evoke separate components of withdrawal reaction. However, this behavior may not be coordinated. For example, glutamic acid, injected into the cephalopedal sinus in the dose of 0.1 mg/g of an animal's weight, induces the withdrawal of tentacles and foot and mucus secretion, but the snail can not fully withdraw into its shell because of the absence of coordination (Stepanov, Smirnova, and Sapronov 1994). The ethological analysis of snail defensive behavior may serve as one of the tests for screening of different environmental pollutants. Two elements of a defensive behavior are suitable for a detailed physiological analysis. First is the defensive reflex of pneumostome closure. This reflex in snails is controlled by the pneumostome circular muscle. The muscle contraction consists of three partly independent components. The first component is the tonic contraction. It depends on the background motor neurons impulse activity. The second type is spontaneous contractions. These contractions are mainly caused by spontaneous synchronous discharge of motor neurons. The third type is a reflex contraction elicited by sensory stimulation. The most suitable method for recording the pneumostome movements proved to be a differential photoelectric device (Stepanov and Poszinsky 1982). A pneumostome opening is transilluminated with light through a glass fiber bundle. When the pneumostome opening is opened, a red spot of an irregular shape appears on the shell opposite to the pneumostome. A photodiode in the lightproof cylinder is fixed to the shell. It receives the light passing through the pneumostome opening plus an environmental light. The second photodiode receives only the environmental light. A differential amplifier picks out only the signals from the pneumostome opening movement. The device is valuable if environmental light is changing. That may be the case if, for example, an action associated with turning off a light is investigated. It was found that the defensive behavior can be initiated when the intensity of the light is stepwise decreased. The hungry state of the animal suppresses the tonic type of circular muscle contraction. A pneumostome is opened to its maximal dimension. This was used to evaluate the level of maximum opening. After the level of the closed pneumostome is also recorded on a pen recorder, the difference between the two levels gives the maximal value of the amplitude of a behavioral response in a given snail. A ratio of amplitude of reflex closure to the maximal amplitude gives the relative amplitude as a percentage of maximal response. This approach provides an

Figure 4.1 Experimental setup for behavioral experiments. The animal is fixed by its shell to a holder (H), but in such a manner that it can move freely on a plastic ball (B). I: indifferent carbon electrode, T: electrically driven tapper, S: electrode for manual application of electric shock to the skin.

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opportunity to compare the responses in different behavioral states (Stepanov, Smirnova, and Sapronov 1994). The withdrawal of optic tentacles constitutes an independent component of withdrawal behavior. A method for quantitative recording of this behavioral response has been suggested (Stepanov and Poszinsky 1982). A polyethylene ball, floating in an aluminum basin, is covered with aluminum foil. The aluminum basin is connected with the negative pole of a DC constant current source. The active electrode (stainless steel wire) is connected with the positive pole and is positioned manually on the skin of the animal. The current range is 1-20 microamperes. The current intensity must be chosen individually for every snail. It has to exert the full contraction of stimulated tentacle and the hardly visible contraction of a contralateral one. After a withdrawal the tentacle begins to protrude. At a certain moment an eye appears on the tip of the tentacle. This appearance is clearly visible. An experimenter can record the time interval between the touching of the tentacle with the electrode and the appearance of the eye. This time interval was called the tentacle reflex time. It is interesting to mention that a histogram of tentacle reflex time is asymmetrical and that the distribution is not normal. The left side is short, and the right side resembles a tail (this shape of the histogram was found both in Helix and in Achatina). Any influences move the histogram along the time axis or changes its shape. The tentacle withdrawal reflex may be used as an objective and quantitative criterion of any influences on the central nervous system. A component of the phototactic behavior may be regarded as defensive behavior. Snails prefer darkness, and this preference can be quantitatively estimated in a special chamber, which is designed in the following way. Half of the chamber is covered with a lightproof cover, and the second part of the chamber receives light from an electric bulb containing a heat filter. An animal or a group of animals at the beginning of the experiment is situated in the middle of the bright part of the chamber. Every 30 minutes the number of animals that crawl into the dark compartment is counted, over 4-hour period. It was shown that if both parts of the chamber are equally illuminated, the animals are distributed equally in the whole chamber, meaning that there is no preference for any part of the chamber. The redistribution of more than 85% of animals into the dark compartment, as was shown, points to a strong dark preference. As the percentage of animals moving to the dark compartment was found to be exponentially fitted, so regression analysis may be used. It is interesting to note that even after removal of the eye such preferences remained (Stepanov and Sidelnikov, unpublished data). This finding suggests that extraretinal receptors found in the mantle and skin of snails mediates this behavior. Feeding Behavior Terrestrial snails are usually generalized herbivores faced with the problem of locating food and assessing its nutritional value or possible harm. Foraging snails

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detect food odors from a distance of 20-50 cm via olfactory receptors located at their tentacles. Helix inverts its lips and protracts small tentacles (rhinofores) to taste the food. In the feeding behavior of snails, appetitive and consummatory phases are easily observed. The appetitive phase contains active locomotion in the direction of food and light touches by either pair of tentacles. We consider the behavior after the contact of rhinophores with food as the consummatory phase, which contains lifting of the head, contact with food by the lips, and rhythmic buccal mass movements, resulting in scraping the food. The final decision to begin feeding is based on the chemosensory stimulation received at the lips. It will be described in the following sections that Helix is capable of associating the odor of food with a subsequent electric shock. It is possible to record the duration of the appetitive phase and the latency of the consummatory phase in the setup described above (Figure 4.1). A piece of food (carrot, cabbage, apple, etc.) weighing 2-4 g was attached to the stainless steel wire, which may be used as an electrode. The food was presented to the animal's mouth at a distance of 2-5 mm. The hungry snail captures the piece of the food with its mouth and pulls the food from the needle. The time interval from the presentation of food to obtaining it from the needle can be measured by an experimenter. It was found during a session consisting of 5-10 presentations of food that the above-mentioned time interval decreased exponentially. Experimental data are well fitted with the mathematical model, which describes the reaction of a firstorder linear system to a steplike external stimulus (Stepanov 1983):

where X is a number of a trial, Y is a time interval, B3 is the coefficient reflecting an initial level of the feeding status, B4 is the coefficient reflecting the asymptotic level of the feeding behavior, and B2 is the coefficient reflecting the velocity of the system transition from initial level to asymptotic one. The coefficient B3 reflects the food preference. The less B3, the more preferable this kind of food is to the animal. In Helix pomatia, B3 for carrot was less than for cabbage. Figure 4.2 shows the performance of snails given 10 trials with an intertrial interval of 6-10 minutes. Curve 1 represents the time needed to capture a piece of cabbage. The coefficients of the mathematical model are as follows: B2 = 0.62, B3 = 87, B4 = 18. Curve 2 represents the time needed to capture a piece of carrot. The coefficients of the mathematical model are as follows: B2= 0.60, B3= 46, B4= 16. The coefficients B3 differ significantly (p < 0.001), indicating that B3 can be used to reflect food preference. This result confirmed the experimental observations on snail feeding behavior in a terrarium, which indicated that animals preferred to eat carrot. Later it was shown that the feeding behavior of Achatina fulica also fits with the abovementioned mathematical model (Stepanov, Nikolaev, and Borodkin 1991). During the consummatory phase of feeding behavior, defensive behavior changes; 15 minutes after the first chewing movement, a generalized defensive

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Figure 4.2 Performance of snails in a food capture task. Curve 1 represents the time needed to capture a piece of cabbage. Curve 2 represents the time needed to capture a piece of carrot.

reaction can be elicited by a weaker stimulus (Shevelkin 1989). It is interesting to mention that the neurochemical basis of feeding behavior of snails and mammals can be very close. For example, a peptide pentagastrin stimulated snail's feeding behavior (Sudakov and Kozyrev 1986). Exploratory behavior It is difficult to distinguish between the appetitive phase of feeding, during which the snail locomotes actively, and exploratory locomotion. During all types of locomotory activity, even satiated snails contact the substrate with lips and tentacles, occasionally trying to eat it. It should be noted that satiated snails do not usually locomote, but rather stay immobile in shells. An interesting case of exploratory behavior can be easily seen in response to a weak repeated tactile stimulation. Instead of withdrawal, the snail turns its head to the stimulated place and tries to actively contact the stimulating object. High humidity and water elicit a high level of locomotory activity in snails, which can be regarded as exploratory

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at least in part. Sometimes they travel quite a long distance in such conditions. Sexual Behavior The sexual behavior of Helix is far from dull. Courtship in these hermaphroditic snails lasts for about 8 hours and consists of 12 distinct stages (Lind 1976). The highlight of courtship is the reciprocal shooting of calcareous "love darts" from and into both members of the mating pair. A short time later the penises are everted and reciprocal copulation ensues. Prior to dart shooting, the degree of eversion can be reliably classified into six successive stages from its size and shape (Adamo and Chase 1988). A group of neurons in the metacerebral lobe of the cerebral ganglion was identified as responsible for releasing the love dart and everting the penis (Chase 1986). Normally, in the behavioral hierarchy of Helix, avoidance behavior suppresses feeding and exploratory locomotion. Recently it has been shown that courtship can suppress feeding (Adamo and Chase 1991). Locomotion is suppressed during copulation, but avoidance responses are not suppressed significantly. Information about the behavioral repertoire of Helix is necessary for investigation of behavioral plasticity, which will be described in the following sections. NONASSOCIATIVE MODIFICATIONS OF BEHAVIOR Habituation and sensitization of a behavioral response are two forms of modification of behavior that, by definition, are not associative. Habituation refers to a decrement of responsiveness due to repetition of a stimulus. Habituation should be distinguished from fatigue, when the response is not restored by the presentation of a novel stimulus. Sensitization is a form of nonassociative learning in which an animal learns to strengthen its defensive reflexes and to respond with escape to a variety of previously weak or neutral stimuli after it has been noncontingently exposed to potentially dangerous or noxious stimuli. In behavioral experiments carried out in a specially designed snail setup (Figure 4.1), we investigated plastic properties of the avoidance reaction in Helix. Repeated tactile skin stimulation of moderate intensity elicits certain dynamics of avoidance response amplitude. Normally, the response to the second and third stimuli was greater than the first response in a series. After this short period of amplitude increase, which can be considered to be sensitization, a decrease of response amplitude to successive repeated stimuli was observed. We tried to establish whether these changes in amplitude are plastic, by using the parametric features described by Thompson and Spencer (1966). Sixty-five adult animals were used for these experiments, and the results are similar for 85%-90% of snails. In 10%-15% of cases the results differed from the average, but we presume that this difference, which sometimes disappeared during the experimental session, is due to some uncontrolled changes by the experimenter

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in the functional state of the animal. In the majority of the experiments the decrement of the response amplitude was more rapid if: (1) intensity of the stimuli is increased; (2) interstimulus interval is decreased. Extrastimulus (3) or a rest period (4) restores the amplitude. Effectiveness of the extrastimulus declines with several applications (5). Response amplitude decreases more rapidly during the successive series in one experimental session (6). Change of the stimulation site of more than 2 mm led to partial restoration of response to repeated stimulation (7). Decrease of the interstimulus interval led to a slower decrement of the response amplitude (8). A necessity for amplitude restoration (to a given value), a period of rest is significantly increased if stimulation is continued after full extinction of the response. Therefore, all parametric features of habituation are fulfilled in Helix, and we can call the decrement in question habituation. Habituation persisted throughout the experimental session, but never was seen 2-3 days after the elaboration. A noxious electrical shock applied to the foot of Helix, evokes not only restoration of habituated response of tentacles withdrawal and pneumostome closure but also an increase above the initial value in some cases (Balaban 1983). This suggests that sensitization can be independent from habituation. ENVIRONMENTAL CONDITIONING It was shown in a marine snail Aplysia that sensitization persists from minutes to weeks, depending on the number and intensity of the sensitizing stimuli (Pinsker, Hening, Carew, and Kandel 1973). In the Pavlovian school the test for existence of environmental conditioning is obligatory, especially in cases when nonassociative processes are studied (Kupalov, Voevodina, and Volkova 1964; Pavlov 1927). Environmental (context) conditioning is a form of associative learning in which the reinforcing stimulus is made contingent on environmental properties. Several series of experiments aimed to investigation of existence of long-term sensitization and environmental conditioning in terrestrial snails are described. In the experimental setup, the snail was tethered by its shell in a manner allowing it to crawl on a ball that rotated freely in a 0.01% solution of NaCl (Figure 4.1). The ball was laced with bare stainless steel wire to complete an electrical circuit between the animal's foot and a carbon electrode placed in the water. Electric shock was delivered using a 1-10 mA, 0.5 s current through a macroelectrode applied manually to the dorsal surface of the snail's foot. Punctate mechanical stimuli were applied with calibrated von Frey hairs, permitting delivery of pressures ranging from 6 to 68 g/mm2. The snails were injected with 5,7-DHT (dihydroxytryptamine creatinine sulphate from Sigma) in a volume of 0.1 ml, in doses of 15 mg/kg dissolved in physiological solutions containing 1 mg/ml ascorbic acid as antioxidant. Control animals received an injection of the same volume of vehicle. After several preliminary series, the behavioral response, the intensity (25

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gm/mm), and the location of tactile stimulation were chosen. Tactile stimulation of the middle part of the foot appeared to be sensitive to previously applied noxious stimuli, and it was chosen for investigation of long-term effects. Each snail was exposed for 20 minutes daily to the experimental setup. Only snails from two experimental groups received 2 electrical shocks per day for 5 days. The shocks were explicitly unpaired with testing tactile stimulation. Three days after completion of sensitizing treatment (animals were fed during 3-day periods of rest), the responsiveness to the same tactile stimuli was compared in control and experimental snails. An experimenter blind to the experimental histories of animals applied the tactile stimulus to the skin of the foot and measured the withdrawal amplitude in percentages of the maximal withdrawal, taken as 100%. Testing was performed in the experimental setup and also in the nonreinforced environment, on the glass lid of a terrarium in which the animals were kept between sessions. To reduce possible effects of recent handling, the test was administered no sooner than 5 minutes after the subjects had been placed in the environment. Only actively locomoting animals were tested. Five tests per day for 2 to 3 days were scored for each animal. No shocks were delivered during the test sessions. Six days after the completion of sensitizing treatment and testing sessions, 4 control and 4 experimental snails were injected with 5,7-DHT, while others were injected with vehicle. Three days after the injection, a second testing session was performed using the same procedure as described before. Before noxious reinforcement, no significant difference in amplitudes of tentacle withdrawal to the testing tactile stimuli existed. Three days after a 5-day session, during which experimental snails received 2 shocks per day, testing of responsiveness performed in the setup used for sensitizing revealed a significant increase of the median response amplitude in sensitized animals. The difference between control groups and the difference between sensitized groups of animals were not significant. The amplitude of withdrawal was greater in the context previously paired with the shock than the response of the snails that did not receive the shock and were tested in the same setup. Our next step was to compare responses of control and shocked snails in another environment. Similar results were obtained with the same animals on the glass lid of the terrarium in which animals were maintained continuously between training sessions. Testing was carried out for three days, alternating with testing in the experimental setup. Besides a change in the surface texture, intensity of light was lower, and the snail was not fixed by its shell during testing on the glass. No significant difference in responsiveness was found between control groups or experimental groups. In fact, snails displayed a heightened defensive reaction only in the environment that had a history of pairings with shock. This outcome is consistent with the assumption that the snails can differentiate the environment in which shocks were scheduled to occur. The specificity of that enhancement is extremely important because it allows us to rule out a sensitizing effect of the shock as the sole result of sensitization training.

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As to the effect of 5,7-DHT injection, it was shown previously in Helix that after 5,7-DHT treatment (ablating the serotonergic neurons) both the sensitization of the withdrawal reaction and associative aversive conditioning are impaired (Balaban, Vehovszky, Maksimova, and Zakharov 1987). The feeding behavior of 5,7-DHTinjected intact animals was visually normal, as were the electrophysiological responses of the investigated neurons to single feeding and noxious stimuli. This is indicative of the fact that the absence of associative learning of this type is not due to changes of responsiveness of the neurons taking part in the feeding and aversive behavior, respectively. Further, 5,7-DHT treatment after elaboration of aversive conditioning in Helix does not impair the conditioned responses (Balaban et al. 1987). This result suggests that 5-HT-containing cells participate in associative learning during the consolidation phase of the conditioned reflex but are not necessary during its reproduction. In the present work we tested the possibility of the involvement of 5-HTcontaining neurons, which modulate the network underlying avoidance responses (Zakharov and Balaban 1991), in environmental conditioning. Injection of 5,7DHT led to disappearance of the effects of training. Only responses of vehicleinjected sensitized snails differed significantly from responses of snails from other groups in both environments. The difference between vehicle-injected sensitized snails and 5,7-DHT-injected snails also was significant. This result suggests that 5HT-ergic neurons are necessary for the reproduction and/or maintenance of environmental conditioning. ASSOCIATIVE MODIFICATIONS IN BEHAVIOR The history of attempts to elaborate conditioned responses in snails started with the experiments of E. Thompson (1917) using the pond snail Physa gyrina. He associated tactile stimulation of foot with food presentation, and after 250 paired trials in 2 days tactile CS elicited feeding responses in 39.6% of cases compared to 3.3% before conditioning. The changes were maintained for 4 days, and no controls with nonpaired presentation or differential conditioning were made. In Russia, experiments in Physa acuta were carried out by V. Sokolov (1959), who paired light with water application of noxious 0.2% KC1 solution. Conditioned responses (snails moved to a dark compartment) were noted after 8 paired presentations and became frequent after 30 trials. However, this reflex was not stable, and fast extinction was seen. The first report on conditioning in terrestrial snails was published in 1976. The pneumostome closure evoked by strong noxious stimuli was used as an unconditioned response in these experiments (Litvinov, Maximova, Balaban, and Masinovsky 1976). As the conditioned stimuli, weak noxious stimuli were used that normally do not evoke pneumostome closure: tactile stimulation, local heating of the foot epithelium, or tapping on the shell. Elaboration was slow, and more than 100 trials were necessary for conditioning. However, the conditioned stimulus

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(CS) used was of the same modality as the reinforcement; therefore, the question about nonassociative sensitization as the mechanism of observed changes in behavior was not elucidated. Pavlov wrote that in principle any stimulus initiating any response may serve as the conditioned stimulus (Pavlov 1951). The only requirement is that this stimulus should not initiate the same reflex that is initiated by the unconditioned stimulus (UCS). In the experiments of O. A. Maksimova (Maksimova 1979), a tactile stimulus was used as the CS, and food as the UCS. An animal had to turn to the right when CS was delivered in order to receive a piece of food. Before learning, the conditioned stimulus elicited only withdrawal reactions. After 100-150 paired trials, snails began to react to tactile stimulus with feeding behavior (details are published in a book: Maksimova and Balaban 1983), turning to the right side, where they usually found food. Conditioning with food reinforcement is easy to elaborate, but such reinforcement cannot be given on a preparation suitable for intracellular investigation. Therefore, another behavioral approach was chosen. Food-Aversion Conditioning The primary purpose of behavioral experiments is to generate rapid and obvious modification in motor responses, reproducible in neurophysiological preparation, and to demonstrate specificity of elaborated behavioral modifications for the pairing of conditioned and unconditioned stimuli. The withdrawal reaction as an unconditioned response meets these criteria, and the underlying neuronal circuitry has already been investigated in Helix (Balaban 1983; Zakharov and Balaban 1991). Aversive learning appeared to be one of the most suitable paradigms for investigation of learning in snails, because it usually concerns two competitive behavioral acts, one of which changes dramatically because of the pairing of two stimuli. To be suitable for neurophysiological investigation, learning should occur rapidly, and as in a study on the carnivorous mollusc Pleurobranchaea (Davis and Jillette 1978; Mpitsos and Collins 1978), we reinforced food presentation by strong electric shock, which evoked generalized withdrawal reaction of the snail (Maksimova and Balaban 1983). A similar procedure was used in the experiments of Stepanov and Lokhov (1986). Snails chosen for experiments were deprived of food for a week prior to conditioning. A day before conditioning, the snails were tested for their feeding behavior. Subjects who refused food were excluded from the experiments. During 5 days, 1 session per day was performed with 10 trials per session. When a food piece (carrot, cabbage, etc.) is offered to an animal at a distance of 4-5 mm from the mouth, the snail begins to search for the food (the appetitive phase of feeding behavior). It lowers its optic tentacles and tastes the food. Then the animal opens its mouth, captures the food piece, and pulls it away from the needle (consummatory phase). The time interval between the presentation of the food and a hungry snails removing it from the needle is 20-50 sec. This time never exceeds 1 minute

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in active animals. The reinforcement consists in turning on the current when the animal captures the food piece. The intensity of the current is selected in such a way as to evoke the withdrawal of tentacles and a half of the anterior portion of the foot. The pneumostome often does not react to this stimulation. It is necessary to use a moderate current intensity to avoid a generalized escape reaction. In these conditions every animal begins to avoid the food at the end of the first session. The criterion of the conditioned reaction in a trial was food avoidance for 3 minutes after food presentation. The food avoidance behavior manifests itself in different ways. At first, a snail ceases to lower its optic tentacles. Then the animal raises the most anterior part of its foot up to prevent contact of its lips with the food. Further, the snail withdraws both pairs of tentacles and the anterior part of the body. Finally, the animal crawls away from the needle with the food after the first contact with it. In other words, a fully conditioned animal behaves as a satiated one. It is important to emphasize that an experimenter can observe and estimate the individual conditioning of every subject. It is enough for this purpose to compute a percentage of conditioned reactions (CR) as a number of food refusals during a session divided by 10 (because 10 trials per a session are used) and multiplied by 100. This percentage increases from the first session to the fifth session. Properties of Conditioning and Two-Way Conditioning Intensity of Reward In full accordance with Pavlov's (1927) data, intensity of reward in our experiments had great influence on speed of elaboration of conditioned reflex. In a series of experiments in which we used a weak reinforcement, the dynamics of elaboration were quite different. We consider the reinforcement as weak if it elicits only tentacles and head withdrawal, while strong reinforcement, used in some experiments, elicited complete withdrawal of the body in the shell and release of mucus. It was possible to deliver up to 20 trials per day to one snail if a weak reinforcement was used. In all other respects the experiments were similar to experiments with a strong reinforcement. In the cases when the snail did not take food for 150 sec, no reinforcement was given, thus encouraging the snail not to respond. Unpaired control received the same number of food presentations and reinforcements. It appeared that usage of a weak reinforcement requires about 60-100 paired trials for exceeding the 60% level of conditioned responses, and only 5-15 trials when a strong reinforcement was used. But in both cases all stimuli should not be given in one session (day); no less than 3 days should pass from the beginning of the experiment. Optimal timing is 5-8 days with 1-2 paired trials for experiments with strong reinforcement, and 10-20 trials for experiments with weak reinforcement. It means that, independently of intensity of reward some optimal timing exists. Retention of Conditioned Reaction In a major part of learning experiments in snails, we tested responsiveness

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during the first week after the end of a learning session that lasted 5-10 days. Average percentage of aversive reactions (withdrawal and refusal) during the first week was 85%-90%. Measuring the level of responses in trained snails 2, 4 and 6 weeks after the training showed that even after 6 weeks the percentage of conditioned responses was about 60% (Maksimova and Balaban 1983). This result was consistent throughout several independent series of experiments in 28 snails. Therefore, the duration of behavioral changes is long enough to consider it as a long-term modification of behavior. Differential Conditioning Besides employing unpaired controls, which was conventional for all experiments, in some series we used a different odor (or type of food), which was presented without the reinforcement. At first, when the nonreinforced odor was presented, a conditioned response can be seen in about 50% of the presentations. Very quickly, however, this percentage decreased to 10%-15%. An example is given in Figure 4.3 of such an experiment in which pneumostome closure was monitored using a light-beam and a photodiode. In this experiment the presentation of watermelon odor (cotton was soaked in watermelon juice and presented at a distance of 5 mm from the tentacles) before pairing did not evoke any withdrawal response (upper trace, Figure 4.3). After 10-20 pairings with a moderately intense noxious stimulus, only a transient response is seen (second trace, Figure 4.3). After several hundred pairings the conditioned withdrawal response to food in hungry snails is long lasting and stable (third trace, Figure 4.3), but presentation of an unpaired odor (carrot) evokes no response (lower trace, Figure 4.3). Differential conditioned response confirms specificity of the elaborated behavioral changes. Extinction and Spontaneous Recovery Active extinction of conditioned aversion response to food is possible in one day. In a series of experiments, 5 conditioned snails were presented with food offered at small intervals (3-5 min). After 30 presentations the number of aversive responses decreased to 50%, and continuation of presentation of food led to zero aversive reactions. It should be remembered that the snails are hungry and feeding motivation is high. Presentation of the same food to the same animals 24 hours after the extinction trials revealed spontaneous recovery of the conditioned response, but the rate of extinction was higher. At 72 hours after the second extinction series, the level of conditioned responses to the first 10 stimuli was even higher than 24 hours after learning (about 70%), and extinction was rapid. These results are in full conformity with results obtained in vertebrates (Pavlov 1927) and allows us to consider the observed modifications of behavior as classical conditioning. Two-way Conditioning One of the most interesting paradigms introduced in science by Pavlov's student

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Figure 4.3 Pneumostome closure responses in the snail during paired presentation of watermelon odor (tilled dot) and moderately intense noxious stimulus (triangle, upper trace) across training trials. The upward shift in the trace represents pneumostome closure. The unfilled dot represents the presentation of carrot. Carrot odor was not paired with the noxious stimulus.

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E. Asratjan (1970) is "two-way conditioning." The operational definition of twoway conditioning is clear and simple: after elaboration of a conditioned feeding response in dogs to light by repeated contingent presentation of light and feeding reinforcement, presentation of food only elicits before normal feeding an anticipation of light stimulus—eye-blinking and turning of the head to the light source. In our opinion, this notion of formation of associative connection not only between the conditioned stimulus (CS) preceding the unconditioned stimulus (UCS) but in the reverse direction as well undermines a part of Pavlovian theory that presumes that a connection is formed only when the CS precedes the UCS. Discussion of this point was taboo in Russian literature until recently, but the phenomenon of two-way conditioning was abundantly described in the literature and is considered a necessary criterion for an animal capable of associative learning. Therefore, we carried out several series of experiments on the behavioral and cellular level aimed at demonstrating the possibility of the formation oftwo-way connections in snails. In fact, all experimentation turned to testing what will happen in the animal with elaborated aversive conditioning, if a noxious stimulus (similar to one used as a reinforcement, but weaker) is applied. It appeared that weak noxious (tactile) stimuli, which before learning evoked no response or withdrawal, after elaboration of aversive conditioning elicited activation of exploratory activity and feeding behavior. Such behavioral response never was observed before learning. In responses of identified cells involved in feeding the absence of response to noxious stimuli changed to activation of the pattern accompanying feeding (Balaban, Maksimova, and Galanina 1985). These results suggest that two-way conditioning can be formed in snails. Results presented in this section confirm the suggestion that Helix can associate sensory inputs and adaptively modify its behavior for a period of time substantially greater than the time needed for protein turnover, thus suggesting involvement of genomes. Mathematical Model of Conditioning During a search for an adequate mathematical model of conditioning dynamics, the above-mentioned reaction of the first-order linear system to the stepwise external stimulus has been found to be good enough to fit all experimental data (Stepanov 1983):

This model is one of the best initial approximations of the real physiological process of learning. All regression coefficients may be interpreted in physiological terms. B3 is the initial level before the beginning of conditioning, and B4 is an asymptotic level of a conditioned state. B2 is the velocity of an achievement of the asymptotic level. It is necessary to pay attention to B3. Nearly all previous mathematical models of learning were based on the probability theory approach. It is

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well known that probability values must lie in the interval between 0 and 1. However, many experimental data could not be significantly fitted if B3 had to be equal to zero. This model allows B3 to receive any value—positive, zero, or negative. The negative values of B3 point to an inhibitory influence on the elaboration of the conditioned reflex, and the positive values provide information about facilitation of learning taking place prior to conditioning itself. This method not only gives an opportunity to calculate coefficient values but also computes a confidence interval for every coefficient (Stepanov 1983). Moreover, if an experimenter uses a group of animals he can calculate the variance of measurement error and, with the help of Fisher's F-criterion, be convinced of statistically significant fitting of experimental data. Food avoidance conditioned reflex was found to be fitted significantly by this model. The analysis of the three coefficients gave additional information about the learning process. For example, if a preferred food was chosen as a conditioned stimulus, then B3 values were negative. If non-preferred food was used as a conditioned stimulus, then B3 values were close to zero. B3 corresponded well to real behavior of the animal. If another kind of food was used as the differential stimulus (for example, cabbage as CS carrot as DS), then the asymptotic level (B4) was greater in comparison with these coefficients than in animals conditioned without the differential stimulus exposure. Figure 4.4 shows an application of the model to this situation. The coefficients of the mathematical model in the first curve are: B2 = 0.078, B3= 0, B4= 98; for the second, B2 = 0.078, B3= 0, B4 = 91; and for the third, B2 = 0.049, B = -32, B4 = 93. The coefficients B3 for curves 1 and 2 differ significantly (p < 0.05). The coefficients B2 and B3 of curve 3 differ significantly from the same coefficients of the curves 1 and 2 (p < 0.05). It is interesting to note that during the first session there were food refusals during differential stimulus exposure. However, the animal did not refuse DS at all to the end of conditioning (5th session) and some weeks later. This result indicates the continuation of memory formation after the end of conditioning itself. This may be a sign of a biochemical process involvement in the memory consolidation stage. This process strengthens only the temporal connections between CS and US. At the same time the temporal connection between DS and US is inhibited and decreased. This model allows us to estimate the level of memory retention some time after original learning. As a rule, food avoidance of CS is maintained up to one month. When a second conditioning experiment was undertaken with the same CS, the coefficient B3 received positive values. This value can be used as a quantitative parameter of conditioned reflex maintenance and stability. Conditioned Tentacle Withdrawal It is necessary to mention that food avoidance conditioning strongly depends on the level of satiation. An experimenter must carefully control all details of food consumption. As mentioned above, the initial level of feeding motivation must be

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Figure 4.4 The dynamics of food avoidance learning in Helix fitted to the mathematical model. 1: conditioned stimulus—carrot, differentiated stimulus—cabbage; 2: conditioned stimulus—cabbage, no differentiated stimulus; 3: conditioned stimulus—carrot, no differentiated stimulus.

created by 7-10 days of food deprivation. During conditioning the animal should not receive any food at all; the differential stimulus becomes more preferred because it allows animals to eat the food used as DS. During any testing for memory consolidation and retention, animals must receive restricted amounts of food. Another reflex, which is not dependent on food motivation, is conditioned tentacle withdrawal. An interesting version of this kind of learning, used first in the lab of G. Kerkut (Emson, Walker, and Kerkut 1971), was designed by G. Christoffersen (Christoffersen 1981). He described the main properties of this reflex. In our experiments, a snail's optic tentacles are extended when an animal is crawling on the surface of a ball. The first touch of the optic tentacle tip with the wire electrode initiates a rapid and complete tentacle withdrawal into the body cavity. Several seconds later the tentacle protrudes and at some moment the eye, clearly visible as a black dot, appears on the tentacle's upper surface. Each time when the eye appears on the tentacle surface an experimenter touches the tentacle

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with the electrode. As the animal tends to hold its tentacle contracted longer and longer, the time of the response becomes larger and larger, and the number of electric shocks becomes less and less. A session lasts 80, minutes and the number of electric shocks during every 4 minutes is counted. The exponential decrease of the number of stimulations during every session was found to fit significantly with the regression model (Stepanov, Kuntzevich, and Lokhov 1989). For this reflex the coefficient B3 means the initial value of stimulation prior to the beginning of conditioning. In other words, it means the reflex time before conditioning. B4 is the asymptotic level of the reflex time, and B2 is the velocity of a transition from the initial to the asymptotic level; the elaboration of the conditioned reflex takes place during every session. The memory fixation displays itself in a lowering down of curves from session to session. This was due to a decrease of B3 and sometimes B4. Figure 4.5 presents an application of the model to this situation. The intersession intervals were 24, 96, and 192 hours between every consecutive session. Curve 1 represents the first session. The regression coefficients are as follows: B2 = 0.28, B3 = 34.9, B4 = 8.3. The regression coefficients of session 2 are: B2= 0.29, B3 = 32.3, B4= 6.5; of session 3: B2= 0.17, B3 = 14.6, B4= 5.0; and of session 4: B2= 0.20, B3 = 16.7, B4= 3.5. The coefficients B2 of the second and the third curve and of the third and the fourth curves differ significantly (p < 0.001). The coefficients B3 of the second and the third curves and of the third and the fourth curves differ significantly (p < 0.001). The coefficients B4 of the first and the second curves and of the second and the third curves and of the third and the fourth curves differ significantly (p < 0.01, p < 0.05, p < 0.02). The advantage of the tentacle conditioned reflex is an opportunity to analyze the short-term memory formation during a session and the long-term changes reflected during an intersession interval. It is important to note that the length of the intersession interval is a crucial parameter for long-term memory formation. Christoffersen used intervals of 24, 96, and 192 hours between consecutive sessions. In our experiments, when sessions were run every day, only the short-term memory formation during every session could be observed (Stepanov et al. 1989). The memory consolidation during the intersession interval was absent, and learning curves were superimposed on each other. This result suggests that the memory consolidation needs rest and the absence of neuronal activity in the reflex arc. It is interesting that Pavlov himself used 2-3 day intervals between sessions of classical salivary feeding reflex elaboration in dogs (Pavlov 1951). That may be one of the very important features of memory "fixation" process. The retention of tentacle conditioned reflex lasts more than 3 weeks. As in the case of food avoidance, the coefficient B3 gives a quantitative parameter for reflex retention. There is one feature inherent to this reflex. If current intensity was chosen in the range that was enough to excite unilateral tentacle withdrawal only, then the second tentacle could be used as a control. All tentacle learning dynamics during a 20-minute session were found to be very similar to one another. This feature enlarges the opportunities to use this reflex.

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Figure 4.5 An application of the model to the instrumental conditioning of left tentacle withdrawal reflex in Helix after 1,2,3, or 4 training sessions.

HUMORAL COMPONENT OF LEARNING In spite of a large number of investigations, the problem of the role of chemical substances in the maintenance of memory and, consequently, memory transfer has not been solved (Mitchell, Beaton, and Bradley 1975). An investigation of the existence of a humoral component of conditioned reflexes in the snail Helix lucorum was therefore undertaken (Stepanov, Lokhov, Satarov, Kuntsevich, and Vartanian 1987a, 1987b). It was presumed that this problem might be easier to solve using mollusks because there is the possibility of linking behavioral experiments with neuronal ones performed on the identified neurons. An experiment of that kind was undertaken with the left and right tentacle withdrawal conditioned reflex. The idea of the experiment was that any condi-

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tioned animal at the same time is both donor and recipient. Five days after the last left tentacle conditioning session, the right tentacle first session was performed. The right tentacle learning curve was found to be lower in comparison with the first session curve of the left tentacle. The facilitation of contralateral tentacle learning could be due to the action of some chemical substance secreted into the blood during the first tentacle conditioning. A facilitation of learning was also observed in experiments in which donors were conditioned as usual and naive recipients were conditioned after being injected with trained donors' blood (Stepanov 1991). Their blood in the volume of 200-300 mcl was collected after every session. The part of the snail's foot, hidden under the shell in the normal position of the animal, was found to be nearly insensible to the insertion of a surgical needle, if the shell is turned along its longitudinal axis to the right side until this foot, which is lighter, comes out from under the shell. The needle can be inserted at a 45° angle into the left side