Excitation and Inhibition: Synaptic Morphology

EXCITATION AND INHIBITION

Synaptic Morphology

KOJI UCHIZONO, M.D.

Professor of Physiology, Faculty of Medicine, University of Tokyo, Tokyo

ELSEVIER SCIENTIFIC PUBLISHING COMPANY

Table of Contents

Cover image

Title page

Copyright

Preface

Discussion on the Differentiation of Excitation and Inhibition

Chapter 1: Introduction

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Chapter 2: Development of Concept of Excitation and Inhibition

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Chapter 3: S-F Hypothesis: Morphological Correlates of Excitation and Inhibition

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Chapter 4: Mechanism of Synaptic Excitation

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A ELECTROPHYSIOLOGY

B MORPHOLOGY OF EXCITATORY SYNAPSES

C EXCITATORY TRANSMISSION

Chapter 5: Mechanism of Inhibition

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A GENERAL REMARKS

B MECHANISM OF POSTSYNAPTIC INHIBITION

C MORPHOLOGY OF POSTSYNAPTIC INHIBITION

D CLASSIFICATION OF SYNAPSES INTO S-TYPE AND F-TYPE

E INHIBITORY TRANSMITTER SUBSTANCE

F MECHANISM OF PRESYNAPTIC INHIBITION

G PHARMACOLOGY OF PRESYNAPTIC INHIBITION

Chapter 6: Physiological Classification of Neurons

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A CRITERIA OF NEURON CLASSIFICATION

B CEREBELLUM

C HIPPOCAMPUS

D SPINAL CORD

E CRUSTACEA

Chapter 7: The Release Mechanism of Transmitters

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Chapter 8: Synaptic Vesicles as a Basis of Transmitter

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Chapter 9: Membrane Recycle

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Chapter 10: Transmission in the Sympathetic Ganglion

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Chapter 11: Transmitter Substance

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A ACETYLCHOLINE AS AN EXCITATORY TRANSMITTER

B ACETYLCHOLINE AS AN INHIBITORY TRANSMITTER

C GABA AND GLYCINE

D STRUCTURAL CHARACTERISTICS OF GABA- AND GLYCINE-SYNAPSE

E CATECHOLAMINE AND SEROTONINE

F TRANSMITTERS IN THE SENSORY SYSTEM: DORSAL ROOT PEPTIDE AND ACETYLCHOLINE

G CYCLIC AMP AND CEREBELLUM

H CYCLIC AMP AND ITS ROLE IN SYNAPTIC TRANSMITTER

Chapter 12: Criticism of S-F Hypothesis

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Electron Micrographs Indicative of Synaptic Differentiation in Various Parts of the Central Nervous System

Introduction to Electron Micrographs Indicative of Synaptic Differentiation in Various Parts of the Central Nervous System

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References

Author Index

Subject Index

Copyright

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© First edition, 1975 by IGAKU SHOIN LTD., 5-24-3 Hongo, Bunkyo-ku, Tokyo. All rights reserved. No part of this book may be translated or reproduced in any form by print, photoprint, microfilm, or any other means without written permission from the publisher.

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Preface

The problem of excitation and inhibition has been the central theme of neurophysiology since its foundation about a century ago. Since that time the mechanisms of excitation have been extensively studied by many workers in various fields of neurophysiology, having brought about much progress to this discipline. Mechanisms of excitation have been remarkably clarified at the cellular level and in terms of ions and molecules. However, studies on the mechanisms of inhibition have been much delayed, probably because the phenomena of inhibition do not show up positively in any ways. Excitation has its positive counterpart like contraction of muscle or depolarization of nerve, favoring the analytical investigations of neuronal activities.

In a simple model of neuromuscular preparation the excitation of nerve and muscle is positively expressed by the action potentials and/or contraction of muscle. Even with the ingenuity of SHERRINGTON he could not reveal any positive function directly associated with inhibition. During the SHERRINGTON’S time the notion of inhibition remained as a simple loss or disappearance of excitation. No reality of inhibition has been obtained in positive terms. The innate mechanisms of inhibition have remained vague during the past fifty years until ECCLES in 1960 dramatically succeeded in recording the negative potentials, hyperpolarization or negative potential changes: IPSPs.

ECCLES has laid a milestone in the history of physiology of synapses by establishing the synaptic potentials of excitation and inhibition. Extensive electrophysiological investigations by ECCLES and his associates have brought about tremendous progress in the elucidation of the mechanism of inhibition.

My interest has been focussed on the mechanism of excitation and inhibition expressed by electrical signs. However, my inquiry has not been satisfied until the reality of excitation and inhibition has been elucidated in terms of both structure and function. When I moved from Tokyo to Salt Lake City to work with Dr. HUNT, then at University of Utah, he so kindly recommended me to study fine structure of excitatory tissues under Dr. H. S. BENNETT, University of Washington, Seattle, U.S.A. I moved from Salt Lake City to Seattle to satisfy my inquiry into the relationship between function and structure of the nervous system.

Prof. BENNETT is a pioneer, noted electron microscopist in the world. At that time more than twenty scientists, young and old, were always gathered around him, from all over the world, producing an exciting academic climate. He is renowned by his pioneering works in ultrastructure of muscle and nerve. He will especially be remembered as a co-discoverer of the synaptic vesicles with Prof. E. De ROBERTIS, University of Buenos Aires, Argentina. I have been priviledged to join Prof. BENNETT’S laboratory with my fellow scientists in Seattle, some of whom had been the leading figures in the field of electron microscopy. It was a turning point in my personal history of researches. Since that time I have tried to build a bridge between physiology and morphology, my long cherished idea, but the task was not an easy one. It took me already fifteen years to realize only a small part of my dream. Interdiciplinary field seems to be wide open, but the work to cultivate it beatifully and meaningfully is quite a new discipline. A couple of years later, I was asked to move to University of Tokyo. It was opportune for me to combine two different disciplines, physiology and morphology, remodeling the old laboratory.

During the past fifteen years I have devoted myself to construct a new bridge which would lead us to an unprecedented field. I have been priviledged to have fellow physiologists and electron microscopists around me in my own laboratory. I do not think that my dream, even a part of it, has been realized, but I do say that the lane which we are treading would be a right one. The task to clarify the mechanism of excitation and inhibition, the most basic functions of the nervous system, has occupied my interest.

A possibility remains that the higher nervous activities may be correlated with these fundamental activities of the basic structure of the nervous system, synapse.

In this book a new hypothesis is proposed which emphasizes the relationship between structure and function of synapses. The S-F hypothesis, simple and immature it might be, will give us a new bastion to open a new frontier. The hypothesis seems to have provoked a discussion in some fields of neurophysiology, neuroanatomy and neurochemistry.

I would like to express my most sincere thanks for my fellow scientists who worked with me for the past decades. The book would not have been produced if the publisher would not have been interested in and sympathetic for my works. The book was produced by the real collaboration of the author and the publisher, Igaku Shoin Ltd. This author shall not forget the financial aid offered to him by the Japanese government for the past years. I would like to specifically mention the names of some of those who have worked with the author for many years. The work was supported by the excellent technical skill of Mr. E. KISHIMOTO, secreterial help by Miss M. NAKAJIMA and photographic skill of Miss M. OKUNOGI.

September, 1974

K. UCHIZONO, M. D.

Discussion on the Differentiation of Excitation and Inhibition

1

Introduction

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This chapter presents an introduction to the notion of inhibition and excitation in neuroscience. It was Decartes who first introduced the notion of inhibition in the physiology of nervous system. The idea of inhibition is a counterpart of the excitation in the physiology of the nervous system. The notion of excitation has been the central idea of neurophysiologists and neuroanatomists for the past century. They have been exclusively concerned with the excitatory activity of neurons, nerve fibers, and muscles without paying any special attention to the inhibitory action of the nervous system. Sherrington’s ingeneous insight into the physiology of central nervous system (CNS) gave rise to two opposite conceptions concerning the reflex activity, excitation, and inhibition of the CNS of mammals. The term synapse was first introduced by Sherrington (1897) into the physiology of the CNS. The development of synaptology as a major neurobiological discipline may be traced to two conceptual formulations, namely, the reticular theory and the neuron doctrine.

It was DECARTES who first introduced the notion of inhibition in the physiology of nervous system. The idea of inhibition is a counterpart of the excitation in the physiology of the nervous system. The notion of excitation has been the central idea of neurophysiologists and neuroanatomists for the past century. Fortuitously they have been exclusively concerned with the excitatory activity of neurons, nerve fibers and muscles without paying any special attention to the inhibitory action of the nervous system. It is interesting to note that, despite CAJAL’S commanding understanding of the structure of nervous system, he failed to recognize the existence and accordingly the importance of inhibitory neurons. He thought that the nervous system consisted exclusively of neurons specialized for excitatory action. It was pointed out by ECCLES (1964) that the CAJAL’S diagrams and those of his student, LORENTE DE NÓ (1934) showed no inhibitory synaptic action. SHERRINGTON’S ingeneous insight into the physiology of central nervous system (CNS) gave rise to two opposite conceptions concerning the reflex activity, excitation and inhibition of the CNS of mammals.

The term synapse was first introduced by SHERRINGTON (1897) into the physiology of the CNS. The development of synaptology as a major neurobiological discipline may be traced to two conceptual formulations, the reticular theory and the neuron doctrine. Nineteenth century histologists, upon examining the CNS with the light microscope, came to the idea that the nerve fibers in the CNS made profuse branching and these branches gave rise to profound anastomosing syncytial networks without interruption. This is the reticular theory which was first proposed by GERLACH (1871) who assumed that the nerve cells were situated at the nodes of the reticular structures. GOLGI (1885) adopted the same notion and expanded it to the general structure of the CNS. His conviction in the reticular theory was so strong as to make one believe that he was a real founder of the notion of the reticular structure of the CNS. His (1886, 1889) and FOREL (1887) proposed the idea that each nerve cell was isolated unit and its axon and branches did not anastomose, but merely entered into close contact in some way or other. It is historically remarkable that the most powerful opponent of the reticular theory was Ramón y CAJAL (1888, 1890 a, b, c) who, applying intensively GOLGI technique to the study of the structure of the CNS, had reached the same conclusion as proposed by the latter. It might be useless to cite all histologists who followed CAJAL to trace the history of impact between two schools, reticular theorists and neuron theorists. It was the neuron theory that insisted that nerve cells must enter into functional connection with one another by contiguity, but not by continuity. The notion of continuity of the neuronal networks was proposed by the reticular theorists who insisted that the nervous system was the endless meshworks with syncytial connections in the CNS. With the support of GOLGI, the reticular theory lingered on until as recently as 1925 when HELD (1905, 1909) was compelled to write in defense of the neuron theory. In view of this long lasting support for the reticular theory, CAJAL (1934) was also compelled to defend the neuron theory by examining critically the whole controversy between two rival theories. In spite of his unchallengeable criticisms, the old reticularists continued to believe in their hypothesis until as recently as 1940 when BOEKE (1940) reviewed the questions of neuroanatomy of the time. The observation of neurofibrils which were discovered by classical light microscopists in mid 18th century and have been investigated until the present time gave rise to the neurofibril hypothesis of the nervous system which held that neurofibrils were continuous from cell to cell and vital in impulse transmission. Throughout the first third of the 20th century a controversy was rampant over whether the neurofibrils were or were not continuous from cell to cell, and whether they were or were not involved in conduction or transmission in the nervous system. At the end of this period it was finally agreed that these fibrous structures in nerve were real and not fixation artifact (BOZLER, 1927; WEISS and WANG, 1936). By the advent of the electron microscope, however, it was definitely clarified that the neurofibrils were not continuous from cell to cell but were disrupted by 200 Å cleft at the synapse. Thus finally the reticular theory had to disappear. BODIAN (1942, 1952) and NONIDEZ (1944) must be cited as final unassailable defenders of the neuron theory, because they obtained one of the most convincing evidences of neuron theory from degeneration experiments. They showed that when the nerve fibers were sectioned the distal parts of the fibers degenerated up to the end of the fibers, but the degeneration did not spread beyond. This fact clearly demonstrated that in some way or other discontinuity exists in the CNS and a neuron is an isolated functional unit. It was as late as 1954 that the neuron theory was decisively established morphologically by the aid of the electron microscope, because the resolving power of the light microscope was inadequate to disclose the fine structure of the synapse at the level that was required to explain the electrophysiology of transmission of nerve impulses. It was the advent of the electron microscopy that unquestionably established the neuron theory, the reticular theory being completely abolished. At two hundreds Angstroms cleft between presynaptic and postsynaptic membranes was revealed by PALADE and PALAY (1954), PALAY (1956), De ROBERTIS (1956), De ROBERTIS and FRANCHI (1956), who completely wiped out all doubts about the structural discontinuity of the synapse. Structural assymmetry at the synapse was confirmed electron microscopically and the functional polarity of it was also revealed electrophysiologically. The impulses conduct unidirectionally at the synapse. Two way conduction takes place in the nerve fibers but not at the synapse. The higher resolving power provided by the electron microscope has allowed us to study the synapse at a level which was not attainable with the light microscope. We are now in the stage where we can obtain meaningful correlations between structure and function of the nervous system. The development of sophisticated techniques for intracellular recording of the nervous activity in the 1950’s led to an important advance in the concepts of neuronal functions. It became possible to penetrate a microelectrode into the cytoplasm of neurons through the excitable membrane without grossly damaging to the cell. It was revealed that the inside of the neuron is about 80 millivolts negative to the outside. It also became clear that this potential difference is the results of the selective distribution of ions across the semipermeable membrane. Excitatory and inhibitory synaptic potentials were found to consist of small depolarization and hyperpolarization of the postsynaptic membrane respectively caused by the different kinds of transmitter substances which are released from the nerve endings of the presynaptic fibers at the time of the arrival of nerve impulses.

The term synapse was merely introduced by SHERRINGTON (1897) to denote the specific physiological activity at a supposed junctional site. On a priori grounds it is unnecessary that a set of distinct structural specialization should be present. There is no theoretical necessity to identify specialized structure at synapses because communications between nerve cells might occur at morphologically non-specialized interface by ephaptic transmission or some other kinds of mechanisms. But, electrophysiology and electron microscopy surpassed such intriguing arguments, identifying morphological and functional entity of synapses unequivocally. The facts that synapses clearly exhibit certain well-defined structure and that these structural specializations can reflect the functional nature of the junction have allowed neurobiologists to argue both from function to structure and vice versa. Specification of the mode of transmission at a given synapse is understood and explained beautifully on the basis of ultrastructural findings. There is no doubt that the ultrastructural methods will provide us with useful information about the nervous organization and function in domains inaccessible to the microelectrode.

There is no doubt that the well defined disconitinuity exists at a synapse. About 200Å cleft separates the presynaptic from the postsynaptic element as shown in Fig. 1. Opposing pairs of arrows indicate the presynaptic and postsynaptic membane invaded by narrow extracellular space. This is a morphological correlate of a synapse which had been postulated by SHERRINGTON (1897) as a functional entity. Electron microscopy has finally established an unassailable basis for the concept of synaptic transmission.

Fig. 1 Discontinuity of the elements of the nervous system.

Two large and small synapses separated by a glial foot (Gl) filled with glial fillaments and a soma of postsynaptic neuron are indicated by opposing pairs of arrows. Each synapse contains many synaptic vesicles and a large synapse is equipped with a fairly large number of mitochondria (M) beside synaptic vesicles (SV). Peripheral part of a neuron is filled with endoplasmic reticulum. M: Mitochondria SV: Synaptic vesicles Gl: Glial filaments

2

Development of Concept of Excitation and Inhibition

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This chapter describes development of the concept of excitation and inhibition. It was Sherrington (1925) who first established experimentally the notion of inhibition in the nervous system. Prior to him, some ideas concerning the inhibition had been proposed by some physiologists. René Decartes was the first who introduced the notion of l’esprit animal and tried to explain the oculomotor activity based on the excitation and relaxation of the eye muscle. Weber and Weber (1845) were believed to be the first to find experimentally the phenomenon of inhibition. They showed that the stimulation of the vagus nerve slowed down and temporarily arrested the heart beats. These experiments were conducted more than a century ago. They recognized the phenomenon of inhibition at such early days considering the level of development of physiology of that time. At the same time, Biederman (1887) also found that there were two types of nerves in the crayfish, of which the one makes the claw muscle contract and the other makes it relax. These counteracting or antagonizing functions of the nervous system, however, were all of peripheral origin and the stimulation of