Durham E-Theses Investigations into the structure and function of nerve and skeletal muscle of anisoptebous odonata, with special reference to aeschnid nymphs Malpus, C. M. How to cite: Malpus, C. M. (1968) Investigations into the structure and function of nerve and skeletal muscle of anisoptebous odonata, with special reference to aeschnid nymphs, Durham theses, Durham University. Available at Durham E-Theses Online: http://etheses.dur.ac.uk/8803/ Use policy The full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that: • a full bibliographic reference is made to the original source • a link is made to the metadata record in Durham E-Theses • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders. Please consult the full Durham E-Theses policy for further details. Academic Support Oce, Durham University, University Oce, Old Elvet, Durham DH1 3HP e-mail: [email protected] Tel: +44 0191 334 6107 http://etheses.dur.ac.uk 2 INVESTIGATIONS. INTO THE STRUCTURE AND FUNCTION OF NERVE. AND SKELETAL. MUSCLE OF ANISOPTEHOUS ODONATA, WITH SPECIAL REFERENCE TO AESCHNID NYMPHS by C. MV MALPUS, B.SC.. (Dunelm) GREY: COLLEGE:. Being a thesis presented in candidature for the degree of Doctor of Philosophy of the University of Durham. January 1968 PUBLICATION A preliminary report of some of this work, comprising the subject matter of Chapter k and part of Chapter 7, has been published under the title of "Electrical and Mechanical Responses of the skeletal muscle of a primitive insect"' in Nature 215, 991 - 992. The. majority of Chapter 6 has been accepted for publication by Comparative Biochemistry and Physiology under the title "Fatigue-dependent facilitation of the slow skeletal muscle response of dragonfly nymphs". FIGURE 1. A late-instar larva of a species of Aeschna. probably A.juncaea. Ventral surface.. X k. L • V CONTENTS CHAPTER PAGE 1. GENERAL INTRODUCTION 1 2. THE EXPERIMENTAL ANIMAL AND ITS PREPARATION FOR EXPERIMENTATION 10 Introduction 10 The anatomy of the mesothoracic leg 14 Analysis of the haemolymph 17 The formulation and testing of an experimental saline ... ... ... .«. ••• 21 The excised preparation ... ... ... ... 24 Observation and display of electrical and mechanical responses 29 The glass micro-electrode ... ... ... ... ~*>h Stimulation techniques 38 3. THE PROPERTIES OF THE MUSCLE FIBRE MEMBRANE ... 44 Introduction ... ... ... 44 Methods 47 Results 48 Discussion 52 4. THE NORMAL PATTERN OF MUSCLE RESPONSE 56 Introduction 56 Methods 6l Results 62 Discussion 68 CHAPTER PAGE 5. THE VARIATION OF THE SIZE OF THE SLOW ACTION POTENTIAL 72 Introduction ... 72 Methods 73 Results and calculations 73 Discussion ... 82 6. THE FATIGUE OF THE SLOW RESPONSE 90 Introduction 90 Methods 92 Results ... ... •*• ... 93 Discussion 100 7. THE MECHANICAL SLOW RESPONSE AND ITS RELATIONSHIP TO ELECTRICAL EVENTS 102 Introduction 102 Methods 103 Results 104- Discussion Ill 8. THE EFFECT OF VARIATION OF DIVALENT CATIONS ON THE SLOW MUSCLE RESPONSE 117 Introduction ... ... ... 117 Methods 120 The effect of changes in calcium 121 The effect of changes in magnesium 129 Discussion ... ... ... ... ... 131 PAGE 9. GENERAL DISCUSSION AND CONCLUSIONS 139 APPENDIX A.- Additional Tables 1^9 APPENDIX B. Theory of the determination of membrane constants 163 APPENDIX" C. The correlation coefficient ... 16? APPENDIX D. Note on the calculation of facilitation 170 APPENDIX E. The use of the electronic computer 17^ SUMMARY 182 REFERENCES 187 CHAPTER 1 GENERAL. INTRODUCTION -.1. CHAPTER 1 GENERAL INTRODUCTION The science of quantitative neuromuscular physiology is approximately 20 years old, dating from the. invention of the intra-cellular micro-electrode (Graham & Gerard, 19^6;. Ling & Gerard, 19^9). In spite of the numerical superiority of the invertebrate animals it is perhaps not surprising that the great majority of experimental work has been carried out either on vertebrate animals or on invertebrate tissues such as the giant axons of squids and other molluscs. It was already clear by the early 1950s that hopes of a unified theory of the action of excitable tissues were to be disappointed, with the discoveries by such workers as Fatt & Katz (in crustaceans) and Hoyle (in insects) that, invertebrate muscle membranes do not show all-or-nothing electrical responses to neural stimulation, and that some types do not even show an active membrane response. As a result of these and other discoveries, the fields of vertebrate and invertebrate neuromuscular physiology have to a large extent separated, with many common techniques, but with few concrete theoretical links'. EKBI It is now clear that the overwhelming majority of synaptic junctions of animals with discrete nervous systems operate, through chemical mediation; only in a few animals does any possibility of essentially ephaptic transmission exist;, the neuromuscular junction of skeletal muscle is, of course, simply a chemically- mediated synapse, that is to say the presynaptic impulse causes release of a chemical agent or transmitter substance which stimulates the post-synaptic membrane to produce a post-synaptic impulse. This mechanism, and the evidence for it, are fully set out by Katz (196l). It has also become apparent that the basic mechanisms that underlie nerve action in all animals are. similar; the ionic hypothesis (Hodgkin, 1951) can be. applied to giant axons of squids as well as to the. myelinated, nerve fibres of frogs and mammals, and recent work indicates similar processes in the cockroach (Narahashi, 1965);. even in herbivorous insects, which have apparently hostile, ionic concentrations in their haemolymph, similar processes are. at work, although details of the mechanism whereby the charge is transported across the membrane during activity may be different, and active components in extra-axonal sheaths may also be involved (Treherne, 1966;, Treherne & Maddrell, 196?).- It is in the mechanisms of muscle action that the great differences between vertebrate and invertebrate lie. These differences seem to originate from two causes, the relative inexcitability and leakiness of the invertebrate muscle fibre membrane (e.g. Fatt & Katz, 1933) and the virtual absence in invertebrates of all-or-nothing responses - either mechanical or electrical (Hoyle, 1957i Wiersma, 196l). Early workers on invertebrate material, such as Pringle (1939) and Wiersma & van Harreveld (1938) established the presence of two distinct types of muscle- responses;, these were defined as "fast" (or "twitch') and "slow" (or "tonic"), the definitive factor being the time- course characteristics of the mechanical response. With the advent of the intracellular recording technique it has become apparent that many invertebrate muscle fibres exhibit both types of response, being innervated by more than one axon per muscle fibre (see, e.g. Hoyle, 1957; Wiersma, 1961). In vertebrates both types of response can also be found, but occur in completely separate muscle fibres; multiple axon innervation of a single fibre is not found. The picture which has emerged of neuromuscular transmission in vertebrate twitch muscle fibres (the vast majority of vertebrate fibres) shows a pattern which is fairly uniform within the vertebrates, but unlike any responses found elsewhere. Katz (1961, 1966) gives a full description of the standard transmission type found in vertebrate twitch systems. The non-propagated responses of vertebrate slow fibres, first described by Kuffler & Vaughan Williams (1953a & b), are much more like those of invertebrates, and for this reason are often thought primitive. Typical vertebrate slow responses are shown by Bach-y-Eita & Ito (1966). Unlike the twitch fibres, slow fibres of vertebrates are usually multiterminally innervated, i.e. the innervating axon synapses with the muscle fibre at many points, or end-plates. This type of multiple innervation is normal in invertebrate fibres, which are often multi-axonally innervated as well. The similarity between vertebrate slow muscle fibres and the fibres of invertebrates is underlined by the recent discovery that vertebrate slow muscle fibres have a membrane capacity and resistance several times greater than do vertebrate twitch fibres (Adrian & Peachey, 1965)f invertebrate muscle fibres also have relatively large membrane capacities and resistance (e.g. Fatt & Katz, 1953). Within the phylum Arthropoda no coherent picture of neuromuscular transmission has appeared comparable to that seen in vertebrates. It is generally thought that the arthropods, a rather diffuse grouping, represents an adaptive radiation and evolution of types from a basic metamerically-segmented annelid-like form or forms. If this is true, it is perhaps not surprising that a multi• plicity of modes of physiological action has evolved. In the case of transmission of nerve impulses it is becoming apparent that mechanisms have been evolved, where necessary, to adapt axonal activity in detail, rather than fundamentally, where hostile environments are provided by, e.g., exotic haemolymphs (Treherne & Maddrell, 1967). In the case of the action of the muscle membrane, however, present knowledge shows such a variety of forms that the conclusion that some of the basic mechanisms have changed, or have evolved separately, is inevitable. Two major arthropod groups